national centre for biological sciences tata institute of...

national centre for biological sciences tata institute of fundamental research

Savita Ayyar, PhD Head, Research Development Office Ref:TFR:NCB:RDO:11 22 March 2011 Dr. T.S.Rao Adviser Department of Biotechnology Ministry of Science and Technology Block–2 (8th Floor), CGO Complex, Lodi Road, New Delhi – 110003 Tel.: 011-24364065, Fax : 011-24362884 [email protected] Dear Dr Rao, Sub: Project titled "Lynx in organization and dynamics of nicotinic acetylcholine receptor complexes" I am happy to forward the above titled grant entitled on behalf of Prof Satyajit Mayor, Dr Julie Miwa and Prof Henry Lester to the DBT. This grant proposal has been written in response to the recent call for proposals under the Indo-US Brain Research Collaborative Partnerships Programme. It has already been submitted to the NIH via their online application system. I believe that you will find the proposal complete in all respects. I shall be happy to provide any further information in case you need it. Thanking you, Yours sincerely,

Dr Savita Ayyar Head, Research Development Office National Centre for Biological Sciences [email protected] GKVK, Bellary Road. Bangalore 560 065. India

Phone 91 80 23666001/1/18/19 Fax 91 80 23636662

www.ncbs.res.in

State Application Identifier

Applicant Identifier

1. * TYPE OF SUBMISSION 4. a. Federal Identifier

5. APPLICANT INFORMATION * Organizational DUNS:* Legal Name:

Department: Division:

* Street1:

Street2:

* City:

* State:

* ZIP / Postal Code:* Country:

Person to be contacted on matters involving this application* First Name: Middle Name:

* Last Name: Suffix:

* Phone Number: Fax Number:

Email:

6. * EMPLOYER IDENTIFICATION (EIN) or (TIN):

7. * TYPE OF APPLICANT:

Other (Specify):

Women Owned Socially and Economically DisadvantagedSmall Business Organization Type

If Revision, mark appropriate box(es).

9. * NAME OF FEDERAL AGENCY:

A. Increase Award B. Decrease Award C. Increase Duration D. Decrease Duration

E. Other (specify):

10. CATALOG OF FEDERAL DOMESTIC ASSISTANCE NUMBER:

* Is this application being submitted to other agencies?

TITLE:

11. * DESCRIPTIVE TITLE OF APPLICANT'S PROJECT:

2. DATE SUBMITTED

3. DATE RECEIVED BY STATEAPPLICATION FOR FEDERAL ASSISTANCE

SF 424 (R&R)

County / Parish:

Province:

Prefix:

What other Agencies?

Pre-application Application Changed/Corrected Application

03/22/2011

009584210

California Institute of Technology

1200 E. California Blvd.

Office of Sponsored Research

Pasadena Los Angeles

91125-0001

Lucy

Molina

626-795-4571

[email protected]

951643307

Lynx in organization and dynamics of nicotinic acetylcholine receptor complexes

Yes No

USA: UNITED STATES

CA: California

O: Private Institution of Higher Education

National Institutes of Health

626-395-2372

New Resubmission

Renewal Continuation Revision

8. * TYPE OF APPLICATION:

OMB Number: 4040-0001 Expiration Date: 06/30/2011

b. Agency Routing Identifier

12. PROPOSED PROJECT:* Start Date * Ending Date09/01/2011 08/31/2013

* 13. CONGRESSIONAL DISTRICT OF APPLICANT

CA-029

14. PROJECT DIRECTOR/PRINCIPAL INVESTIGATOR CONTACT INFORMATION* First Name: Middle Name:


Position/Title:

* Organization Name:


* Street1:

Street2:

* City:



* Email:

* State:

County / Parish:

Province:

Prefix:

Miwa

MJulie

91125-0029

MC 156-29

Biology

Pasadena

1200 E. California Boulevard

Senior Research Fellow


626-564-8709

[email protected]

CA: California

USA: UNITED STATES

626-395-6221

APPLICATION FOR FEDERAL ASSISTANCESF 424 (R&R) Page 215. ESTIMATED PROJECT FUNDING 16. * IS APPLICATION SUBJECT TO REVIEW BY STATE EXECUTIVE

ORDER 12372 PROCESS?

a. YESa. Total Federal Funds Requested

17. By signing this application, I certify (1) to the statements contained in the list of certifications* and (2) that the statements herein are true, complete and accurate to the best of my knowledge. I also provide the required assurances * and agree to comply with any resulting terms if I accept an award. I am aware that any false, fictitious. or fraudulent statements or claims may subject me to criminal, civil, or administrative penalities. (U.S. Code, Title 18, Section 1001)

19. Authorized Representative

* First Name: Middle Name:


* Position/Title:

* Organization:


* Street1:

Street2:

* City:

* State:



* Email:

* Signature of Authorized Representative * Date Signed

20. Pre-application

* The list of certifications and assurances, or an Internet site where you may obtain this list, is contained in the announcement or agency specific instructions.

County / Parish:

c. Total Federal & Non-Federal Funds

18. SFLLL or other Explanatory Documentation

Province:

b. Total Non-Federal Funds

Prefix:

445,500.00

0.00

445,500.00

0.00

* I agree

Lucy

Molina

Contract and Grant Analyst


Research Administration


Mail Code 201-15

Pasadena Los Angeles

CA: California

USA: UNITED STATES 91125-0001

626-395-2372 626-795-4571

[email protected]


View AttachmentDelete AttachmentAdd Attachment

Add Attachment Delete Attachment View Attachment

DATE:

THIS PREAPPLICATION/APPLICATION WAS MADE AVAILABLE TO THE STATE EXECUTIVE ORDER 12372 PROCESS FOR REVIEW ON:

PROGRAM HAS NOT BEEN SELECTED BY STATE FOR REVIEW

PROGRAM IS NOT COVERED BY E.O. 12372; OR

Lucy Molina 03/22/2011

b. NOd. Estimated Program Income

424 R&R and PHS-398 SpecificTable Of Contents Page Numbers

SF 424 R&R Face Page------------------------------------------------------------------------------------------ 1

Table of Contents--------------------------------------------------------------------------------------------- 3

Performance Sites--------------------------------------------------------------------------------------------- 4

Research & Related Other Project Information------------------------------------------------------------------ 5

Project Summary/Abstract (Description)---------------------------------------- 6

Public Health Relevance Statement (Narrative attachment)---------------------------------------- 7

Facilities & Other Resources---------------------------------------- 8

Equipment---------------------------------------- 10

Other Attachments---------------------------------------- 12

1__biosketches___sup_ltrs_total_amd1003353140---------------------------------------- 12

collaboration_plan_par_11_0991003353128---------------------------------------- 32

Research & Related Senior/Key Person-------------------------------------------------------------------------- 33

Biographical Sketches for each listed Senior/Key Person---------------------------------------- 35

PHS 398 Specific Cover Page Supplement------------------------------------------------------------------------ 45

PHS 398 Specific Modular Budget------------------------------------------------------------------------------- 47

Personnel Justification---------------------------------------- 50

PHS 398 Specific Research Plan-------------------------------------------------------------------------------- 54

Specific Aims---------------------------------------- 55

Research Strategy---------------------------------------- 56

Vertebrate Animals---------------------------------------- 62

Multiple PI Leadership Plan---------------------------------------- 64

Bibliography & References Cited---------------------------------------- 65

Resource Sharing Plan---------------------------------------- 67

PHS 398 Checklist--------------------------------------------------------------------------------------------- 68

Table of Contents Page 3

Principal Investigator/Program Director (Last, first, middle): Miwa, Julie, M

County:

* ZIP / Postal Code:

* Country:

* State:

* City:

Street2:

Organization Name:

Project/Performance Site Location(s)

Project/Performance Site Primary Location

* Street1:

Province:


0095842100000

1200 E. California Blvd

MC 156-29

Pasadena

CA: California

USA: UNITED STATES


I am submitting an application as an individual, and not on behalf of a company, state, local or tribal government, academia, or other type of organization.

DUNS Number:

CA-029* Project/ Performance Site Congressional District:

Project/Performance Site Location 1

USA: UNITED STATES

* ZIP / Postal Code:

* Country:

Province:

* State:

* City:

Street2:

* Street1:

Organization Name:

County:

DUNS Number:

* Project/ Performance Site Congressional District:

I am submitting an application as an individual, and not on behalf of a company, state, local or tribal government, academia, or other type of organization.

Additional Location(s) View AttachmentDelete AttachmentAdd Attachment

Performance Sites Page 4


1. * Are Human Subjects Involved?

IRB Approval Date:

Human Subject Assurance Number:

2. * Are Vertebrate Animals Used?

IACUC Approval Date:

Animal Welfare Assurance Number

4.b. If yes, please explain:

4.c. If this project has an actual or potential impact on the environment, has an exemption been authorized or an environmental assessment (EA) or environmental impact statement (EIS) been performed?

4.d. If yes, please explain:

6. * Does this project involve activities outside of the United States or partnerships with international collaborators?

6.b. Optional Explanation:

7. * Project Summary/Abstract

11. Equipment

8. * Project Narrative

12. Other Attachments

RESEARCH & RELATED Other Project Information

Is the IACUC review Pending?

If no, is the IRB review Pending?

2.a. If YES to Vertebrate Animals

3. * Is proprietary/privileged information included in the application?

4.a. * Does this project have an actual or potential impact on the environment?

6.a. If yes, identify countries:

1 2 3 4 5 6

9. Bibliography & References Cited

10. Facilities & Other Resources

Yes No1.a If YES to Human Subjects

Yes No

Yes No

If yes, check appropriate exemption number.

Yes No

04/12/2010

A3426-01

Yes No

Yes No

Yes No

Yes No

Yes No

India

View AttachmentDelete AttachmentSummary_Abstract_PAR_11_099_JM1003353 Add Attachment

View AttachmentDelete AttachmentAdd Attachmentpublic_health_narrative_HAL1003353120

View AttachmentDelete AttachmentAdd AttachmentBibliography___Refs_Cited_PAR_11_099_

View AttachmentDelete AttachmentAdd AttachmentFacilities__Resources_Complete1003353

View AttachmentDelete AttachmentAdd AttachmentEquipment_Total1003353110.pdf

View AttachmentsDelete AttachmentsAdd Attachments

Is the Project Exempt from Federal regulations?

5. * Is the research performance site designated, or eligible to be designated, as a historic place? Yes No

5.a. If yes, please explain:

Other Information Page 5


Project Summary Title: Lynx in organization and dynamics of nicotinic acetylcholine receptor complexes Channelopathies are diseases of ion channel dysfunction, arising either in channel proteins themselves or in associated proteins. They can arise either genetically or via autoimmune reaction. Mis-regulation of nicotinic acetylcholine receptors (nAChRs) is linked to neural disorders, including schizophrenia, some epilepsies, nicotine addiction, myasthenia gravis, Alzheimer’s disease, and Parkinson’s disease. Common findings are that the number and localization of nAChRs are altered during in the disease state, and that restoration of their correct number and distribution can ameliorate disease phenotypes. Localization in synaptic vs extrasynaptic areas is also a crucial aspect of nAChR function. Lynx proteins are widely expressed regulators of nicotinic receptor function. Removal of the lynx1 gene results in nAChR hypersensitivity, enhanced learning, and extended critical periods for binocular selectivity, but also susceptibility to neurodegeneration. Genetic, biochemical, and physiological studies indicate direct or indirect interactions between α7 and α4β2 nAChRs. Lynx proteins have (a) GPI anchors and (b) structural and functional similarities to the soluble nAChR ligands, α-bungarotoxin and related toxins from snakes and snails. GPI-anchored membrane proteins can influence the distribution and mobility of surface receptors, through their preferential association with well-ordered, cholesterol-rich domains and nanodomains that accumulate signaling molecules and cytoskeletal components at the plasma membrane. These molecular events can produce an altered microenvironment, influencing receptor distribution and lifetime at the cell surface. Therefore, this project investigates the effect of lynx-nAChR interactions on the mechanisms of nAChR trafficking to the plasma membrane, the distribution of nAChRs on the plasma membrane, the mechanisms of internalization of surface nAChRs, and the underlying interactions with cytoskeletal and trafficking machinery.

Project Description Page 6


The worldwide health burden of neural disease is projected to increase 5-fold by year 2050, to >$1trillion/year. Optimal therapies do not yet exist for several neural diseases that involve nicotinic receptors: Alzheimer’s disease, cognitive dysfunction, schizophrenia, nicotine addiction, some epilepsies, and myasthenia gravis. Nicotinic receptors are also promising targets for therapeutics, for instance in Parkinson’s disease and mood disorders.

Public Health Relevance Statement Page 7


Facilities, other resources, and equipment at Caltech Laboratory:

About 2000 sq ft of contiguous space are available for this project on the 3rd floor of the Kerckhoff building at Caltech. The cell culture and nucleic acid molecular biology facilities are adequate for the project.

Clinical: N/A Animal: The Office of Laboratory Animal Resources at Caltech OLAR has space and facilities adequate to

handle the contemplated strains of mice. OLAR’s Web site is http://www.its.caltech.edu/~olar/procedures/breeding.html. The NIH does not allow reviewers to examine “private” Web sites, because users can be traced by their IP addresses. Therefore we will supply a pdf of the OLAR Web site if the reviewers request.

One of OLAR’s divisions is the Transgenic Animal Facility at Caltech, directed by Ms. Shirley Pease. Although no new strains are proposed in this application, Ms. Pease has ~ 20 yr of experience with these techniques; she has generated all of our previous knock-in and transgenic lines. Ms. Pease will work with us to generate any backcrosses to C57Bl/6 mice.

Computers:

The lab has Windows computers in each rig (See Equipment section). Networked computers (“GABA”, “Serotonin”, “Galanin”, and “Glutamate”) will be available to this project for data analysis, communications, and word processing. The project uses Matlab and its Optimization Toolbox. Caltech has site licenses for both.

Office:

All professionals on this project have office space. Other:

Electronics shops, machine shops and computer consultants adequate for supporting the project. The Caltech Biological Imaging Center is a world-renowned research for advanced imaging, image analysis algorithms, and other techniques that may prove useful in this project.

Specialized equipment in the Caltech lab (see Equipment section)

Facilities Page 8


National Centre for Biological Sciences (NCBS) Facilities

NCBS has access to several world-class research facilities, details of which can be accessed at http://www.ncbs.res.in/research_infrastructure.

Internet technology support: NCBS has centralized IT support for all its personnel. IT provides services for the installation of software and hardware, maintenance of computer systems and the administration of network facilities within NCBS. Hands on technical support is available for Windows, Apple and Linux based systems and applications. In addition, Linux based high performance computing clusters are also available.

Library: The library is housed on three levels: the ground floor has a large, well-lit reading room and journal stacks and textbooks are available on the other floors. The library holds approximately 5000 books, 9500 bound volumes, 575 CD/DVDs and subscribes to 140 scientific journals in each year. More than 200 books and 1000 bound volumes are added on average to its collection every year. Electronic subscriptions are available for online access to many major and specialized journals and journal articles from unsubscribed titles are available quickly via online purchases.

Facilities Page 9


Caltech Equipment The proposed project will employ several advanced rigs for imaging and electrophysiology. “Golgi” and “Katz” do not presently have electrophysiology; the Aims do not require electrophysiology on these rigs. However, if required, we have installed electrophysiology on these rigs in the past and can do so again.

*we have extensively modified these instruments

Optics → mixed → Electrophysiology Oocytes

Rig & location

FS 200 Crellin

“Golgi” 327 K

“Katz” 325A K

“Hartline” 325A K

“Erlanger” 323 K

“Kandel” 325 K

Specific Aim -- 1 1 1

Primary use

Flexstation:

Molecular Devices

Fluorescent Plate reader

Cultures & slices

Epi-fluorescence

TIRF

Whole-cell patch, TIRF

Whole-cell patch; Single

channels

OpusXpress

8 channel parallel

oocyte ephys

Microscope Nikon C1 si*

Confocal

Olympus IX71* 1.45 & 1.65NA lenses

Olympus IX70*

Nikon Diaphot

300

Light source or

Lasers

Argon 488/514;

Diode 440; DPSS 561

Argon 488/514 nm

Arc lamp &

Argon 488

Arc lamp

Image recording

PMTs. spectral array, FLIM

Andor iXon+ CCD; focus hold

Video camera

Video camera

Physiology Manipulators -- -- Two Sutter Burleigh

Drug application

Robot pipettes

Temperature-controlled chamber

Temperature-controlled chamber

Dynaflow

Picospritzer on

Piezoelectric manipulator

Robot pipettes

Electrode Amplifiers -- -- Axon

Multiclamp

Two Axopatch

200

Axon Geneclamp

Software Molecular devices Nikon Andor* pCLAMP 9 pCLAMP 10 pCLAMP 9

Equipment Page 10


National Centre for Biological Sciences (NCBS) Equipment

Shared (between 30 independent PIs) facilities at NCBS include a well-managed Central Imaging and Flow cytometry Facility (CIFF; equipped with one Transmission Electron Microscope (TEM) and an Atomic Force Microscope (AFM); live cell imaging facilities at the nanoscale including 8 confocal microscopes, one near field scanning optical microscope (NSOM), and a STED microscope, as well as six different flow cytometers), an Animal House for the upkeep of transgenic and wildtype mice, rats, Xenopus and Zebrafish, a Mass Spectrometry facility, a Mouse genetics facility for the generation of custom transgenic mice, a transgenic fly facility for the generation of custom transgenic Drosophila, and mechanical, electrical and electronics workshop. Researchers at NCBS also have access to additional research facilities via the Centre for Cellular and Molecular Platforms, details of which can be accessed at http://www.ccamp.res.in/.

Equipment Page 11


BIOGRAPHICAL SKETCH Provide the following information for the Senior/key personnel and other significant contributors.

Follow this format for each person. DO NOT EXCEED FOUR PAGES.

NAME Ying Wang

POSITION TITLE Postdoctoral Scholar in Biology

eRA COMMONS USER NAME (credential, e.g., agency login) wangyiusc EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, include postdoctoral training and residency training if applicable.)

INSTITUTION AND LOCATION DEGREE (if applicable)

MM/YY FIELD OF STUDY

University of Science and Technology of China, Hefei, Anhui, China B.S. 07/2004 Biological Sciences

University of Southern California,Los Angeles,CA Ph.D. 08/2009 Neuroscience University of Southern California,Los Angeles,CA postdoc 10/2009 Neuroscience California Institute of Technology postdoc present Neuroscience

A. Personal Statement I have considerable experience with working on alpha 7 nicotinic acetylcholine receptors (nAChRs) and have published work on the mechanisms involved in the assembly and surface expression of alpha7 nAChRs. I am currently a postdoctoral scholar in Dr. Henry Lester’s laboratory and I have constructed a suite of fluorescently labeled alpha 7 nAChR constructs that are suitable to be used in this proposal. I routinely use these constructs for high-resolution imaging experiments in living cells, using techniques such as TIRF microscopy and spectrally-resolved confocal imaging. These experiments are done in conjunction with labeled a-bungarotoxin experiments to measure the ratio of surface to total levels of receptor in the cell. I will be performing the alpha 7 nAChR experiments outlined in specific aim 1. B. Positions and Honors

ACTIVITY/OCCUPATION

BEGINNING DATE

(mm/yy)

ENDING DATE

(mm/yy) FIELD INSTITUTION/COMPANY SUPERVISOR/

EMPLOYER

Graduate Teaching Assistant Graduate Research Assistant

08/04

05/05

05/05

08/09

Biology Neuroscience

University of Southern California University of Southern California

Zuo-Zhong Wang, Ph.D. Zuo-Zhong Wang, Ph.D.

Postdoctoral Scholar Postdoctoral Scholar

08/09

01/10

10/09

present

Neuroscience Neuroscience

University of Southern California California Institute of Technology

Jeannie Chen, Ph.D. Henry A. Lester, Ph.D.

Professional Societies: Society for Neuroscience (SFN) C. Selected Peer-reviewed Publications 1) Wang, Y., Yao, Y., Tang, X.-Q., and Wang, Z.-Z. (2009). Mouse RIC-3, an endoplasmic reticulum

chaperone, promotes assembly of the alpha7 acetylcholine receptor through a cytoplasmic coiled-coil domain. J Neurosci 29:12625-12635.

2) Linnoila, J., Wang, Y., Yao, Y., and Wang, Z.-Z. 2008. A mammalian homologue of Drosophila tumorous imaginal discs, Tid1, mediates agrin signaling at the neuromuscular junction. Neuron 60(4):625-41.

D. Research Support N/A

1__biosketches___sup_ltrs_total_amd1003353140 Page 12


BIOGRAPHICAL SKETCH Provide the following information for the key personnel and other significant contributors in the order listed on Form Page 2.


NAME Tim Indersmitten

POSITION TITLE Postdoctoral Scholar in Biology California Institute of Technology eRA COMMONS USER NAME

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training.)


YEAR(s) FIELD OF STUDY

University of Nijmegen, The Netherlands B.A. 2000 Psychology University of Nijmegen, The Netherlands M.S. 2003 Neuro & rehab Psych University of California, Los Angeles Ph.D 2010 Neuroscience California Institute of Technology Postdoc Present

A. Personal Statement I will be performing the patch-clamp electrophysiological and pharmacological studies outlined in this proposal. I am an experienced electrophysiologist who has a strong background in neuroscience as it relates to plasticity and neurological disease. My current project involves the pharmacological characterization of alpha 7 nAChR mutants using patch-clamp electrophysiology in transiently transfect mammalian cells. B. Positions and Honors:

07/2004 – 2010 Graduate Student Researcher, Departments of Physiology, Neurology and Physiological Sciences, UCLA

03/2003 – 06/2004 Research Specialist, Brain Behavior Laboratory, Dept. Psychiatry – Neuropsychiatry,

University of Pennsylvania Hospital

ACADEMIC AND PROFESSIONAL HONORS 12/2007 – 05/2009 Representative for the Neuroscience Interdepartmental Program

Committee. University of California, Los Angeles 10/2006 – 05/2007 Vice President of the Biological Sciences Council University of California, Los Angeles 10/2006 – 05/2007 Graduate Student Association Representative for the Biological Sciences University of California, Los Angeles

2001 C. Publications:

1. Coba MP, Komiyama NH, Kopanitsa MV, Nithianantharajah J, Stanford LE, Tuck EJ, Skene N, Indersmitten T, Saksida LM, Bussey TJ, O’Dell TJ, & Grant SGN. (in review). TNiK complexes link multiple schizophrenia genes and mechanisms.

2. Grant SGN, Komiyama NH, Indersmitten T, Watabe A, Moody T, & O’Dell TJ (in press). Beta-Adrenergic Receptor Activation Rescues Theta Frequency Stimulation Induced LTP in Mice Expressing C-Terminally Truncated GluN2A NMDA Receptor Subunits.

3. Carlisle HJ, Luong TN, Medina-Marino A, Schenker L, Khorosheva E, Indersmitten T, Gunapala K, Steele K, O’Dell TJ, Patterson PH, & Kennedy MB (in press). Mice with a deletion of densin-180 have reduced neuronal metabotropic glutamate receptors and display schizophrenia-like behaviors.



4. Kohler CG, Loughead J, Ruparel K, Indersmitten T, Barrett FS, Gur RE, & Gur RC. (2008). Brain activation during eye gaze discrimination in stable schizophrenia. Schizophrenia Research. 99(1-3): 286-293.

5. Turetsky BI, Kohler CG, Indersmitten T, Bhati MT, Charbonnier D, & Gur RC. (2007). Facial emotion recognition in schizophrenia: when and why does it go awry? Schizophrenia Research. 94(1-3): 253-263.

6. Verma R, Davatzikos C, Loughead J, Indersmitten T, Hu R, Kohler C, Gur RE, & Gur RC. (2005). Quantification of facial expressions using high-dimensional shape transformations. Journal of Neuroscience Methods. 141(1): 61-73.

7. Indersmitten T, & Gur RC. (2003). Emotion processing in chimeric faces: hemispheric asymmetries in expression and recognition of emotions. Journal of Neuroscience. 23(9): 3820-3825.

D. Research Support N/A





NAME Larry Wade

POSITION TITLE Principal-technical staff, Analytical Chemistry Group Jet Propulsion Laboratory, California Institute of Technology

eRA COMMONS USER NAME (credential, e.g., agency login) EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, include postdoctoral training and residency training if applicable.)



California Institute of Technology Ph.D. 08/2010 Molecular Biology & Biochemistry

A. Personal Statement I have obtained considerable experience in optics, and invented new methods for super-resolution microscopy. I also developed variable-angle TIRF microscopy for the purpose of distinguishing nicotinic receptors in the endoplasmic reticulum from those on the plasma membrane. I will provide technical advice and oversee the TIRF measurements outlined in this proposal, and work with Rell Parker, a graduate student under Dr. Miwa’s direction and/or post-doctoral fellow Ying Wang on the TIRF experiments. B. Positions

ACTIVITY/OCCUPATION

BEGINNING DATE

(mm/yy)

ENDING DATE

(mm/yy) FIELD INSTITUTION/COMPANY SUPERVISOR/

EMPLOYER

Principal technical staff

1991

present

Analytical Chem

Jet Propulsion Lab California Institute of Technology

.

Visitor in Applied Physics

2000 2004 Applied Physics California Institute of Technology

Honors Awarded NASA Exceptional Technology Achievement Medal 2010 Professional Societies:

International Cryogenic Cooler Conference Board Member and Program Committee 1998-2004 SPIE Space Telescopes Conference Program Committee 1996 and 1998 Space Cryogenics Conference Chairman 1997 Chaired numerous conference sessions on cryogenics and cryocoolers at International Cryogenic

Cooler, Cryogenic Engineering, and Space Cryogenics conferences. Member American Physical Society Member Biophysical Society Member American Society for Cellular Biology Member Society for Neuroscience C. Selected Peer-reviewed Publications Planck Collaboration, Planck Early Results: The Planck mission, sub Astro & Astrophys, 2011. Planck Collaboration, Planck Early Results: The thermal performance of Planck, sub Astro & Astrophys, 2011.



Planck Collaboration, Planck early results: First assessment of the Low Frequency Instrument in-flight performance, sub Astro & Astrophys, 2011. Planck Collaboration, Planck early results: first assessment of the High Frequency Instrument in-flight performance, sub Astro & Astrophys, 2011. Planck Collaboration, Planck Early Results: The Low Frequency Instrument data processing, sub Astro & Astrophys, 2011. L. A. Wade, C. P. Collier and S. Fraser, Single-Biomolecule Resolution Imaging with an Optical Microscope, Proc. Microsc. Microanal. 11 (Suppl 2) (2005): 146-147. L. A. Wade, I. R. Shapiro, Z. Ma, S. R. Quake, and C. P. Collier, Correlating AFM Probe Morphology to Image Resolution for Single-Wall Carbon Nanotube Tips, Nano Lett., 2004, 4, 725-731. L. A. Wade, I. R. Shapiro, Z. Ma, S. R. Quake, and C. P. Collier, Single-molecule Fluorescence and Force Microscopy Employing Carbon Nanotubes, Nanotech 2003, 2003, 3, 317. L. A. Wade, C. A. Lindensmith, Low-Power, Zero Vibration 5K Sorption Coolers for Astrophysics Instruments, Advances in Cryogenic Engineering, AIP Conference Proceedings, AIP, Melville, New York, Volume 47, 2002, pg 1225-1232. Hydrogen Sorption Cryocoolers For The Planck Mission, Advances in Cryogenic Engineering 45, Plenum Press, New York (2000), pp 499-506. D. Research Support N/A





NAME Parker, Rell Lin

POSITION TITLE Graduate Student in Biology California Institute of Technology eRA COMMONS USER NAME (credential, e.g., agency login)

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, include postdoctoral training and residency training if applicable.)



University of California, Berkeley BA 05/06 Molecular and cell Biology

University of California, Davis In progress Veterinary medicine

NIH, NINDS, Bethesda, MD HHMI-NIH Research Scholar

07/09 Cell biology and neuroscience

California Institute of Technology, Pasadena, CA In progress Neuroscience

Please refer to the application instructions in order to complete sections A, B, C, and D of the Biographical Sketch. A. Personal Statement I will study the role of lynx1 in the trafficking and membrane localization of nAChRs and as a chaperone of nAChRs. As an undergraduate at UC Berkeley I studied molecular and cellular biology, with an emphasis in neuroscience. I then attended veterinary school at UC Davis, where I received training in anatomy, physiology, pharmacology and pathology while completing the first two years of veterinary school. This experience has given me insight into animal models of disease. Following the second year of veterinary school, I completed a year-long fellowship at the NIH through the HHMI-NIH research scholars program. While at the NIH, I studied the cell biology of hereditary spastic paraplegia. These studies gave me experience in studying the cell biology of disease, using molecular biology and cell culture techniques. While at Caltech, I have taken a microscopy lecture and lab, which has helped prepare me for the proposed studies. B. Positions and Honors 2008-2009 HHMI-NIH Research Scholar 2009-present Graduate Student, California Institute of Technology Academic and Professional Honors 2007 Students Training in Advanced Research Fellowship, UC Davis C. Publications Park SH, Zhu P-P, Parker RL, Blackstone C. 2010. Hereditary spastic paraplegia proteins REEP1, spastin, and atlastin-1 coordinate microtubule interactions with the tubular ER network. Pages 1097-1110. J Clin Invest. PMID: 20200447 Renvoisé B, Parker RL, Yang D, Bakowska JC, Hurley JH, Blackstone C. 2010. SPG20 Protein Spartin is Recruited to Midbodies by ESCRT-III Protein Ist1 and Participates in Cytokinesis. Molecular biology of the cell. PMID: 20719964 D. Research Support None





NAME Garima Singhal

POSITION TITLE Senior Research Scholar

eRA COMMONS USER NAME (credential, e.g., agency login) EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, include postdoctoral training and residency training if applicable.)



International College for Girls, Jaipur, Rajasthan, India B.Sc 2002-2005 Biotechnology

National Brain Research Centre, Manesar, Haryana, India M.Sc 2005- 2007 Neuroscience

National Centre for Biological Sciences, Bangalore, Karnataka, India P.hD. 2007 -

present Cell biology

A. Personal Statement My work involves an amalgamation of molecular techniques such as molecular cloning, immunocytochemistry, tissue culture, transactivation assays, biochemistry techniques and imaging (microscopy). etc. Guide: Dr. Prabodha Kumar Swain; Assistant Professor, NBRC. My research has given me experience using molecular biology and cell culture techniques, which has helped prepare me for the proposed studies.

B. Positions and Honors

Other Experience and Professional Memberships RESEARCH PROJECT IN MASTERS The title of my thesis is “Studies on the differential regulation of the Human Short Wave (Blue) Opsin promoter by Maf family of transcription factors”. Guide: Dr. Prabodha Kumar Swain; Assistant Professor, NBRC

Honors Biophysical Society International Student Travel Award for 55th Annual meeting held in Baltimore, MD, USA. CSIR (Council for Scientific and Industrial Research, Government of India) fellowship, 2007- present. Department of biotechnology(DBT), Government of India, fellowship during Master’s (2005-2007), through

NBRC (National Brain Research Centre), India Gargi Award, Government of Rajasthan, India, 2000-2002.

C. Selected Peer-reviewed Publications (relevant to application)

NA

D. Ongoing Research Support

CSIR (Council for Scientific and Industrial Research, Government of India) fellowship, 2007- present, annually renewed.





NAME Nishi, Rae

POSITION TITLE Professor

eRA COMMONS USER NAME (credential, e.g., agency login) nishir EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training.)



Stanford University, Stanford, CA BS 1975 Biological Sciences University of California, San Diego PhD 1980 Biology Harvard Medical School, Boston, MA Postdoctoral

Training 1980-1984 Neurobiology

A. Personal Statement My training and experience is well suited for serving on the external advisory committee for Drs. Miwa and Major on this project investigating the role of lynx proteins in trafficking of nicotinic acetylcholine receptors (nAChRs). As a developmental neurobiologist, I have long-studied the development of the cholinergic and cholinoceptive neurons of the avian ciliary ganglion. My work established that nicotinic signaling through alpha 7 subunit containing nAChRs induces cell death during normal development of ciliary ganglion neurons. We then discovered 6 lynx-like genes in the Gallus genome, of which 3 were expressed in the ciliary ganglion. We have shown that one of these genes is the avian ortholog of prostate stem cell antigen (PSCA), a molecule that is another member of the lynx protein family. Recently, my lab has started to investigate the role of lynx-proteins in controlling nAChR responsiveness in the adolescent murine forebrain and frontal cortex. B. Positions and Honors Positions 12/01/10 – present Chair, Steering Committee, Neuroscience, Behavior and Health Initiative, University of

Vermont, Burlington, VT 7/01/05 – present Director, Neuroscience Graduate Program, University of Vermont, Burlington, VT 6/01/04 - 7/30/07 Co-Director, Summer Neurobiology Course, Marine Biological Laboratory, Woods Hole,

MA 7/1/01 – present Professor, Dept. Anatomy & Neurobiology, University of Vermont 7/01/97 - 6/30/01 Professor, Dept. of Cell & Developmental Biology, Oregon Health Sciences University,

Portland, OR 7/01/92 - 7/01/97 Associate Professor, Dept. Cell & Developmental Biology, Oregon Health Sciences

University, Portland, OR 9/01/86 - 6/30/92 Assistant Professor, Dept. Cell Biology & Anatomy; and Affiliate Scientist, Vollum

Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland, OR

7/01/84 - 9/01/86 Instructor, Dept. Neurobiology, Harvard Medical School, Boston, MA 7/01/80- 6/30/84 Postdoctoral fellow, Dept. Neurobiology (with David Potter), Harvard Medical School,

Boston, MA Awards and Honors 10/01/09 – present Member, NDPR Study Section, NIH 11/08 – 11/09 President, Association of Neuroscience Departments and Programs 7/19/09-7/21/09 Chair, Neuroblastoma Peer Reviewed Medical Research Program, Congressionally

Directed Medical Research Programs, DOD, Reston, VA 2000-2004 Member, NINDS Neuroscience Training study section (NST) 2005- 2007 Councilor, Association for Neuroscience Departments and Programs



C. 5 most relevant peer reviewed publications in chronological order (from 47) Nishi R, Stubbusch J, Hulce JJ, Hruska M, Pappas A, Bravo MC, Huber LP, Bakondi B, Soltys J, Rohrer H.

2010. The cortistatin gene PSS2 rather than the somatostatin gene PSS1 is strongly expressed in developing avian autonomic neurons. J Comp Neurol 518:839-850 (NIHMSID #192753; PMC in process)

Hruska M, Keefe J, Wert, D, Tekinay AB, Hulce JJ, Ibanez- Tallon I, and Nishi R. 2009. Prostate stem cell antigen functions as an endogenous lynx1-like prototoxin that antagonizes alpha 7 containing nicotinic receptors and prevents programmed cell death of parasympathetic neurons. J Neurosci 29:14847-14854 (PMC2848080)

Hruska M, Ibanez-Tallon, I, and Nishi R 2007 Cell autonomous inhibition of alpha7- nicotinic receptor signaling rescues ciliary ganglion neurons from cell death, J Neurosci 27:11501-11509.

Bunker G. and Nishi R. 2002. Developmental cell death in vivo: rescue of neurons independently of changes at target tissues. J Comp Neurol 452:80-92.

Reiness CG, Seppa MJ, Dion DM, Sweeney S, Foster DN and Nishi R. 2001 Chick ciliary neurotrophic factor is secreted via a non-classical pathway, Molec Cell Neurosci 17: 931-944.

10 other relevant publications Straub JA, Saulnier Sholler GL, Nishi R. 2007. Embryonic sympathoblasts transiently express TrkB in vivo and

proliferate in response to brain-derived neurotrophic factor in vitro. BMC Dev Biol 7:10 Lee VL, Sechrist J.W., Bronner-Fraser M, and Nishi, R. 2002. Neuronal differentiation from post-mitotic

precursors in the ciliary ganglion. Dev. Biol. 252:312-323. Lee VL., Smiley GG and Nishi R 2001. Cell death and neuronal replacement during formation of the avian

ciliary ganglion. Dev. Biol., 233: 437-448. Darland D and Nishi R 1998. Activin and follistatin influence expression of somatostatin in the ciliary ganglion

in vivo. Devel. Biol. 202: 293-303 Link BL and Nishi R. 1998. Development of the avian iris and ciliary body: mechanisms of cellular

differentiation during the smooth-to-striated muscle transition, Devel. Biol. 203:163-176 Koshlukova S, Finn T, Nishi R, and Halvorsen SW 1996. Identification of functional receptors for ciliary

neurotrophic factor on chick ciliary ganglion neurons. Neuroscience 72: 821-832 Darland D, Link B, and Nishi R. 1995. Developmental expression of activin A and follistatin in targets of ciliary

ganglion neurons suggests a function in the regulation of neurotransmitter phenotype. Neuron 15: 857-866 Heller S, Finn TP, Huber J, Nishi R, Geissen M, Püschel AW and Rohrer H. 1995. Analysis and expression of

the chick GPA receptor suggests multiple functions in neuronal development. Development 121: 2682-2693.

Coulombe JN, Schwall R, Parent AS, Eckenstein FP, and Nishi R 1993. Induction of somatostatin immunoreactivity in cultured ciliary ganglion neurons by activin in choroid cell conditioned medium. Neuron 10: 899-906

Leung DW, Parent AS, Cachianes G, Esch F, Coulombe JN, Nikolics K, Eckenstein FP and Nishi R. 1992. Cloning, expression during development, and evidence for secretion of a trophic factor for ciliary ganglion neurons. Neuron 8:1045-1053

D. Research Support. Ongoing Research Support 1RC1DA028173 09/30/09-09/29/11 NIH/NIDA “Adolescent Brains, Nicotine and Endogenous Prototoxins,” This is a multidisciplinary Challenge Grant that integrates animal behavior (conditioned place preference with nicotine) using transgenic prototoxin knock out mice, morphometry of the brain to track and quantify cholinergic and noradrenergic projections to EYFP labeled neurons in cortex, and studies of human subjects using objective cognitive testing and subsequent genotyping of individuals for alleles of prototoxin genes to acquire a greater understanding of the sensitivity of adolescent brains to nicotine together with the long-term consequences of nicotine exposure on the wiring and neurochemistry of the brain. Role: PI.



1R01DA017784-01-05A2 07/01/05- 06/30/11 NIH/NIDA “Nicotinic Receptors in Neural Development” The aims of this project are to determine whether a7 subunit containing nicotinic acetylcholine receptors are inducing cell death in the ciliary ganglion, and to determine how the endogenous alpha-bungarotoxin like molecule lynx regulates this process. This grant is presently on a one year, no cost extension. The present application is designed to renew this R01. Role: PI. Relevant Past Research Support RO1NS25767-01-17 4/1/89- 7/31/06 NIH/NINDS "Mechanisms of trophic molecules in neuronal development" This project, which was funded for 17 years, examined the trophic interactions between ciliary ganglion neurons and their target tissues in the eye. Over the years of this study, we purified and cloned chicken ciliary neurotrophic factor (CNTF), which we showed is the only CNTF that is secreted and that overexpression of chCNTF rescued neurons from cell death; we identified activin and follistatin as key regulators of somatostatin-like immunoreactivity in choroid neurons through studies in vitro and in vivo; we demonstrated that cell death occurs throughout neurogenesis of the ciliary ganglion, and that a post-mitotic pool of undifferentiated precursors was able to replace neurons that die. Role: P.I.





NAME Darwin K. Berg

POSITION TITLE Professor of Biology

eRA COMMONS USER NAME (credential, e.g., agency login) [email protected] EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training.)



University of California, Berkeley, CA B.A. 1965 Molecular Biology

University of California, Berkeley, CA Ph.D. 1969 Molecular Biology

University of California, Berkeley, CA Postdoc. 1970 Molecular Biology

Harvard Medical School, Boston, MA Postdoc. 1973 Neurobiology

A. Personal Statement

For a number of years, our group has studied nicotinic cholinergic pathways as a model for how synapses

form and pathways function. Recently we have become intrigued with the fact that nicotinic signaling appears

early in postnatal development and drives spontaneous waves of excitation when major networks are only

beginning to form in the CNS. Until recently, the significance of these phenomena was completely unknown.

We now have preliminary results indicating a profound role for nicotinic signaling in promoting glutamate

synapse formation in vivo and perhaps broadly shaping neural networks. This could be a major breakthrough

not only for understanding early events governing development in the nervous system but also for addressing a

fundamental biomedical challenge, namely determining the ways in which early nicotine exposure alters

nervous system development in a long-lasting way. Our group has the interest, expertise and facilities to

address these questions. We employ imaging, electrophysiology, genetic manipulation in vivo, molecular and

biochemical approaches, and EM analysis in our lab. In addition, we have ongoing collaborations with

colleagues on campus and at the nearby medical school and Salk Institute who share out interests and offer

additional approaches. Elucidating how nicotinic cholinergic signaling helps shape the development of neural

networks will be a major contribution to the field. Demonstrating how these events are distorted by nicotine

exposure to change the functional outcome will be an important from a biomedical perspective.

B. Positions and Honors.

Positions and Employment

1973-1975 Instructor, Dept. of Pharmacology, Harvard Medical School, Boston

1975-1979 Assistant Professor, Dept. of Biology, University of California, San Diego

1979-1985 Associate Professor, Dept. of Biology, University of California, San Diego

1985-2004 Professor, Dept. of Biology, University of California, San Diego

1994-1996 Chair, Dept. of Biology, University of California, San Diego

2004- Distinguished Professor, Division of Biological Sciences, University of California, San Diego

Other Experience and Professional Memberships

1972- Member, Society for Neuroscience

1972- Member, American Association for the Advancement of Science

1975- Member, American Heart Association

1981-1985 NIH Peer Review Committee (Study Section), regular member

1989-1999 Planning Committee for Gordon Conference on Neural Plasticity, member

1992-1998 Planning Committee for the Keystone Conference on Synapse Formation & Function, member

1984-1985 Associate Editor, Journal of Neuroscience

1989-1994 Associate Editor, Neuron



1992- NIH Special Study Sections, Ad hoc member and Chair

1997-2000 Member, Society for Neuroscience Ad Hoc Committee for Dev. of Women's Careers in Science

1998-2002 NIH Peer Review Committee (Study Section), regular member

2001-2007 Associate Editor, Journal of Neuroscience

2001- Panel of Mentors, Society for Neuroscience

2003-2004 Selection Committee Member, Society for Neuroscience Young Investigator Award

2004- Board Member, Kavli Institute for Brain & Mind Research

2005-2008 Council Member, Society for Neuroscience.

2005-2008 Audit Committee Chair, Society for Neuroscience

2009- Associate Editor, Frontiers in Synaptic Neuroscience

2009- Honorary Board of Editors, Patient Intelligence

2010- Treasurer-Elect, Society for Neuroscience

Honors

1964 Phi Beta Kappa

1965 B.A. with Distinction

1965-1966 Rosenberg Predoctoral Fellow

1966-1968 NIH Predoctoral Trainee

1969-1970 NSF Predoctoral Fellow

1971-1973 NIH Postdoctoral Trainee

1973-1975 Medical Foundation Postdoctoral Fellow

1981 Guggenheim Sabbatical Fellow

1987-1994 Javits Neuroscience Investigator Award

2008 Fellow, American Association for the Advancement of Science

C. Selected Peer-reviewed Publications (From 115 peer-reviewed original-research articles & 19 reviews)

Five papers most relevant for ongoing projects (chronologically):

1) Coggan JS, Bartol TM, Esquenazi E, Stiles JR, Lamont S, Martone ME, Berg DK, Ellisman MH, Sejnowski

TJ (2005) Ectopic neurotransmitter release at a neuronal synapse. Science 309:446-451. (PMID:

16020730)

2) Liu Z, Neff RA, Berg DK (2006) Sequential interplay of nicotinic and GABAergic signaling guides

neuronal development. Science 314:1610-1613. (PMID: 17158331)

3) Neff RA, Conroy WG, Schoellerman JD, Berg DK (2009) Synchronous and asynchronous transmitter

release at nicotinic synapses are differentially regulated by postsynaptic PSD-95 proteins. J Neurosci

29:15770-15779. (PMID: 200116093)

4) Campbell NR, Fernandes CC, Halff AW, Berg DK (2010) Endogenous signaling through 7-containing

nicotinic receptors promotes maturation and integration of adultborn neurons in the hippocampus. J

Neurosci 30:8734-8744. (PMID: 20592195; PMCID: PMC2905643)

5) Fernandes CC, Berg DK, Gomez-Varela D (2010) Lateral mobility of nicotinic acetylcholine receptors on

neurons is determined by receptor composition, local domain, and cell type. J Neurosci 30:8841-8851 (and

Journal cover). (PMID: 20592206; PMCID: PMC2913715)

Additional publications of relevance for current projects (in chronological order)

6) Zhang Z-W, Vijayaraghavan S, Berg DK (1994) Neuronal acetylcholine receptors that bind -bungarotoxin

with high affinity function as ligand-gated ion channels. Neuron 12:167-177. (PMID: 7507338)

7) Zhang Z-W, Coggan JS, Berg DK (1996) Synaptic currents generated by neuronal acetylcholine receptors

sensitive to -bungarotoxin. Neuron 17:1231-1240. (PMID: 8982169)



8) Shoop RD, Chang KT, Ellisman MH, Berg DK (2001) Synaptically-driven calcium transients via nicotinic

receptors on somatic spines. J Neurosci 21:771-781. (PMID: 11157063)

9) Liu Q-S, Kawai H, Berg DK (2001) -Amyloid peptide blocks the response of 7-containing nicotinic

receptors on hippocampal neurons. Proc Natl Acad Sci (USA) 98:4734-4739. (PMID: 11274373)

10) Chang K, Berg DK (2001) Voltage-gated channels block nicotinic regulation of CREB phosphorylation and

gene expression in neurons. Neuron 32:855-865. (PMID: 11738031)

11) Shoop RD, Esquenazi E, Yamada N, Ellisman MH, Berg DK (2002) Ultrastructure of a somatic spine mat

for nicotinic signaling in neurons. J Neurosci 22:748-756. (PMID: 11826104)

12) Conroy WG, Liu Z, Nai Q, Coggan JS, Berg DK (2003) PDZ-containing proteins provide a functional

postsynaptic scaffold for nicotinic receptors in neurons. Neuron 38:759-771. (PMID: 12797960)

13) Liu Z, Tearly AW, Nai Q, Berg DK (2005) Rapid activity-driven SNARE-dependent trafficking of

nicotinic receptors. J Neurosci 25:1159-1168. (PMID: 15689552)

14) Zhang J, Berg DK (2007) Reversible inhibition of GABAA receptors by 7-containing nicotinic receptors

on vertebrate postsynaptic neurons. J Physiol (Lond) 579:753-763. (PMID: 17204496)

15) Liu Z, Conroy WG, Stawicki TM, Nai Q, Neff RA, Berg DK (2008) EphB receptors co-distribute with a

nicotinic receptor subtype and regulate nicotinic downstream signaling in neurons. Molec Cell Neurosci

38:236-244. (PMID: 18403216)

C. Research Support

Ongoing Research Support

NIH RO1NS012601-36 (P.I., Darwin K. Berg) 05/01/06-04/30/11

NIH/NINDS

Signal Transduction at Neuronal Nicotinic Synapses

The major goals of this project were to examine the interactions of nicotinic and GABAergic

signaling in autonomic and CNS pathways, evaluate nicotinic contributions to filopodial fate on CNS

neurons, and identify the roles of ric-3, neto-1 and other receptor-associated proteins in determining

nicotinic receptor levels and location on neurons.

Role: PI

ARRA Administrative Supplement:

NIH 3RO1NS012601-34S1 (P.I., Darwin K. Berg) 08/01/09-06/30/11

NIH/NINDS

Signal Transduction at Neuronal Nicotinic Synapses (ARRA Administrative Supplement)

The major goals are to accelerate and extend studies outlined in the parent grant above, NIH

R01NS012601-34.

Role: PI

NIH RO1NS035469-16 (P.I., Darwin K. Berg) 05/01/09-03/30/14

NIH/NINDS

Nicotinic Control of Non-Nicotinic Synapses in the Hippocampus.

Major goals of this project are to examine the effects of nicotinic activity on hippocampal neuron

development, testing how nicotinic activity shapes the fate and integration of adultborn neurons, and

how the outcome is corrupted by nicotine . Other goals address the ability of acute nicotine exposure to

reverse GABAergic signaling in adults, transiently making it excitatory and changing network function.

Role: PI

TRDRP 19XT-0072 (P.I., Darwin K. Berg) 07/01/10-6/30/12



Tobacco-Related Disease Research Program

Using Adultborn Neurons to Combat Nicotine Relapse

This is a pilot project examining the effects of chronic nicotine on adultborn neurons, assessing possible

consequences for nicotine relapse behavior, and initiating experiments to evaluate pharmaceutical strategies

for intervention.

Role: PI

Completed Research Support (within the last 5 yrs)

TRDRP 16RT-0167 (P.I., Darwin K. Berg) 07/01/07-6/30/10

Tobacco-Related Disease Research Program

Nicotinic Manipulation of the Hippocampus

The major goals of this project were to examine the ability of nicotine to influence activity in the

hippocampus and to assess the consequences of early nicotine exposure for subsequent development from

the perspective of addiction.

Role: PI

Industrial Grant (UCSD #2004-0309) (P.I., Darwin K. Berg) 10/1/04-9/30/7

PMERP: “Nicotinic Receptors on Hippocampal Neurons”

The major goals of this project were to examine 7-containing nicotinic receptors on rat hippocampal

neurons, determining how the receptors are regulated by BDNF, how the surface receptor population

depends on adjacent intracellular receptor pools, and how these interact at GABAergic synapses.

Role: PI





Barrantes Francisco José Head, Natl. Res. Council of Argentina-Bahia Blanca Head, Institute of Biochemical Research, Bahia Blanca

rtfjb1 EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training.)



Faculty of Medicine, Univ. of Buenos Aires, Argentina M.D. 1968 Medicine Univ. of Buenos Aires, Argentina Ins

Ph. D. Re

1973 Acetylcholine receptor fluore Dept. Biochem., Univ. Illinois at Urbana-Champaign

Max-Planck-Institut Biophys. Chemistry, Göttingen Institut Pasteur, Paris, France

Fellow OAS Postdoc

EMBO Fellow

1973 1974-1977

1974

Fluorescence spectroscopy Acetylcholine receptor Acetylcholine receptor

A. Professional Positions: 1969-1971.Research Fellow, Natl. Res. Council of Argentina (CONICET), Inst. Cell Biology, Faculty of

Medicine, Univ. Buenos Aires (Prof. E. De Robertis). 1973. Research Fellow. Department of Biochemistry, School of Chemical Sciences, Univ. of Illinois.

Urbana-Champaign (Prof. G. Weber), U.S.A. 1974-1977. Member of Research Staff. Department of Molecular Biology, Max-Planck Institute of

Biophysical Chemistry, Göttingen, W. Germany. (Dr. Thomas Jovin). 1978-1982. Joint Head of the Membrane Group (Gruppe 14, BNS) together with Drs. Erwin Neher and Bert

Sakmann at the Max-Planck-Institute of Biophysical Chemistry, Göttingen, W. Germany. 1990-1991. Human Frontier Research Fellowship. MRC, Laboratory of Molecular Biology, Cambridge, U.K. 1987. Visiting professor. Dept. Neurobiology, Weizmann Institute of Science, Rehovot, Israel. 1986-1987. Visiting professor. Dept. Neurobiol.& Behavior, SUNY at Stony Brook, N.Y., U.S.A. 1986 Fellow. The Neurosciences Institute, Rockefeller University, New York, U.S.A. 1983- Director of the Institute of Biochemistry, Univ. Nac. del Sur/CONICET, Bahía Blanca. 1983-2009 Full Professor, Biological Chemistry, Department of Biochemistry. Univ. Nac. del Sur. 1994- Highest rank research career investigator, CONICET. 1999. Visiting Professor, Biochemistry Dept., Univ. Bath, England. 1993.1996. Visiting professor, Dept. of Biochemistry /Molecular Biology, Univ. of Extremadura, Spain. 1998- Professor and Chairholder, UNESCO Chair of Biophysics & Molec. Neurobiology. 2007- Director, CONICET-Bahia Blanca (12 institutes). 2009- Adjunct Professor. National Center for Biological Sciences (NCBS), TIFR, Bangalore, India.

Awards and other Professional Activities. 1971, Award of the Argentinian Association for the Advancement of Sciences; 1987, "Bernardo Houssay Award" from the Argentinian Scientific Research Council (CONICET); 1989, Third World Academy of Sciences Award in Biology; 1990, Fellow, John Simon Guggenheim Memorial Foundation; 1991, Guest Research Fellowship, Royal Society, London, U.K.; 1994-96, Member, Commission on Cell and Membrane Transport, IUPAB. 1994, Member, Committee for Aid for Neurochemistry (CAN) of the Internat. Soc, Neurochemistry (ISN); 1995-1997, Chairman, CAN; Alexander von Humboldt Research Award, Germany. 1999-2001, President, Argentine Society for Neurochemistry; 2002, Awarded Internat. Sarojini Damodaran International Trust, India; 2003, recipient of one of the five Konex Awards, Argentina; 2005, Fulbright Scholar (sabbatical Harvard Med. School); 2004, De Robertis Medal and 2004 De Robertis Lecture; 2006, Chancellor’s Award in Neurosciences, Univ. Louisiana; Elected member of Council, IUPAB (2006-2012); 2006, Prémio União Latina, Lisbon, Portugal, with Prof. Dr. M. Prieto; 2008, Miguel Lillo Medal. Society for Biology, Tucuman; 2009, 2009 Senior Award ,National Academy of Exact, Physical and Natural Sciences of Argentina.

Membership in Academies. 1993, Corresponding Fellow, Third World Academy of Sciences; 1993, Member, Arg. Association Adv. Sciences; 1993, Fellow, Third World Academy of Sciences; 1994- Member, Latin American Academy of Sciences; 1998, Corresponding Member, Brazilian Academy of Sciences; 1999, Elected Member,



National Academy of Medicine, Buenos Aires; 2004, Member, European Academy of Sciences and Arts; 2005, Foreign Fellow, Indian National Science Academy (INSA); 2006-2012, Member of the Council, Latin American Academy of Science (ACAL). Currently Vice-President, TWAS.

Editorial activities. Currently, member of the Editorial board of the following journals: 2007- The Open Structural Biology Journal; 2008-, Biochemica et Biophysica Acta (BBA), Biomembranes. 2009- Biophysical Reviews (IUPAB); 2010- Associate Editor, Frontiers in Membrane Physiology and Biophysics (Frontiers in Physiology). B. Selected publications (out of more than 200). Gutiérrez-Merino, Bonini de Romanelli, I., Pietrasanta, L. and Barrantes, F.J. Preferential distribution of the

fluorescent phospholipid probes NBD-phosphatidylcholine and rhodamine-phosphatidylethanola mine in the

exofacial leaflet of acetylcholine receptor-rich membranes from T. marmorata. Biochemistry 34 (1995) 4847-55. Antollini, S.S., Soto, M.A., Bonini de Romanelli, I., Gutiérrez Merino, C.. Sotomayor, P. and Barrantes, F.J.

Physical state of bulk and protein-associated lipid in nicotinic acetylcholine receptor-rich membrane studied by

Laurdan generalized polarization and fluorescence energy transfer. Biophys. J. 70 (1996) 1275-1284. Barrantes, F.J. (1998). The nicotinic acetylcholine receptor: Current views and future trends. Neuroscience

Intelligence Unit. Springer Verlag, Berlin/Heidelberg and Landes Publishing Co., Georgetown, TX 226 pp. Bouzat, C., Roccamo, A.M., Garbus, I. and Barrantes, F.J. Mutations at lipid-exposed residues of the acetylcholine

receptor affect its gating kinetics. Molec. Pharmac. 54 (1998) 146-153. Bouzat, C., Barrantes, F.J. and Sine, S. Nicotinic receptor fourth transmembrane domain. Hydrogen bonding by

conserved threonine contributes to channel gating kinetics. J. Gen. Physiol. 115 (2000) 663-671. Barrantes, F.J., Antollini, S.S., Blanton, M.P. and Prieto, M. Topography of nicotinic acetylcholine receptor

membrane-embedded domains. J. Biol. Chem. 275 (2000) 37333-37339. Barrantes, F.J. Transmembrane modulation of nicotinic acetylcholine receptor function. Curr. Opinion Drug Disc.

& Develop. 6 (2003) 620-632. de Almeida, R. F. M., Loura, L. M . S., Prieto, M., Watts, A. Fedorov, A. and Barrantes, F. J. Cholesterol

modulates the organization of the M4 transmembrane domain of the muscle nicotinic acetylcholine receptor.

Biophys. J. 86 (2004) 2261-2272. Barrantes, F. J. (2004). Structural basis for lipid modulation of nicotinic acetylcholine receptor function. Brain

Res. Brain Res. Reviews 47, 71-95. [cover page article] Xu,Y., Barrantes, F.J., Luo, X., Chen, K., Shen, J. & Jiang, H. Conformational Dynamics of the Nicotinic

Acetylcholine Receptor Channel: A 35-ns Molecular Dynamics Simulation Study. JACS 127 (2005) 1291-1299. Antollini, S. and Barrantes, F.J. (2007). Laurdan studies of membrane lipid-acetylcholine receptor protein

interactions. Chapter 36. In A.M. Dopico, Ed.: Methods in Membrane Lipids. The Humana Press Inc. Barrantes, F.J. (2007). Cholesterol effects on nicotinic acetylcholine receptor. J. Neurochem. 103, 72-80. Borroni, V. Baier, C.J., Lang, T., Bonini, I. White, M.W.,Garbus, I. and Barrantes, F.J. (2007). Cholesterol

depletion activates rapid internalization of diffraction-limited acetylcholine receptor domains at the cell

membrane. Molec. Membr. Biol. 24, 1-15. Kellner, R. Baier, J., Willig, K.I., Hell, S.W. and Barrantes, F.J. (2007). Nanoscale organization of nicotinic

acetylcholine receptors revealed by STED microscopy. Neuroscience 144, 135-143. doi: Farías, G.G., Vallés, A.S, Colombres, M., Lukas, R., Barrantes, F.J. and Inestrosa, N.C. (2007). Wnt-7a Induced

presynaptic colocalization of 7-nicotinic acetylcholine receptors and adenomatous polyposis coli in

hippocampal neurons. J. Neurosci. 27, 5313-5325. Liu, X., Xu, Y., Li, H., Wang, X., Jiang, H. and Barrantes, F.J. (2008). Mechanics of channel gating of the

nicotinic acetylcholine receptor. PLoS Computat. Biol. 4, 100-110. [cover page article] Liu, X., Xu, Y., Wang, X., Barrantes, F.J. and Jiang, H. (2008). Unbinding of nicotine from the acetylcholine

binding protein: Steered molecular dynamics simulations J. Phys. Chem. B 112: 4087-4093. Kumari, S., Borroni, V., Chaudhry, A., Chanda, B. Massol, R., Mayor, S. and Barrantes, F.J. (2008). Nicotinic

acetylcholine receptor is internalized via a Rac-dependent, dynamin-independent endocytic pathway. J. Cell Biol.

181, 1171-1193.



Wenz, J.J. and Barrantes, F.J. (2008). Resolution of complex fluorescence spectra of lipids and nicotinic

acetylcholine receptor by multivariate analysis reveals protein-mediated effects on the receptor's immediate lipid

microenvironment. PMC Biophysics 1(6), 1-12. Vallés, A.S., Roccamo, A.M. and Barrantes, F.J. (2009). Ric-3 chaperone-mediated stable cell-surface expression

of the neuronal α7 nicotinic acetylcholine receptor in mammalian cells. Acta Pharmacol. Sinica 30, 818-827. Baier, C.J., Gallegos, C.E., Levi, V. and Barrantes, F.J. (2010). Cholesterol modulation of nicotinic acetylcholine

receptor surface mobility. Eur. Biophys. J. 39:213–227. Fantini, J. and Barrantes, F.J. (2009). Sphingolipid/cholesterol regulation of neurotransmitter receptors

conformation, function and trafficking. Biochim. Biophys. Acta Biomembranes 1788, 2345–2361. Wenz, J.J., Borroni, V. and Barrantes, F.J. (2010). Statistical analysis of high-resolution light microscope images

reveals effects of cytoskeleton-disrupting drugs on the membrane organization of the nicotinic acetylcholine

receptor. J. Membr. Biol. 235:163-175. Bermúdez, V., Antollini, S.S., Fernández Nievas, G.A., Aveldaño, M.I. and Barrantes, F.J. (2010). Partition profile

of the nicotinic acetylcholine receptor in lipid domains upon reconstitution. J. Lipid Res. 51:2629-2641. Barrantes, F.J., Borroni, V. and Vallés, S. (2010). Neuronal acetylcholine receptor-cholesterol crosstalk in

Alzheimer’s disease. FEBS Lett. 584, 1856-1863. Barrantes, F.J. (2010). Cholesterol effects on nicotinic acetylcholine receptor: Cellular aspects. In: Cholesterol

binding proteins and cholesterol transport. Ed. R. Harris. Springer Verlag. Subcell Biochem. 51, 467-487. Zheng, C., Yang, K., Wang M.-Y., Vallés, S., Lukas, R.J., Barrantes, F.J. and Wu, J. (2010). The anticonvulsive

drug lamotrigine blocks neuronal α4β2-nicotinic acetylcholine receptors. J. Pharmac.Exptl. Therap. 335:401–

408. Borroni, M.V. and Barrantes, F.J. (2011). Amyloid precursor protein in central and peripheral cholinergic

synaptopathies. Alzheimer's Dis.Res. J. (in press) Borroni, V. and Barrantes, F.J. (2011) Cholesterol modulates the rate and mechanism of acetylcholine receptor

internalization. J. Biol. Chem. (in press). Levitan, I. and Barrantes, F.J. Eds. (2011) Cholesterol Regulation of Ion Channels and Receptors. John Wiley &

Sons.

C. Research Support. 1. Studies of the nicotinic acetylcholine receptor in native membranes and in cellular systems. PICT2008 – No. 1003 BID 1201 OC/AR Ministry of Science & Technology of Argentina. Role: PI 2. High resolution nonlinear optical microscopy of acetylcholine receptor membranes. Ministry of Science & Technology of Argentina / Max-Planck-Institute of Biophysical Chemistry, Göttingen, Germany (F. J. Barrantes – Stefan Hell). The goal of this project is study the cell surface localization, distribution and dynamics of the AChR -using high-resolution fluorescence microcopy techniques (nanoscopies). Role: PI 3. (completed) FIRCA I-R03-TW01225-01.01/04/00-3/31/04 United States Department of Health and Human Services (DHHS), Fogarty International Center. Role of protein-lipid interactions in AChR gating. The overall aim of this project was to explore the effect of lipids on the function of the AChR, with particular emphasis on steroid-receptor interactions. Role: Co-Pi with Dr. Michael White, Drexel University. 4. (completed). NIH grant R01DA015389 01/01/06-01/31/09. Dr. Ronald Lukas, PI, Neurobiology, Barrow Neurological Institute, St. Joseph's Hospital & Medical Center, Phoenix, AZ. Extended exposure to nicotine induces long-lasting changes in numbers and function of diverse AChR subtypes. Study of α-7 and α-4/ -2 neuronal acetylcholine receptors in clonal cell lines, with particular emphasis on anticonvulsive drugs and the Wnt-system. Role: Subcontracted as collaborator. 5. Ongoing research project with Prof. S. Mayor, NCBS, Bangalore, India, on AChR trafficking. Supported by TWAS and TIFR Associate Professorship. Role: Co-PI. 6. Philip Morris USA/Philip Morris International. 2008/2011. Characterization of the α7-type acetylcholine receptor in vascular endothelial cells. Role: PI.



Dear Julie, I am delighted to serve on the advisory committee for your proposal outlined in PAR-11-099 (R21) and entitled "Investigating the role of lynx proteins in the trafficking of nicotinic receptor complexes", with Dr. Julie M. Miwa and Dr.Satyajit Major as the principal investigators. This is an important project with exciting possibilies, given your recent findings with lynx. Attached is an NIH-style copy of my Biosketch. Happy to help in any way I can. Best regards, Darwin Darwin K. Berg Neurobiology Section, Biology Division University of California, San Diego 9500 Gilman Drive La Jolla, CA 92093-0357 Phone: 858-534-4680 Fax: 858-534-7309 Email: [email protected]

Subject: Biosketch for PAR-11-099 From: "Darwin Berg" <[email protected]> Date: Sat, March 19, 2011 11:26 am To: "[email protected]" <[email protected]> Priority: Normal



DEPARTMENT OF ANATOMY AND NEUROBIOLOGY UNIVERSITYOF VERMONT COLLEGE OF MEDICINE 149 BEAUMONT AVE. BURLINGTON, VT 05405 (802) 656-4504 Julie M Miwa, PhD California Institute of Technology Biology 1200 E. California Blvd., MC156-29 Pasadena, CA 91106 Dear Julie: I will be very happy to serve on the advisory committee for your proposal outlined in PAR-11-099 (R21) for the U.S. - India Bilateral Brain Research Collaborative Partnerships entitled "Investigating the role of lynx proteins in the trafficking of nicotinic receptor complexes" that you are submitting with Dr.Satyajit Major. As you know, I have been working with lynx protein family members for quite some time now and your work in the role that these proteins play in nicotinic acetylcholine receptor trafficking is very intriguing. Very sincerely,

Rae Nishi, Ph.D. Professor, Anatomy and Neurobiology



Subject: Pancho 18 March/19 March From: Francisco Barrantes <[email protected]> Date: Sat, 19 Mar 2011 12:01:52 -0700 (PDT) To: [email protected], Julie Miwa <[email protected]>, Satyajit Mayor <[email protected]> Dear Julie, Henry, and Satyajit (Jitu) Thank you for the invitation to serve on the advisory committee as a "great Spanish philosopher" (quote dates back I believe to 2009...) in the Lynx project. I gladly consent. Our lab is ready to contribute technical expertise, as well as data in fields that complement the proposed project, with the PALM/GSDIM super-resolution microscope built in Bahia Blanca for the decade-long ongoing collaboration with Bangalore - now about to expand to the US West coast...-nice! Our moving to Buenos Aires later this year will certainly facilitate traffic in all directions. I am organizing a meeting in Buenos Aires at the end of 2013. This will be an excellent meeting time for workers on the project. Travel for the Indian group will be supported by the Argentina-collaboration, and I understand that you will find non-Federal sources for the Caltech group's travel. Cheers Pancho



Collaboration plan

Communications We plan monthly teleconferences, including all participants in the project as well as the Advisory Committee. Agendas will be sent in advance, and minutes will be distributed after the meeting. In addition to the purely technical facilities detailed here, we wish to state that all participants have experience with the social skills, clear speaking, patience, and punctuality required for a productive teleconference. The PIs understand their responsibilities in encouraging new participants to speak up and to speak clearly. 1. Our major communications channel is Skype, which all participants use regularly. We have verified that screen sharing in Skype proceeds rapidly enough for good videos. At Caltech, the Lester lab conference room has several microphones, a camera, a flat-panel TV, and other arrangements that (very economically) provide an experience approaching a corporate telepresence room. 2. The Caltech dropbox handles files up to 2 GB. Anybody, anywhere, can send a file: http://dropbox.caltech.edu/cgi-bin/dropbox.cgi 3. Some institutions (the NIH) block Skype, and Skype functions poorly when some participants use phones. The Caltech group has a Cisco WEBEX account; this is an internationally accepted method for high-quality professional meetings that include telephones, computers, video, and presentations. Our office manager is skilled in using WEBEX for the monthly teleconferences in our NIDA-sponsored National Co-operative Drug Development group.

Personal meetings We plan four such meetings. Most of these meetings will occur during travel that also benefits other projects under way in both labs. Therefore the marginal cost to PAR-11-099 will be small. February 25-29, 2012. Biophysical Society Meetings, San Diego late 2012. Bangalore, India. This meeting will be funded exclusively by the proposed project, and will take place because we believe that it instantiates the spirit of PAR-11-099. February 2-6, 2013. Biophysical Society Meetings, Philadelphia Final retreat, Meeting organized by F. Barrantes, Buenos Aires, Argentina.

Advisory Committee Three scientists with relevant expertise agree to serve on the Advisory Committee for this project.: Francisco Barrantes, Bahia Blanca and Buenos Aires. See his letter and Biosketch. Darwin Berg, University of California at San Diego. See his letter and Biosketch. Rae Nishi, University of Vermont. See her letter and Biosketch.

collaboration_plan_par_11_0991003353128 Page 32


Province:

PROFILE - Project Director/Principal Investigator

Prefix: * First Name: Middle Name:


Organization Name: Division:

Position/Title: Department:

* Street1:

Street2:


* E-Mail:

Credential, e.g., agency login:

* Project Role: Other Project Role Category:

* Zip / Postal Code:* Country:

* State:

County/ Parish:* City:

Attach Current & Pending Support

RESEARCH & RELATED Senior/Key Person Profile (Expanded)

*Attach Biographical Sketch

Julie M

Miwa

Senior Research Fellow



Pasadena

Biology

MC 156-29

miwajulie

CA: California

USA: UNITED STATES 91125-0029

626-395-6221 626-564-8709

[email protected]

PD/PI

Biosketch_Miwa_031711a1003353 View AttachmentDelete AttachmentAdd Attachment



Degree Type:

Degree Year:

Province:

PROFILE - Senior/Key Person





* Street1:

Street2:


* E-Mail:



* State:


PhD

ADr.

Bren Professor of Biology


Los Angeles

MC 156-29

Division of Biology

USA: UNITED STATES

lesterha

626-564-8709

Henry

Lester


Pasadena

CA: California

91125-0029

626-395-4946

[email protected]

* Project Role: PD/PI Other Project Role Category:

1

Degree Type:

Degree Year:

Biosketch_Lester_Pubs___OS__P*Attach Biographical Sketch Add Attachment Delete Attachment View Attachment

Attach Current & Pending Support Add Attachment Delete Attachment View Attachment

Key Personnel Page 33


RESEARCH & RELATED Senior/Key Person Profile (Expanded)

Province:

PROFILE - Senior/Key Person





* Street1:

Street2:


* E-Mail:



* State:


Dr.

National Centre fro Biological Sciences

IND: INDIA

mayors

0091-80-23636662

Satyajit

Mayor

GKVK Campus Bellary Road

Bangalore 560065

0091-80-23666001

[email protected]

* Project Role: PD/PI Other Project Role Category:

2

Degree Type:

Degree Year:

Biosketch_Mayor1003353138.pdf*Attach Biographical Sketch Add Attachment Delete Attachment View Attachment

Attach Current & Pending Support Add Attachment Delete Attachment View Attachment

Key Personnel Page 34


BIOGRAPHICAL SKETCH Provide the following information for the key personnel in the order listed for Form Page 2.


NAME Miwa, Julie Michiko ERA commons: MIWAJULIE

POSITION TITLE Senior Research Fellow

EDUCATION/TRAINING



University of California at Berkeley B.A. 1987 Neurobiology The Rockefeller University Ph.D. 1997 Neuroscience The Rockefeller University Post-doctoral 1997-2002 Molecular Biology Yale University College of Medicine Post-doctoral 2001-2003 Psychiatry California Institute of Technology 2009-present Neuroscience

A. Personal Statement:

The current proposal investigates intracellular mechanisms of lynx action on the receptor, and could uncover mechanisms underlying channelopathies. Lynx proteins are GPI-anchored proteins, which are resident in cholesterol rich lipid domains in the plasma membrane. The association of lynx with nicotinic receptors implicates lynx/receptor interactions in the movement of receptors. We are monitoring receptor mobility and insertion events at the cell surface, as well as investigating mechanisms underlying receptor endocytotic mechanisms. I will direct the Caltech parts of this award (primarily specific aim 1) as well as oversee the execution of the collaboration plan.

I have been investigating the function of accessory components to nAChRs since I was a graduate student at the Rockefeller University. There, I made the initial discovery of lynx toxin-like genes and formed the hypothesis that they act as critical modifiers of receptor function. I deepened my investigations into the biology of this gene with the generation of genetically engineered lynx mouse lines, and the characterization using electrophysiological, behavioral and histological approaches. I also participated in the biophysical characterization of lynx action on nAChRs in vitro. In my current position, I have taken a two-pronged approach to gain further insights into lynx biology. I am investigating the intracellular interactions of lynx and nAChRs, with a focus on intracellular events governing receptor assembly and localization. Surface expression of receptors is the final endpoint of those processes, and will be the focus of this award. In addition, I am studying the significance of lynx in neurological function and disease. With this knowledge we can better understand how to regulate cholinergic function through lynx modulation, and what type of therapeutic benefit come about as a consequence. Research on lynx biology, therefore, has tremendous potential to be relevant for a array of neurological and neuropsychiatric disorders. With an established history of lynx discovery, and as the primary advocate of lynx1 biology in the scientific community, I believe my experience and insights can contribute to the successful completion of this project. B. Positions and Honors: Positions: Graduate Fellow, The Rockefeller University, New York, NY, 1997 Post-doctoral Fellow, The Rockefeller University, New York, NY, 1997-2001

Biosketches Page 35


Post-doctoral Associate, The Rockefeller University, New York, NY, 2001-2002 Visiting Scientist, Yale University College of Medicine, New Haven, CT, 2001-2003 Research Associate, The Rockefeller University, New York, NY 2002-2009 Croll Research Fellow, The Rockefeller University, New York, NY, 2007-2008 Senior Research Fellow, California Institute of Technology, Pasadena, CA, 2009-present Honors: Awards, invited lecture and previewed work: Awards: New Investigator Award, Tobacco Related Disease Research Program- 19KT-0032 2010-2013 Croll Research Fellowship, Croll Research Foundation - 2007-2008 National Institutes of Health Postdoctoral Fellowship - NIH CA09673 National Institutes of Health Predoctoral Fellowship - NIH 5T32 GM07524-18 Research Abroad Travel Fellowship Rockefeller University Invited Lectures: Society for Research on Nicotine and Tobacco conference, Baltimore, MD (2010) Preconference workshop: What do animal models tell us about drug abuse and its treatment? Department of Anatomy and Neurobiology ANNB/COBRE Seminar Series, University of Vermont College of Medicine (2010). Balancing nicotinic receptor function through lynx toxin-like modulators of nicotinic receptor function. Gordon Research Conference on Neural Plasticity, section on Structural Dendritic Plasticity. Newport Rhode Island (1999). A novel toxin-like modulator of nicotinic acetylcholine receptors, lynx1. Neurons and Circuits, Ascona (Monte Verita),Switzerland (1999). A novel toxin-like modulator of nicotinic acetylcholine receptors, lynx1. Previewed work: Hall, ZW (1999) -neurotoxins and Their Relatives: Foes and Friends? Neuron 23, 4-5 PMID:10402186 Higley, M.J. and Strittmatter,S.M. (2010) Lynx for Braking Plasticity, Science 330(6008), 1189-1190 PMID: 21109660 Pamela J. Hines (2010) Lynx Vision. Sci Signaling 30 November 2010: ec365 Castro, J. (2011). Understanding the Brain's "Brake Pedal" in Neural Plasticity, Sci. Am. Feb, 2011. Other experiences and memberships: 2006- present Society for Neuroscience, member 2009- present Society for Research on Nicotine and Tobacco, member 2008-present Co-founder and consultant, Ophidion, Inc. Reviewer, Journal of Neuroscience, Neuron, Nature Medicine, Molecular Pharmacology, Molecular and Cellular Neuroscience C. Selected Peer-reviewed Publications: 1. Miwa, JM, Ibanez-Tallon, I, Crabtree, GW, Sanchez, R, Sali, A, Role, LW and Heintz, N (1999) lynx1, an endogenous toxin-like modulator of nicotinic acetylcholine receptors in the mammalian CNS. Neuron 23, 105-114. PMCID:10402197. 2. Ibanez-Tallon, I, Miwa, JM, Wang, HL, Adams, NC, Crabtree, GW, Sine, SM, and Heintz, N (2002) Novel modulation of neuronal nicotinic acetylcholine receptors by association with the endogenous prototoxin lynx1. Neuron 33, 893-903. PMID:11906696 3. Ibanez-Tallon, I, Wen, H, Miwa, JM, Xing, J, Tekinay, AB, Ono, F, Brehm, P, and Heintz, N (2004) Tethering Naturally Occurring Peptide Toxins for Cell-Autonomous Modulation of Ion Channel and Receptors in vivo. Neuron 41, 305-311. PMID:15294139 4. Miwa, JM, Stevens, TR King, SL, Calderone, BJ, Ibanez-Tallon, I, Xiao, C, Maki Fitzsimonds, R, Pavlides, C, Lester, HA, Picciotto, MR and Heintz, N (2006) The Prototoxin lynx1 acts on

Biosketches Page 36


Nicotinic Acetylcholine Receptors to Balance Neuronal Activity and Survival in vivo. Neuron 51, 587-600. PMID:16950157 5. Drenan, RM, Grady, SR, Whiteaker, P, McClure-Begley, T, McKinney, S, Miwa, JM, Bupp, S, Heintz, N, McIntosh, JM, Bencherif, M, Marks, MJ, and Lester, HA. (2008). In vivo activation of midbrain dopamine neurons via sensitized, high-affinity 6 nicotinic acetylcholine receptors. Neuron 60, 123-136. PMID:18940593 6. Tekinay, AB, Nong, Y, Miwa, JM, Lieberam, I, Ibanez-Tallon, I, Greengard, P, and Heintz, N. (2009) A role for LYNX2 in anxiety-related behavior. Proc. Natl. Acad. Sci. 106, 4477-4482. PMID:19246390 7. Lester, HA, Xiao, C, Srinivasan, R, Son, CD, Miwa, JM, Pantoja, R, Banghart, MR, Dougherty, DA, Goate, AM, and Wang, JC. (2009). Nicotine is a Selective Pharmacological Chaperone of Acetylcholine Receptor Number and Stoichiometry. Implications for Drug Discovery. AAPS, 11, 167-177. PMID:19280351 8. Drenan, RM, Grady, SR, Steele, AD, McKinney, S, Patzlaff, NE, McIntosh, JM, Marks, JM, Miwa, JM., Lester, HA (2010). Cholinergic Modulation of Locomotion and Striatal Dopamine Release is Mediated by α6α4* Nicotinic Acetylcholine Receptors, J. Neurosci., 30(29):9877-9889. PMID:20660270 9. Morishita H, Miwa JM, Heintz N, Hensch TK. (2010). Lynx1, a cholinergic brake, limits plasticity in adult visual cortex. Science 330(6008):1238-40. PMID: 21071629 10. Srinivasan,R, Pantoja, R, Moss, FJ, Mackey, EDW, Son, CD, Miwa, JM, and Lester, HA (2010). Nicotine-induced α4β2 nicotinic receptor upregulation: Stoichiometry, β-subunit trafficking motifs, subcellular compartments and endoplasmic reticulum exit sites. J. Gen. Phys. 137(1):59-79. 11. Miwa, JM, Freedman, R, and Lester, HA. (2011). Neuronal circuits modulated by nicotinic receptors. Neuron, invited review, in the final stages of editing.

D. Research Support:

Julie M. Miwa (P.I.) 07/01/2010-06/30/2013 Tobacco Related Disease Research Program 19KT-0032 New Investigator Award Nicotinic Receptor- lynx interactions Lynx modulators have been shown to have a critical role in the control of nicotinic receptor signaling through direct interactions in stable complexes. The effect of lynx on the assembly and stoichiometry of α4β2 nicotinic receptors are being explored using FRET measurements and live cell imaging in neurons in order to understand the role of lynx proteins in nicotine dependence. Pending Research Support R01 NS-11756 08-01/2011-07/31/2016 Henry A. Lester, (P.I.) Julie M. Miwa (Co-P.I.) Chemical Synapses - Biophysical Studies Structure-function studies, intracellular events, and chaperoning/upregulation of nicotinic receptors and other Cys-loop family members.

Biosketches Page 37


BIOGRAPHICAL SKETCH AND SUPPORT

Henry A. Lester Bren Professor of Biology California Institute of Technology commons name: lesterha

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training.)

Harvard College, Cambridge MA A.B. 1966 Chemistry and Physics The Rockefeller University, New York NY Ph.D. 1971 Biophysics Institut Pasteur, Paris Postdoc 1971-73 Neurobiologie Moléculaire

A. Personal Statement Roughly 70% of my research has concerned nAChRs and other Cys-loop receptors—from genes, through molecules, through mouse models. Now I want to help Julie Miwa and Jitu Mayor understand modifications to nAChRs organization caused by interactions with lynx proteins, for three disease-related reasons given in Significance. I have learned how to apply various techniques to these problems: fluorescence imaging, electrophysiology, unnatural amino acid mutagenesis, targeted mouse mutations, and simulation. The Table presents the numbers and publication period of my papers whose titles or abstracts mention the topics of the proposed research. This experience and focus suits me well to help direct a project in response to PAR-11-099. B. Positions and Honors. Professional History at Caltech 1973-present: Assistant, Associate, and Professor of Biology, and Bren Professor of Biology. Visiting Professorships: Dept of Biological Chemistry, Hebrew University, Jerusalem, Israel (1980-1981). Howard Florey Institute, Melbourne Univ (2005). Current Ed Bds: J Gen Physiol, Physiol Genomics, Channels, Chem Neurobiol, Mol Pharm Major Reviewing Activities: Neurological Sciences Study Section (1981-1982); AHA, L.A. Affiliate, Research Committee (1983-5); Physiology Study Section (1985-1989); NIMH Special Review Committees (1992-1994); Advisory Council, NIMH (2000-2003); NTRC Study Section (2007 - ). Other Advisory Activities: Marine Biological Laboratory, Woods Hole, Neurobiology Course Instructor (1985-1986), Gordon Conf on Ion Channels, Chair(1990); Center for Genetics and Genomics, Brown University (2000); Transporter Project, Mount Sinai Medical School (1999-2004); FASEB Res. Conferences (2000-2004); Center for Molecular Dynamics, Univ of Montana, (2005-); Nicotine Addiction PPG, Univ. Chicago, (2008-). Center Directorships: Silvio Conte NIMH Center for Neuroscience Research, Caltech (1992-2002); NIDA National Cooperative Discovery Group in Smoking Cessation, Caltech-Boulder-Targacept (2005-2015) Named and Keynote Lectures Grass Foundation Visiting Scientist, SFN Hawaii Chapter, 1989; Michigan Chapter, 1990; Birmingham Chapter, 1998. Bristol-Myers Lect, SUNY at Buffalo, 1991. Harvey Lect, 1996; Keynote, Oregon Health Sciences University Graduate Research Forum, 1997. Vanzant Lect, Rice Univ., 1997. DeCamp Lect, Rockefeller Univ., 1998. Keynote, 1999 FASEB Conference on Transporters. Keynote, New England Pharmacologists Ann Meeting, 2000. Keynote, NINDS-NIMH Intramural Retreat, 2000. Nahum Lect, Yale University School of Medicine, 2001. Keynote, NIMH Dynamical Neuroscience Symposium, 2002. Keynote, University of Southern California Neuroscience Retreat, 2005. Keynote, SFN Satellite Symposium: The 5-HT3 Receptor (2005). Murphy Lect, Univ. Wisconsin at Madison, 2006. Distinguished Lect, Dept of

Topic Dates # of papers

fluorescence 1995-2010 27

other optical methods (including light flashes) 1977 – 2004 16

nicotinic receptors 1970-2010 107

nicotine 1972-2010 33

desensitization (mentioned anywhere in the text) 1972-2010 121

neurodegeneration (including PD) 1995-2010 15

ADNFLE 2003-2009 6

simulation 1973-2011 15 (> 100 equations)

Biographical Sketches for each listed Senior/Key Person 2 Page 38


Physiology & Membrane Biology, UC Davis (2006). Fuller Lect, ASPET, 2009. John Brookhart Lecture, Oregon Health Services University, 2009. Keynote, Texas Tech Research Week, 2010. Keynote, Vanderbilt Student Research, 2010. Basic Science Lecture, Society for Research in Nicotine and Tobacco, 2011. Fellowships and Honors: NSF predoctoral (1966-1968); NIH predoctoral (1968-1971); NIH postdoctoral (1971-1973); Alfred P. Sloan Research Fellowship (1974-1976); NINDS Research Career Development Award (1977-1982); NINDS Senator Jacob Javits Investigator (1985-1999), American Academy of Arts & Sciences, 2006; K. S. Cole Award, Biophysical Soc, 2007. President, Biophysical Society (2009-2010). B. Fifteen nice peer-reviewed publications (in chronological order, 254 total; h-index 71). * are most relevant to PAR-11-099 *Lester, H. A. and Chang, H. W. (1977) Response of acetylcholine receptors to rapid photochemically

produced increases in agonist concentration. Nature 266, 373-374. *Wathey, J. C., Nass, M. M. and Lester, H. A. (1979) Numerical reconstruction of the quantal event at

nicotinic synapses. Biophys. J. 27, 145-164. Nerbonne, J. M., Richard, S., Nargeot, J. and Lester, H. A. (1984) New photoactivatable cyclic nucleotides

produce intracellular jumps in cyclic AMP and cyclic GMP concentrations. Nature 310, 74-76. Lester, H. A.. (1988) Heterologous expression of excitability proteins: route to more specific drugs? Science

241, 1057-1063. *Leonard, R. J., Labarca, C. G., Charnet, P., Davidson. N. and Lester, H. A. (1988) Evidence that the M2

membrane spanning region lines the ion channel pore of the nicotinic receptor. Science 242, 1578-1581. *Charnet, P., Labarca, C., Leonard, R. J., Vogelaar, N. J., Czyzyk, L., Gouin, A., Davidson, N. and Lester, H.

A. (1990) An open-channel blocker interacts with adjacent turns of helices in the nicotinic acetylcholine receptor. Neuron 2, 87-95.

Mager, S., Naeve, J., Quick, M., Labarca, C., Davidson, N. and Lester, H. A. (1993) Steady states, charge movements and rates for a cloned GABA transporter expressed in Xenopus oocytes. Neuron 10, 177-188.

*Nowak, M. W., Kearney, P. C., Sampson, J. R., Saks, M. E., Labarca, C. G., Silverman, S. K., Zhong, W., Thorson, J., Abelson, J. N., Davidson, N., Schultz, P. G., Dougherty, D. A. and Lester, H. A. (1995) Nicotinic receptor binding site probed with unnatural amino-acid incorporation in intact cells. Science 268, 439-442.

Doupnik, C.A., Davidson, N., Lester, H.A., and Kofuji, P. (1997) RGS proteins reconstitute the rapid gating kinetics of G-activated inwardly rectifying K+ channels. Proc. Natl. Acad. Sci. USA 94, 10461-10466.

Chiu, C-S., Jensen, K., Sokolova, I., Wang, D., Li, M., Deshpande, P., Davidson, N., Mody, I., Quick, M.W., Quake, S.R., and Lester, H.A. (2002) Number, Density, and Surface / Cytoplasmic Distribution of GABA Transporters at Presynaptic Structures of Knock-in Mice Carrying GAT1-GAT1 Fusions. J. Neurosci. 22: 10251-72.

*Andrew R. Tapper, Sheri S. Mckinney, Raad Nashmi, Johannes Schwarz, Purnima Deshpande, Cesar Labarca, Paul Whiteaker, Michael J. Marks, Allan C. Collins, and Henry A. Lester (2004). Nicotine activation of 4* Receptors is Sufficient for Reward, Tolerance and Sensitization. Science, 306:1029-1032.

*Nashmi R, Xiao C, Deshpande P, McKinney S, Grady S, Whiteaker P, Huang Q, McClure-Begley TD, Lindstrom J, Labarca C, Collins A, Marks M, and Lester HA (2007) Cell specifically upregulated α4* nicotinic receptors: basis for both tolerance and cognitive sensitization. J Neurosci 27: 8202– 8218

*Drenan RM, Grady SR, Whiteaker P, McClure-Begley T, McKinney SR, Miwa J, Bupp S, Heintz N, McIntosh JM, Bencherif M, Marks MA, and Lester HA (2008) In Vivo Activation of Midbrain Dopamine Neurons via Sensitized, High-Affinity α6* Nicotinic Acetylcholine Receptors. Neuron 60:123-136.

*Xiu X, Puskar NL, Shanata JA, Lester HA, and Dougherty DA (2009) Nicotine binding to brain receptors requires a strong cation-pi interaction. Nature 458, 534-537.

*Xiao C, Nashmi R, McKinney S, Cai H, McIntosh JM, and Lester HA (2009) Chronic nicotine selectively enhances α4β2* nicotinic acetylcholine receptors in the nigrostriatal dopamine pathway. J Neurosci 29:12428-39.



D. Research Support for Henry A. Lester 1. Support for nAChR research

R01 NS-11756-34 (Lester, PI; Dougherty, Co-I) 12/1/05-12/30/10 (including NCTE) ―Chemical Synapses - Biophysical Studies‖ Structure-function studies, intracellular events, and chaperoning/upregulation of nicotinic receptors and other Cys-loop family members. All experiments are now performed in clonal cell lines, oocytes, and cultured neurons. NS-11756 Aim 2b concerns lynx-nAChR interactions. Much of the preliminary data in the present application were supported by NS-11756. NS-11756 concerns only interactions between lynx and α4β2, whereas the present application extends these experiments to α7 and muscle nAChRs. Yet this extension is neither incremental nor trivial. The organization, interacting molecules, and disease significance seem to be entirely different among these three nAChRs. NS-11756 concerns interactions between α4β2 nAChRs and lynx within the ER and Golgi. The present application takes advantage of Jitu Mayor’s expertise to extend these studies to the cell surface.

R01 AG-033954-01 (Lester) 01/15/09 – 12/31/13 ―Chronic Nicotine: Cell-Specific Receptor and Circuit Alterations in Basal Ganglia‖ AG033954 attempts to reveal a mechanistic basis for an inadvertent therapeutic effect of chronic nicotine: the inverse correlation between an individual’s history of tobacco use and his/her susceptibility to smoking and Parkinson’s disease. AG-033954 supports experiments on upregulation by chronic nicotine in subthalamic nucleus, substantia nigra pars reticulata, substantia nigra pars reticulata, and dorsal striatum. Please see ―Comment on DA-17279, AG-033954, and NS-11756‖.

R01 DA-017279-06 (Lester) 7/1/10-6/30/15 ―Alpha4 Nicotinic Receptors in Addiction: Mouse Models‖. Experiments on brain slices from midbrain, hippocampus, and amygdala. Experiments with in vivo recording. Fluorescent nAChR subunit knock-in mice.

U19 DA019375 (Lester: Director) 9/1/10-8/31/15 ―Nicotinic Ligands for Smoking Cessation‖, National Cooperative Drug Development Center for Smoking Cessation. Discovery and target selection of nicotinic ligands for smoking cessation. The award includes subcontracts to Michael Marks at Boulder, and to Targacept, Inc. We are selecting and testing among α6- and α4-selective compounds. The Caltech group’s activities include generation of the α6Leu9’Ser mouse (reported in Drenan et al., Neuron 2008) and subsequent behavioral pharmacology.

1RC2 DA-028955-01 10/01/2009 - 9/30/2011 Mice with Functional Fluorescent Nicotinic Receptor Subunits Generates and validate fourteen strains of knock-in mice with fully functional fluorescent mouse nicotinic receptor (nAChR) subunits. Includes a subcontract to Michael Marks at Boulder.

Univ of CA/TRDRP Research Program (Lester) 07/01/08 – 06/30/11 ―Nicotinic Receptors in Nicotine Addiction‖ The TRDRP project studies the biology of α6* nAChRs by generating several BAC transgenic mouse strains with fluorescent, fully functional α6* nAChRs. A renewal application has been submitted (7/1/11-6/30/13).

1 P50 MH086383-01 (Freedman) 09/01/09 – 08/31/14 (Director: R. Freedman, Univ Colorado Denver Health Sciences Ctr.; Lester, PI of Project 5) ―Basic to Clinical Molecular Neurobiology of Nicotinic Receptors in Schizophrenia‖ Upregulation is unlikely to explain how schizophrenics smoke heavily. We will produce a mouse strain harboring human CHRNA7 syntenically in place of mouse chrna7. This will allow us to study differences between the mouse and human gene, to assess the effects of variants in the gene found in schizophrenia, to



determine the optimal time window and dose for treatment during perinatal period, and to help design the safest and most effective human treatment. 2. Support for “neuroengineering” using the GluCl channel system

R21 EY018502 (Lester) 9/30/07 – 8/31/10 ―Parkinson’s Disease: Viral Constructs for Pharmacological Deep-Brain Stimulation‖ To develop a novel gene transfer-based approach as an alternative to deep brain stimulation (DBS) in the treatment of advanced Parkinson’s disease (PD).

McKnight Found Tech Innovations (Lester) 08/01/08 – 07/31/10 ―Ion Channels for Neuronal Engineering‖ To develop the C. elegans GluCl channels into reagents for selective, reversible manipulations of neuronal circuits.

1RC1 MH-088550-01 10/01/2009 - 9/30/2011 ―Avermectin receptors for neuronal engineering‖ Addresses 06-MH-103, New technologies for neuroscience research. Reagents will be generated that can achieve adjustable, reversible, cell-specific, electrophysiologically measurable, and non-antigenic manipulation of neuronal excitability, and of Ca-activated signal transduction within neurons. Submitted applications:

Michael J. Fox Foundation 6/1/2011- 7/30/2013 Marie-Françoise Chesselet and Henry A. Lester, co-PIs. ―Mouse Strains for Specifying Nicotinic Receptor Targets in Parkinson’s Disease Neuroprotection‖ Thy-1 α-synuclein overexpressing mice provide an appropriate model for nigrostriatal dopamine loss caused by a mechanism highly relevant to sporadic PD. This loss begins at ~ 1 yr of age; but deficits begin earlier in weight, motor co-ordination, spontaneous activity, nest building, cortical noradrenaline, and proteinase-K-resistant α-synuclein aggregates. Many cell types express nAChRs, and it is therefore appropriate to test whether nicotine protects non-DA neurons as well as DA neurons. We will experiment with chronic nicotine administration in the male thy-1 α-syn mice. We will also cross the thy-1 α-syn over-expressing strain with three nAChR KO strains (α4 KO, α5 KO, and α6 KO). We will perform chronic nicotine experiments on males of the three new α-syn overexpressing strains (α4 KO, α5 KO, and α6 KO).

Caltech/UCLA Joint Translational Research Program 6/1/2011- 7/30/2013 Marie-Françoise Chesselet and Henry A. Lester, co-PIs. (similar to MJFF application) (duplicate funding will not be accepted)





NAME Satyajit Mayor

POSITION TITLE Professor and Dean

eRA COMMONS USER NAME (credential, e.g., agency login) mayors

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, include postdoctoral training and residency training if applicable.)



Indian Institute of Technology, Mumbai MSc 1980-1985 Chemistry Rockefeller University, NYC PhD 1985-1991 Life Science Columbia University, NYC Post-doctoral training 1991-1995 Cell Biology

A. Personal Statement: The broad aim of Satyajit Mayor’s laboratory is to provide an understanding of the molecular mechanisms of endocytosis in metazoan cells, and study this phenomenon at many scales. At the molecular scale he wants to uncover the molecular players in endocytic processes; at the mesoscopic scale his work attempts to provide a physical description of cell membrane structure and organization process and its material properties; at the cellular scale his work is aimed at synthesising a role for endocytosis in cellular signalling and cell surface homeostasis; at the scale of the tissue he wishes to determine how control of endocytosis impinges on many developmental programs in tissue morphogenesis. Since many of the phenomena originate at the nanoscale, a region where conventional optics is not able to provide information, Mayor, has developed new microscopy techniques to study nanometer scale organization of cellular components1. Using quantitative fluorescence microscopy his laboratory has explored the sorting properties and endocytic pathways of a variety of molecules, including membrane proteins, lipids and lipid-tethered proteins, both in living cells in tissue culture and in vivo. His method of investigation is to study the organization and dynamics of fluorescently-tagged molecules at different scales in living cells; from the nanometer scale in specialized domains in cell membranes to the micron scale, prevalent in mapping endocytic pathways or in specialized regions of the cell surface.

B. Positions and Honors

Positions and Employment

INSTITUTION AND LOCATION POSITION (if applicable)


Woodshole Microscopy Course, Woodshole, Massachusetts, USA

Lecturer October 1994

Imaging

National Centre for Biological Sciences, Bangalore, India

Reader (SE) Assistant Professor

Sept 1995- Feb 2001

Cell Biology

National Centre for Biological Sciences, Bangalore, India

Associate Professor Feb 2001 – Feb 2006

Cell Biology



Other Experience and Professional Memberships S. Mayor currently is Professor and Dean of the Faculty at the National Centre for Biological Sciences. He is on the Governing Board of inSTEM, India first Stem Cell Biology Research Institute. At the NCBS he has helped initiate a Centre, called Centre for Cell and Molecular Platforms (C-CAMP; funded by the Department of Biotechnology, Govt. of India) to help develop, innovate new techniques, and expand the use of platform technologies (Mass Spectrometry, Screening, Genomics, and Fluorescence microscopy and Cytometry). He is also co-chair of the Bangalore Microscopy Course, a yearly affair organized jointly by 100X-Imaging and NCBS that attracts researchers who have pioneered the use of microscopy to solve outstanding problems in biology, to teach microscopy. He also serves on the Scientific Advisory board of two Biotech companies in Bangalore. S. Mayor serves on the Editorial board of several international journals in his field of research: these include Cell, Molecular Biology of the Cell, Traffic, Integrative Biology and Biochemica Biophysica Acta, and as an invited editor for Proceedings of the National Academy of Sciences (USA), J. Cell Science and The Biochemical Journal.

Honors 2010; TWAS Prize in Biology, 2010; EMBO Global Exchange Fellow, 2006-2011, JC Bose Fellowship, DST, 2003; Shanti Swaroop Bhatnagar Award, CSIR 2003; Swarnajayanti Fellowship, Department of Science and Technology, 1999-2004; Wellcome Trust International Senior Research Fellow (Biomedical Research, India), 1992-1995; Helen Hays Whitney Post-Doctoral Fellowship 1988; The Rockefeller-Oxford Student Exchange Program Recipient 1986-1991; Lucille P. Markey Charitable Trust Graduate Fellowship 1984; Visiting Students Research Program Recipient (TIFR)

C. Selected Peer-reviewed Publications (relevant to application) 1. Goswami, D., Gowrishankar, K., Bilgrami, S., Ghosh, S., Raghupathy, R., Chadda, R.,

Vishwakarma, R., Rao, M., and Mayor, S. Nanoclusters of GPI-anchored proteins are formed by cortical actin-driven activity. Cell 135, 1085-1097(2008).

2. Vyas, N., Goswami, D., Manonmani, A., Sharma, P., Ranganath, H. A., VijayRaghavan, K., Shashidhara, L. S., Sowdhamini, R., and Mayor, S. Nanoscale organization of hedgehog is essential for long-range signaling. Cell 133, 1214-1227 (2008)..

3. Kumari, S., Borroni, V., Chaudhry, A., Chanda, B., Massol, R., Mayor, S., and Barrantes, F. J.. Nicotinic acetylcholine receptor is internalized via a Rac-dependent, dynamin-independent endocytic pathway. J Cell Biol 181, 1179-1193 (2008).

4. Altman, D., Goswami, D., Hasson, T., Spudich, J.A., and Mayor, S. (2007). Precise positioning of myosin VI on endocytic vesicles in vivo. PLoS Biol 5, e210.

5. Mayor, S., and Pagano, R.E. (2007). Pathways of clathrin-independent endocytosis. Nat Rev Mol Cell Biol 8, 603-612.

6. Sharma, P., Varma, R., Sarasij, R. C., Ira, Gousset, K., Krishnamoorthy, G., Rao, M., and Mayor, S. (2004). Nanoscale organization of multiple GPI-anchored proteins in living cell membranes. Cell 116, 577-589.

7. Rao, M., and S. Mayor. 2005. Use of Forster's resonance energy transfer microscopy to study lipid rafts. Biochim Biophys Acta. 1746:221-33.

8. Varma, R. & Mayor, S. GPI-anchored proteins are organized in sub-micron sized domains at the cell surface. Nature. 394, 798-810 (1998).

D. Research Support

Ongoing Research Support 1. Department of Biotechnology, Centre for Excellence grant (as a Co-PI) 2009-2014

“Multi-component protein assemblies: Analysis, prediction, systems view and experimental validation” 10% effort.



2. Indo-French (CIFEPRA) Grant (S. Mayor and Ludger Johannes) 2008-1011 “Shiga toxin endocytosis modifiers.” 10 % Effort.

3. Department of Science and Technology, J C. Bose Fellowship (Award for personal support) 2008-2011. 4. Department of Science & Technology, Nanoscience initiative Grant for ‘Nanobiotechnology’; 2005-2010,

awarded to S. Mayor, G. Shivashankar, Y. Krishnan-Ghosh, K. Rao. (10 % Effort)

Completed Research Support

1. Human Frontiers Science Programme, Sept. 2005-2008. awarded to S. Mayor, T. Kurzchalia, Y. Hanun and H. Riezman, „Role of sphingolipids in raft formation‟ $400,000.



PHS 398 Cover Page Supplement

1. Project Director / Principal Investigator (PD/PI)

* Title:

* Street1:

* City:

* Country:

Street2:

County/Parish:

* State:

* Zip / Postal Code:

Prefix: * First Name:

Middle Name:

* Last Name:

Suffix:

2. Human Subjects

Clinical Trial?

* Agency-Defined Phase III Clinical Trial?

* First Name:

Middle Name:

* Last Name:

Suffix:

3. Applicant Organization Contact

Person to be contacted on matters involving this application


Email:

OMB Number: 0925-0001

Province:

Prefix:

Julie

M

Miwa

No Yes

No Yes

Lucy

Molina

626-395-2372 626-795-4571

[email protected]

Contract and Grant Analyst

1200 E. California Blvd.


Pasadena

Los Angeles

USA: UNITED STATES

CA: California

91125-0001

Clinical Trial & HESC Page 45


4. Human Embryonic Stem Cells

* Does the proposed project involve human embryonic stem cells?

If the proposed project involves human embryonic stem cells, list below the registration number of the specific cell line(s) from the following list: http://stemcells.nih.gov/research/registry/. Or, if a specific stem cell line cannot be referenced at this time, please check the box indicating that one from the registry will be used:

Specific stem cell line cannot be referenced at this time. One from the registry will be used.

PHS 398 Cover Page Supplement

YesNo

Cell Line(s):

Clinical Trial & HESC Page 46


PHS 398 Modular Budget, Periods 1 and 2OMB Number: 0925-0001

1.

Start Date: End Date:

Budget Period: 1

* Direct Cost less Consortium F&AA. Direct Costs

B. Indirect Costs

Consortium F&A* Total Direct Costs

Indirect Cost TypeIndirect Cost Rate (%)

Indirect Cost Base ($) * Funds Requested ($)

Cognizant Agency (Agency Name, POC Name and Phone Number)

Indirect Cost Rate Agreement Date Total Indirect Costs

2.

3.

4.

* Funds Requested ($)

09/01/2011 08/31/2012

FY10 On Campus 62.0% MTDC 62 150,000.00 93,000.00

Office of Naval Research, David Godfrey, 703-696-2586

10/01/2010 93,000.00

150,000.00

150,000.00

243,000.00Funds Requested ($)C. Total Direct and Indirect Costs (A + B)

1.


Budget Period: 2


B. Indirect Costs

Consortium F&A

* Total Direct Costs





Funds Requested ($)C. Total Direct and Indirect Costs (A + B)

2.

3.

4.


62 125,000.00FY10 On Campus 62.0% MTDC 77,500.00

Office of Naval Research, David Godfrey, 703-696-2586

77,500.0010/01/2010

09/01/2012 08/31/2013

125,000.00

125,000.00

202,500.00

Modular Budget Page 47


PHS 398 Modular Budget, Periods 3 and 4

1.


Budget Period: 3


B. Indirect Costs







2.

3.

4.

1.


Budget Period: 4


B. Indirect Costs

Consortium F&A

* Total Direct Costs






2.

3.

4.





PHS 398 Modular Budget, Periods 5 and Cumulative

1.


Budget Period: 5


B. Indirect Costs







2.

3.

4.

Cumulative Budget Information


*Section A, Total Direct Cost less Consortium F&A for Entire Project Period

1. Total Costs, Entire Project Period

Section A, Total Consortium F&A for Entire Project Period

*Section A, Total Direct Costs for Entire Project Period

*Section B, Total Indirect Costs for Entire Project Period

*Section C, Total Direct and Indirect Costs (A+B) for Entire Project Period

2. Budget Justifications

275,000.00

275,000.00

170,500.00

445,500.00

$

$

$

$

$

Personnel Justification

Consortium Justification

Additional Narrative Justification

View AttachmentDelete AttachmentAdd AttachmentBudget_Jus_Caltech___India1003





Budget Justification Julie Miwa, Ph.D. Dr. Miwa discovered lynx and has contributed several key first-author papers to describing its biology (see biosketch). Dr. Miwa will oversee most aspects of Aim 1 at Caltech. Dr. Miwa will devote 50% effort to the project (6.0 calendar months). Henry Lester, Ph.D. Henry Lester oversaw most of the recent fluorescence experiments at Caltech and will continue to analyze fluorescence data and construct experimental strategies in Aim 1. Dr. Lester will devote 5% effort to the project (0.60 calendar months). Ying Wang, Ph.D. Dr. Wang has considerable experience with working on alpha 7 nicotinic acetylcholine receptors (nAChRs). She has published work on the mechanisms involved in the assembly and surface expression of alpha7 nAChRs. She is currently a post-doctoral fellow in Dr. Henry Lester’s laboratory and has constructed a suite of fluorescently labeled alpha 7 nAChR constructs that are suitable to be used in this proposal. She routinely uses these constructs for high-resolution imaging experiments in living cells, using techniques such as TIRF microscopy and spectrally-resolved confocal imaging. These experiments are done in conjunction with labeled a-bungarotoxin experiments to measure the ratio of surface to total levels of receptor in the cell. She will be performing the alpha 7 nAChR experiments outlined in specific aim 1. Tim Indersmitten, Ph.D. Dr. Indersmitten will be performing the patch-clamp electrophysiological and pharmacological studies outlined in this proposal. He is an experienced electrophysiologist who has a strong background in neuroscience as it relates to plasticity and neurological disease. His current project involves the pharmacological characterization of alpha 7 nAChR mutants using patch-clamp electrophysiology in transiently transfect mammalian cells. He will be committing 25% effort to this award (3.0 calendar months). Larry Wade, Ph.D. Dr. Wade is an expert in optics, and invented new methods for super-resolution microscopy. He developed variable-angle TIRF microscopy for the purpose of distinguishing nicotinic receptors in the endoplasmic reticulum from those on the plasma membrane. He will provide technical advice and oversee the TIRF measurements outlined in this proposal, and work with Rell Parker, a graduate student under Dr. Miwa’s direction and/or post-doctoral fellow Ying Wang on the TIRF experiments.

Personnel Justification Page 50


Program Director/Principal Investigator (Last, First, Middle): Mayor, Satyajit

DETAILED BUDGET FOR INITIAL BUDGET PERIOD DIRECT COSTS ONLY

FROM THROUGH

09/01/2011 08/31/2012

List PERSONNEL (Applicant organization only) Use Cal, Acad, or Summer to Enter Months Devoted to Project Enter Dollar Amounts Requested (omit cents) for Salary Requested and Fringe Benefits

NAME ROLE ON PROJECT

Cal. Mnths

Acad. Mnths

Summer Mnths

INST.BASE SALARY

SALARY REQUESTED

FRINGE BENEFITS TOTAL

Prof Satyajit Mayor PD/PI 4.0 0

Ms Garima Singhal Grad student 12 4,800 4,800

Unnamed PDRA 12 10,670 10,670

SUBTOTALS 15,470 15,470 CONSULTANT COSTS EQUIPMENT (Itemize) Customized TIRF Microscope for Anisotropy Imaging

125,000 SUPPLIES (Itemize by category)

Molecular Biology consumables for two researchers, tissue culture reagents including media, sera, antibiotics, transfection reagents and drugs and compounds such as a-Bungarotoxin, Latrunculin A

50,380 TRAVEL Travel for PI and posts to visit collaborating lab 9,150 INPATIENT CARE COSTS OUTPATIENT CARE COSTS ALTERATIONS AND RENOVATIONS (Itemize by category)

OTHER EXPENSES (Itemize by category)

CONSORTIUM/CONTRACTUAL COSTS DIRECT COSTS

SUBTOTAL DIRECT COSTS FOR INITIAL BUDGET PERIOD (Item 7a, Face Page) $ 200,000 CONSORTIUM/CONTRACTUAL COSTS FACILITIES AND ADMINISTRATIVE COSTS

TOTAL DIRECT COSTS FOR INITIAL BUDGET PERIOD $ 200,000 PHS 398 (Rev. 6/09) Page Form Page 4



Program Director/Principal Investigator (Last, First, Middle):

BUDGET FOR ENTIRE PROPOSED PROJECT PERIOD DIRECT COSTS ONLY

BUDGET CATEGORY TOTALS

INITIAL BUDGET PERIOD

(from Form Page 4)

2nd ADDITIONAL YEAR OF SUPPORT

REQUESTED

3rd ADDITIONAL YEAR OF SUPPORT

REQUESTED

4th ADDITIONAL YEAR OF SUPPORT

REQUESTED

5th ADDITIONAL YEAR OF SUPPORT

REQUESTED

PERSONNEL: Salary and fringe benefits. Applicant organization only. 15,470 15,470

CONSULTANT COSTS

EQUIPMENT 125,000 20,000

SUPPLIES 50,380 30,380

TRAVEL 9,150 9,150 INPATIENT CARE COSTS OUTPATIENT CARE COSTS ALTERATIONS AND RENOVATIONS

OTHER EXPENSES DIRECT CONSORTIUM/ CONTRACTUAL COSTS SUBTOTAL DIRECT COSTS (Sum = Item 8a, Face Page) 200,000 75,000 F&A CONSORTIUM/ CONTRACTUAL COSTS

TOTAL DIRECT COSTS 200,000 75,000

TOTAL DIRECT COSTS FOR ENTIRE PROPOSED PROJECT PERIOD $ 275,000 JUSTIFICATION. Follow the budget justification instructions exactly. Use continuation pages as needed. See attached.

PHS 398 (Rev. 6/09) Page Form Page 5



BUDGET JUSTIFICATION (Mayor lab) I. Personnel Post1: Principal Applicant: Prof S Mayor (salary not requested) will provide scientific oversight of the project and ensure that space and resources are available for the work proposed. Graduate Student: Currently Ms Garima Singhal who is registered to do her PhD with me is a prime candidate for this position. She has developed the necessary tools to study the AChR receptor and its intracellular dynamics and segregation. Post-Doctoral Fellow: A candidate for this work will be a PhD student with experience in microscopy and biophysics, especially focused on TIRF and FRET microscopy. II. Equipment: For the work on membrane organization we will have to build a new microscope system equipped with TIRF excitation and fluorescence anisotropy imaging. This will require a set of polarization preserving TIRF lasers and a combiner module; an appropriate dual camera-based polarization imaging platform; a motorized high NA objective type TIRF microscope; photobleaching/photoconversion laser module ; a high end computer for image collection and analysis as well as custom-built software based on MicromanagerTM. III. Supplies: The costs requested are for two persons working in cell and molecular biology related areas. These include tissue culture related consumables, such as media, sera, antibiotics, transfection reagents etc, drugs and compounds such as -Bungarotoxin, Latrunculin A and molecular biology and cloning consumables. Also, the costs for getting appropriate filters and fluorescent dyes necessary for the work have been incorporated. IV Travel Funds are requested for one trip per annum of two weeks each for Prof. Satyajit Mayor for coordination of the project work with the team members at Caltech along with subsistence costs for the trip. As well as for supporting one meeting of the Advisory Board in Bangalore. One meeting each for each person working on the project is also requested for updating and presenting results from this work.



PHS 398 Research Plan

1. Application Type:From SF 424 (R&R) Cover Page. The response provided on that page, regarding the type of application being submitted, is repeated for your reference, as you attach the appropriate sections of the Research Plan.

*Type of Application:


2. Specific Aims

3. *Research Strategy

6. Protection of Human Subjects

7. Inclusion of Women and Minorities

8. Targeted/Planned Enrollment Table

9. Inclusion of Children

10. Vertebrate Animals

13. Consortium/Contractual Arrangements

14. Letters of Support

15. Resource Sharing Plan(s)

16. Appendix

1. Introduction to Application (for RESUBMISSION or REVISION only)

Human Subjects Sections

2. Research Plan Attachments: Please attach applicable sections of the research plan, below.

Other Research Plan Sections

5. Progress Report Publication List

11. Select Agent Research

12. Multiple PD/PI Leadership Plan

4. Inclusion Enrollment Report

New Resubmission Renewal Continuation Revision


Specific_Aims_PAR_11_099_JM Add Attachment Delete Attachment View Attachment

Research_Plan_PAR_11_099_fin Add Attachment Delete Attachment View Attachment







Vertebrate_animals_NS11756_ Add Attachment Delete Attachment View Attachment


Multiple_PI_Leadership_state Add Attachment Delete Attachment View Attachment



A1_Resource_sharing_plan100 Add Attachment Delete Attachment View Attachment

Add Attachments Remove Attachments View Attachments

List of Research Plan Attachments Page 54


Specific Aims: The worldwide health burden of neural disease is projected to increase 5-fold by year 2050, to >$1trillion/year, even excluding lost productivity of family and care-givers (2009 estimate, $144B). Optimal therapies do not yet exist to slow the progression of neurological diseases, for instance in Alzheimer’s and Parkinson’s diseases; therefore understanding neurodegenerative mechanisms is a priority. Channelopathies are diseases of ion channel dysfunction, arising either in channel proteins themselves or in associated proteins (1); but ion channels are also promising targets for therapeutics. This project studies the interaction between lynx and nicotinic acetylcholine receptors. This topic exemplifies the dual themes of channelopathy and therapeutic target. Thus, mis-regulation of nicotinic acetylcholine receptors (nAChRs) is linked to neurological and neuropsychiatric disorders, including schizophrenia, some epilepsies, nicotine addiction, myasthenia gravis, Alzheimer’s disease, and Parkinson’s disease. On the other hand, epidemiological evidence indicates an inverse correlation between a person’s history of tobacco use and his susceptibility to Parkinson’s disease. Animal studies indicate nicotinic receptor involvement in some forms of neuroprotection both in vivo and in vitro. And the anticholinesterase drugs, which enhance both nicotinic and muscarinic transmission, represent good available drugs for Alzheimer’s disease—but this disease surely needs improved therapy that actually arrests the neurodegeneration. Lynx is a GPI-linked protein. As a class, GPI-proteins regulate neurotransmitter release, and protein localization at synapses (2). Localization in synaptic, extrasynaptic, and pre-synaptic areas is a crucial aspect of nAChR function. Receptors at cholesterol rich domains are more stable, and accumulate signal transduction and cytoskeletal components(3). Depletion of cholesterol from these domains results in enhanced receptor mobility(2). Specific Aim 1 (mostly at Caltech, Pasadena USA): We will visualize surface receptors at and near the plasma membrane (PM) to test the hypothesis that lynx-nAChR interactions alter the trafficking of nAChRs and their aggregation. We have employed fluorescently tagged, fully functional nAChRs (nAChRFP) to measure several aspects of nAChR biology in clonal mammalian cells as well as in knock-in mice. a. Using TIRF imaging of surface receptors, we will measure the mobility of surface receptors in living mammalian cells and in cultured primary neurons. b. Using pH sensitive fluorescent moieties to measure nAChRs as they arrive at the PM, we will quantify insertion events of individual receptor-carrying vesicles to characterize receptor incorporation into the cell surface. c. Co-labeling with markers of cholesterol / sphingolipid rich domains and cholesterol depletion studies will determine the influence of the GPI-anchor on the mobility and rates of insertion on the cell surface. Specific Aim 2 (mostly at NCBS, Bangalore India): Our previous studies show that internalization of nAChRs can be accelerated upon binding of α-bungarotoxin or anti-nAChR antibodies, and that this process occurs via a unique dynamin- and clathrin-independent pathway. We will test hypotheses that endogenous lynx proteins also affect nAChR endocytosis. a. The use of functional florescent receptors will again provide direct, non-perturbing visualization of nAChRs. We employ FRET based techniques relying on homo-oligomerization of like fluorophores to measure receptor clustering in the presence and absence of the lynx-protein(s). b. We will create fluorescent GPI-anchored lynx-proteins to explore the nanoscale organization of this protein in relationship to other GPI-anchored proteins as well as with nAChR-enriched regions. c. We will test whether lynx alters nACh endocytosis, using several fluorescently labeled markers in combination with RNA interference and high resolution imaging. The project will begin to generate rich data that reveal mechanisms of nAChR exocytosis, distribution at the cell surface, and endocytosis as influenced by interactions with lynx proteins. The results will enable future joint R01 projects that examine lynx-nAChR interactions (a) at the single-molecule level, (b) as influenced by synaptic formation, and (c) in the context of drug discovery for neural disorders. Abbreviations: α4* refers to an α4-containing nAChR , ER endoplasmic reticulum, FRET Fluorescence resonance energy transfer, FP fusion protein, GPI glycosylphosphotidylinositol, KI knockin, KO knockout, LatA Latrunculin A, ly6/uPAR lymphocyte antigen 6/urokinase plasminogen activator, nAChR nicotinic acetylcholine receptor, PM plasma membrane, SEC super-ecliptic pHluorin, TIRF total internal reflection fluorescence, TGN trans-Golgi network.

Specific Aims Page 55


Significance: The worldwide healthcare burden for neurological disorders is staggering; the proportionate share of the total global burden of disease due to neuropsychiatric disorders is projected to rise to 14.7% by 2020. Alzheimer’s disease is the 6th leading cause of death, and the only one of the top 10 causes of death without an effective treatment. Ongoing studies indicate a role of nicotinic receptors in neural disorders(4): Alzheimer’s disease(5-7), Parkinson’s disease(8-11), mood disorders(12), cognitive dysfunction(13), schizophrenia(14-15), nicotine addiction(16) some epilepsies(17-19) and myasthenia gravis(20). More than 50 studies document an inverse correlation between a person’s history of tobacco use and risk of Parkinson’s disease(8-10). Animal models support a neuroprotective effect of nicotine(21-22). In the top 15 most populated countries, Parkinson’s disease is expected to rise from 4.1 to 8.7 million by the year 2030(23).Over 80% of schizophrenics smoke, and extract more nicotine per cigarette than other groups of smokers, possibly by activating α7 nAChRs (24) (25-26). Some of the other genes identified in schizophrenia are involved in the assembly of α7 nAChRs, further supporting a potential link between α7 nAChRs and schizophrenia. Lynx proteins, and the family of ly6/uPAR family members, consist of secreted(27-28) and GPI-linked variants(29-30). The secreted proteins (SLURPs and snake venom neurotoxins), bind to the extracellular face of the receptors and channels(31-32) and modulate their gating properties(33). The structural analogy between lynx and α-bungarotoxin led to previous studies showing that lynx-receptor interactions also influence nAChRs via functional inhibition through competitive or allosteric interactions(34). The Bangalore group showed that α-bungarotoxin-receptor interactions also lead to the removal of the muscle nAChR subtype receptor from the cell surface by a unique endocytic mechanism(35) raising the possibility that the interactions with lynx-proteins at the cell surface may trigger an acute down modulation of the receptor. Concomitantly, monoclonal antibodies against a receptor epitope implicated in myasthenia gravis also trigger endocytic mechanisms to reduce surface levels of the receptor. It is therefore important to interrogate the role of the lynx molecules in modulating endocytic pathways of multiple receptor subtypes. Innovation: Previous studies have not addressed the possibility and functional consequences of intracellular interactions between membrane-tethered lynx and nAChRs during the assembly and transit of receptors to the PM. For many GPI-linked proteins, the Bangalore Group has shown that the GPI-anchor is required for its assembly into cholesterol-sensitive nanoclusters which in turn, are located in close proximity to each other. This is likely to assist in the assembly of membrane lipids into a more ordered structure at the PM there by constructing a membrane domain(36-37). GPI-proteins undergo differential sorting pathways that can influence the transition from the ER to the Golgi, and ultimate expression at the PM. Therefore, in addition to modeling lynx-receptor interaction as an acute modulator of nAChR electrophysiology, these interactions may lead to differential trafficking and localization. As nAChRs modulate neurotransmitter release, the consequence of lynx on nAChR number can considerably affect neuronal communication. Substantial data illuminate the functional role of the GPI-anchor in the innate immune molecule, CD59(38), another member of the ly6/upar superfamily containing lynx1. Determining that the GPI anchor in lynx sorts to cholesterol rich domains would be modestly innovative, given the substantial precedence for other GPI-proteins. On the other hand, finding that receptor sorting is mediated by lynx1, and that this underlies the robust plasticity observed in lynx1KO mice(39-40), would be highly innovative. Also lynx biology could reveal previously undetected cholinergic dependent processes with high therapeutic value: mechanisms that regulate the number, size, and domain structure of surface nAChRs. The Caltech group recently reviewed how both the pathophysiology and (in some cases inadvertent) therapeutic effects of nicotinic receptor ligands depend on selectivity at all levels studied: brain area, neuronal cell subtype, pharmacological subtype, receptor stoichiometry, and—as studied here—subcellular localization and organization (41). We give one recent example. Critical periods are early postnatal times of high synaptic lability, plasticity, brain rewiring, and synaptic rearrangements. Lynx1 KO mice display reopening of the critical period in adult visual cortex; nAChR activation governs this phenotype (40). These data demonstrate that mechanisms exist are in place for plasticity in adulthood, and releasing the molecular brake--lynx--could have relevance to amblyopia, and other cholinergic-dependent diseases. The two research groups have a continuing record of innovation in instrumentation, cell biology, molecular neuroscience, and mouse genetics. We use appropriate novel and innovative imaging techniques to conduct the experiments of this proposal. Approach: Preliminary data support the project’s importance and feasibility: Genetic experiments, employing double KOs of lynx1 and nAChR subunit double mutant mice, indicate a role of nAChRs; lynx1KO degenerative

Research Strategy Page 56


phenotype in aging mice depends on α7 and α4β2 nAChRs (Figure 1). Co-immunoprecipitation experiments show interactions of lynx1 with α4β2, α7 (Figure 1), and muscle nAChRs (data not shown). Studies on the role of the GPI-anchor were performed in a genetic experiment. We expressed lynx isoforms in the brains of three genetically modified mouse lines, including full length and secreted lynx1. The engineered, secreted

variant of lynx1 lacks the C-terminus membrane anchor sequence, but is otherwise identical to the full-length sequence. Over-expression of full length lynx1 had no significant effect on learning, whereas independent lines with secreted lynx1(s-lynx1) showed motor learning enhancements (Figure 2). Purified s-lynx1 had demonstrated activity when applied to α4β2 injected oocytes (29) (Figure 2 inset), distinct from co-injected GPI-anchored lynx1(34). Thus, membrane tethering and the GPI anchor may be important for the proper function of lynx1. The Caltech lab has developed a series of fluorescence-based procedures to understand the role of nicotine as a selective

pharmacological chaperone of acetylcholine receptor number and stoichiometry (SePhaChARNS). These procedures have been best-developed for the α4β2 nAChR(42-43). This section describes preliminary data on the fluorescent procedures showing good applicability to lynx1-nAChR interactions. Now PAR-11-099 provides the opportunity to exploit other fluorescent nAChR subunits that we have also developed and verified functionally but not yet studied with lynx: in this project (α7 and the muscle nAChR), and others not studied here (α3β4, α6β2(44), α5 subunits). To directly measure the effects of the GPI anchor, we visualized cholesterol rich domains where GPI-anchored proteins localize. Cells were transfected with α4GFPβ2 nAChR with and without lynx1 (Figure 2). We cultured primary neuronal cultures to investigate nAChR trafficking mechanisms in the presence (wild-type) and absence (lynx1KO) mouse cortical neurons. Neurons provide a means or separating receptors in the ER from surface receptors, since dendrites contain few ER organelles. We also exploited genetic models, such as lynx1KO mice, to explore receptor localization and dynamic studies in the context of normal, half-dosages and absent levels of lynx1. In these studies α4GFP β2 nAChRs co-transfected with a PM marker, Cs2cherry, indicated more clustering of nAChRs in dendrites in lynx1KO neurons compared to wild-type. In one fluorescence modality, static measurements of receptors were taken in total internal reflection fluorescence (TIRF) mode of α4GFPβ2, enabling us to visualization of fluorescently labeled intracellular

molecules within ~300 nm of the cell-coverslip interface (45-47). We recently improved and quantified the techniques, including use of variable-angle TIRF with a 1.65 numerical aperture (NA) lens (48-49). We simultaneously detect fluorescence emission from GFP and mcherry. The signal from intracellular and organelles and the PM can be distinguished by several methods: near membrane

I N IP I N IPnAChRlynx1

a4b2 a7

Figure 1. Interactions between lynx and nAChR subtypes. Insert, co-immunoprecipitation of lynx and α4β2 nAChR (lanes 1-3) and α7 nAChRs (lanes 4-6). I= input, N=no antibody IP=anti-nAChR (lane 3 and 7). Indicates a direct interaction of lynx and α4β2 and α7 nAChRs. Graph, genetic evidence that lynx acts through α4β2 and α7 nAChRs. The degenerative phenotype in lynx1KO mice is ameliorated in the absence of β2 and α7 nAChRs.(34, 39).

L7-lynx1 lynx1BAC L7- s lynx1 lynx1KO

lynx1 s lynx1 s lynx1 control

Figure 2. Full length GPI-anchored and soluble forms of lynx1. Transgenic mice expressing full length and secreted lynx1 (s-lynx1) demonstrate differential motor learning in rotarod tests. Inset, left: schematic of full length, GPI-anchored lynx1 and s-lynx1. Inset, right, effect of purified s-lynx1 on α4β2 injected oocytes, in response to 1 mM ACh application(29). Bottom: Co-labeling of cholesterol rich domains with cholera toxin 594 and α4cherryβ2 in white. Bar, 10 μm. Caltech data.

Figure 3. Primary neuronal cultures from cortex. α4cherryβ2 nAChRs in lynx1KO (upper) vs. wild-type (lower) mouse neurons. nAChRs from lynx1 KO mouse neurons demonstrated more receptor clustering than those in wild-type neurons. Quantitation revealed no significance in overall fluorescence (data not shown), but demonstrated increased number of receptor clusters (upper graph), and decreased cluster sizes (bottom graph). Caltech data.



ER has a linear, reticulated pattern with little movement, and co-localizes with ER markers such as ER-tracker and genetically encoded markers such as the peptide KDELCherry. TGN vesicle co-localize with galactose-1-phosphate uridylyltransferase (GalT) and with other markers. We analyze the data using ImageJ and Metamorph, including custom routines (50). To quantify the fraction of ER fluorescence, we measured ratios of integrated densities for the whole cell TIRF footprint to ER localized signals (whole cell/ER ratio).The ratio was significantly increased in the lynx transfected cells, indicating that receptors are exiting the ER. This trend was also seen in both N2A and HEK cells. These data contrast with findings in nicotine treated cells, in which nAChRs demonstrate an increase in PM receptors, and a decreased ratio of the cellular footprint/ER. Approach, experimental design: The composite portrait of receptors at the PM will depend on both the rate of assembly and insertion of receptors into the membrane, as well as the rate of endocytosis. Therefore to understand the molecular events leading surface expression of receptors, we will measure of both nAChR arrival and departure. Specific aim 1, overview: -Using TIRF imaging of surface receptors, we will measure the mobility of surface receptors in living mammalian cells and in cultured primary neurons. -Using pH sensitive fluorescent moieties to measure nAChRs as they arrive at the PM, we will quantify insertion events of individual receptor-carrying vesicles to characterize receptor incorporation into the cell surface. -Co-labeling with markers of cholesterol / sphingolipid rich domains and cholesterol depletion studies will determine the influence of the GPI-anchor on the mobility and rates of insertion on the cell surface. -Measurements of surface nAChRs using electrophysiological measurements. Aim 1, experimental design: The sequence of lynx1 conforms to the consensus for GPI attachment (51). Based on this, we will mutate to eliminate the final cleavage step before GPI attachment (GPI-x-lynx1). We are currently constructing both GFP-x-lynx1 and GFP-x-lynx1GFP and lynx1GFP is planned. Other lynx-related reagents available: mammalian expression constructs for lynx1, soluble lynx1, 8 lynx mutants designed to eliminate nAChR binding, and ly6-H. Choice of receptor subtype: We will study α7 and muscle nAChR, and also conduct PM-oriented α4β2 studies not supported by NS11756. nAChRs available: α7 nAChR labeled with GFP, cherry, YFP and CFP; α4 labeled with GFP, cherry, YFP, CFP, and ecliptic-pHluorin (SEC); β2 labeled with GFP, cherry YFP and CFP, and SEC. lynxGFP and the SEC-α7 are planned. Mouse lines: Lynx1KO mice, α4YFP knockin (KI), α4CFP KI, and lynx1KO/α4YFP KI double mutant lines are available. α7GFP KI founders are established once the allele is transmitted, N1 progeny will be crossed to the lynx1KO line, if time permits. Choice of cell type: Most experiments will be conducted in mammalian cell lines; we select among Neuro2A (N2A), Cos-7, HEK, and C2C12 cells for favorable biology and optics. Stable cell lines are available for α4YFP β2 CFP in HEK cells and α7YFP and α7CFP in SHEP cells. Neurons will be cultured from lynx1KO mice, their wild-type littermates, α4YFP nAChR KI/lynx1KO double mutant lines, and α7GFP KO/lynx1KO lines if/when available. Aim 1, experiment #1: We will visualize surface receptors at and near the PM to test the hypothesis that lynx-

nAChR interactions alter nAChR trafficking and aggregation (Figure 4). We have employed fluorescently tagged, fully functional nAChRs (nAChRFP) to measure several aspects of nAChR biology in clonal mammalian cells. We will visualize the PM receptors by using TIRF, including variable angle TIRF using a 1.65 NA lens which distinguishes PM nAChRs from those in the ER (49). We will label cholesterol rich domains with fluorescently labeled cholera toxin (Figure 2) and determine the extent of co-localization between these domains and nAChRs at the PM. Comparisons between lynx and lynx

mutants will be conducted on muscle, α7GFP, and α4GFP β2 nAChRs.

0

2

4

6

8

PM in

tegr

ated

den

sity

(AU,

hun

dred

thou

sand

s)

(13) (20)α4-mcherryβ2

+ lynxα4-mcherryβ2

- lynx

(13) (20)α4-mcherryβ2

- lynxα4-mcherryβ2

+ lynx

0

1

2

3

Inte

grat

ed d

ensi

ty ra

tio(w

hole

foot

prin

t / E

R)

*

Figure 4. Static features of α4β2 nAChRs affected by lynx. α4GFPβ2 imaged in TIRF mode in HEK cells. Lynx1 increases the ratio of footprint to ER, suggesting that receptors exit the ER, and reside in post-ER organelles and/or PM. Error bars depict 99% confidence intervals based on a two-tailed t -test. Caltech data.



Potential problems, aim 1, experiment #1: We balance the transcriptional/translational load on the cells in each condition, using empty vector and GAT1. Ly6H is the same size as lynx1, and is in the same ly6/uPAR family, but neither binds to ly6-H nor affects nAChR function. Therefore, it provides a control load for the GPI attachment and export machinery. Aim 1, experiment #2: Measurement of nAChR dynamics (Figure 5). Kymographs will be constructed for movies from each cellular footprint imaged in TIRF mode, at grid lines across the footprint. Kymographs are quantified to reveal changes due either to motion or to appearance / disappearance. We already note a significant increase in dynamics in the cells transfected with lynx1 as compared to a balanced transfection with

α4GFP β2 alone (Figure 5). Such

Dynamic experiments

will be conducted on α4GFP β2 and α7 nAChRs using lynx1, soluble lynx1,

GPI-x-lynx1 and Ly6-H as a control. Potential problems, aim 1, experiment #2: The major limitation of this experiment: it only partially distinguishes PM nAChRs from ER or other organelles. Because most receptors are retained in the ER, dynamics occurring there may dominate the kymograph. The problem can be avoided by imaging exclusively PM receptors through various methods, such as variable angle TIRF, pH-sensitive fluorophores, and imaging in neurons (discussed in the following sections). Another, less drastic limitation: it covers only pixels near the coverslip, so may under-represent the extent of receptor movement. This systematic bias will not vitiate comparisons among conditions. Another method of analyzing nAChR dynamics would employ particle tracking algorithms, which we used to quantitate such movements of labeled nAChRs (Figure 6), or in conjunction with variable angle TIRF (52). Aim 1, experiment #3, experimental design: We will measure insertion events at the PM using pH-sensitive GFP, super-ecliptic pHluorin (SEC), fused in-frame to the extracellular portion of nAChRs. By controlling the pH of the extracellular solution, it is possible to reduce fluorescence in intracellular organelles and favor the

detection of PM receptors. This has been successfully demonstrated with SEC-α4 plus β2 or β4 (Figure 6) in primary neuronal cultures (we also verified their function with electrophysiology). The use of neurons has the added advantage of allowing the separation of ER from PM at dendrites and axons of neurons, which are devoid of ER. This technique has been used to image the frequency of insertion events, and the intensity of the puncta at each insertion site.

We will measure insertion of SEC nAChRs (α7 and muscle) plus and minus lynx1, and in the wild-type vs. lynx1KO neurons. Potential problems, aim 1, experiment #3: In order to efficiently control the pH in the SEC system, the SEC must be fused to the extracellular portion of the nAChR. SEC-α7 nAChRs may not express at the same levels as α7 nAChRs; we will probably mix WT and SEC constructs to have only 1-2 SEC molecule per pentamer. Measurements of surface α7 nAChRs will be measured using variable angle TIRF, electrophysiology and/or with labeled α-bungarotoxin. Aim 1, experiment #4: To assess the role of the GPI-anchor we will deplete cholesterol, using methyl-β-cyclodextrin on transiently transfected and stable cell lines to determine the influence of the GPI-anchor on the mobility and rates of insertion on the cell surface. We will co-label with markers of cholesterol / sphingolipid rich domains using labeled cholera toxin, CD59GFP, and PrPcGFP. Mammalian cells will be transfected with Cherry-linked nAChRs, and imaged in TIRF (figure 4, 5). Co-transfections will include lynx1, soluble lynx1, ly6-H, or GPI-anchor mutants. In neurons, α4cherryβ2 and α7cherry receptors, and SEC-α4 β2 will be transfected into wild-type and lynx1KO neuronal cultures. Further, in the second year, α4YFPKI / lynx1KO neuronal cultures will be available.

0

10

20

30

1 2

% α4gfp β2 nAChRs

-lynx1 +lynx1-lynx1 +lynx1

Figure 5. Dynamic features of α4β2 nAChRs are altered by the presence of lynx1. A, Single frames from TIRF movies in living COS-7 cells transfected with α4GFPβ2 with (left), and without (right) lynx1. Lines drawn over the footprint were used to generate kymographs of cells without (upper right) and with lynx1 (mid right). Graph: Quantitation of events in kymographs from receptor vs receptor plus lynx1 indicates altered dynamics in lynx containing cells. Caltech data.

β4-wt β4-R348C

(6) (6)

Ins

min

-1(1

0 μ

m)-1

0

20

40

60

80

100

*

Figure 6. Measurement of nAChR insertion events, visualized using pH sensitive fluorescent proteins, SEC-α4* plus WT or ALS-associated β4. Caltech data of Chris Richards



Aim 1, experiment #5, electrophysiological receptor analyses: To verify findings of alterations in surface expression of nAChRs, we will measure ACh puff-induced currents in whole-cell patched cells co-transfected with α7 and muscle nAChRs with lynx1 and lynx1 mutants in HEK cells. Lester’s biosketch shows our experience. We will verify appropriate pharmacology by blocking with appropriate antagonists: mecamylamine (all nAChRs), DHβE (α4β2), and MLA (α7). Potential problems, aim 1, experiment #5: Measurements of maximal current can be distorted by agonist blocking at high doses of agonist. This can be magnified for receptors with high EC50. Also, faster desensitization (as has been reported for the effects of lynx1) can also distort the EC50 and can appear as a reduction in maximal current. We will minimize desensitization by using (a) a piezoelectric manipulator to retract the drug electrode between drug applications and (b) Applications <500 ms. Approach overview: specific aim 2: We will utilize new microscopies developed in the Bangalore lab (53-54) to image molecular interactions at the nanoscale. Based on the visualization of homo- and hetero-FRET in widefield, confocal and TIRF mode, we will explore the nanoscale organization of labeled functional subtypes of the receptor as well as its co-ocalization with the GPI-anchored lynx-proteins. The nanoscale architecture of the lynx-protein will also be explored, and its spatial relationship with respect to different receptor subtypes will be established. This will be followed up with studies on the influence of the endocytic propensity of the receptors due to their engagement with the α-bungarotoxin-like lynx-proteins. Aim 2, experiment #1: Widefield or TIFR-based homo-FRET methods allow us to directly visualize molecular

clustering at the surface of adherent cells. We will explore the repertoire of functional florescent receptors available in this collaboration to probe the nanoscale organization of nAChRs subtypes, directly. Specifically we

will express multiple labeled receptor isotypes in HEK1 cell as described in Figure 5, and observe receptor oligomerization using homo-FRET methods utilized by the Bangalore laboratory. These methods and the TIRF method have been utilized in several publications. Briefly, excitation by polarized light generates polarized emission from fluorophores; slowly tumbling fluorophore such as the FPs preserve their emission polarization. However, due to energy transfer, the extent of the polarization is drastically reduced. This loss in fluorescence polarization provides a quantitative measure of the extent of FRET between like fluorophores(55).

Potential problems, aim 2, experiment #1: It is possible that if we have

homomeric nAChRs, and more than one

labeled subunits per receptor we are likely to

see homo-FRET due to intra-receptor (inter-subunit) FRET, and this will be difficult to distinguish from inter-receptor FRET. To ensure that we understand the reasons for the reduced anisotropy we will utilize time-domain anisotropy measurements where the decay rates of the anisotropy directly report on the inter- fluorophore distances (Figure 7) It is likely that inter-receptor vs intra-receptor will differ. Caltech experiments show that FRET between adjacent versus non-adjacent subunits exhibits different efficiencies which are readily quantified using donor recovery after photobleaching, pixel-resolved sensitized emission(48, 56), or more recently in unpublished data, fluorescence lifetime imaging microscopy. Inter-receptor distances are likely to differ from these, and their contributions may be assessed by expressing the labeled receptor in the presence of unlabeled receptor where the probability of having a single labeled receptor molecule per oligomeric form is enhanced. However, we expect that in the steady-state anisotropy measurements, if there is a stable composition of labeled subunits (one or more), we will obtain an average anisotropy for the isolated labeled receptor which will be greatly

Figure 7: CHO cells expressing FR-GPI were labeled with PLB-TMR and fluorescence intensity (grayscale) and anisotropy image (pseudocolored according to defined LUT) recorded on a line scanning confocal system (48).

Figure 8: A. cartoons of possible rotational motion (red arrows) for the fluorophores (PLF: fluorescent folic acid analog) or GFP. B, expected time-resolved anisotropy decay profiles for dilute GFP-fluorophores (top) immobilized in a viscous medium, such as membrane, or freely rotating in an aqueous solution (blue line). Fluorophores undergoing FRET exhibit an additional, faster anisotropy decay (36). Banglore data.



reduced when it encounters labeled neighbors. In any case, the muscle nAChR that incorporates a single labeled β1-subunit will be an appropriate subject for this measurement. Aim 2, experiment #2: a) We will utilize the GPI-anchored lynx1FP proteins constructed at Caltech to explore the nanoscale organization of this protein. We will study its organization in relationship to other GPI-anchored

proteins, in particular the ly6-HFP protein, utilizing our existing methods(36). We will first explore how the homo-FRET signal from lynx1FP is affected by the presence or absence of ly6-H GPI-anchored protein expression in nAChR negative cells such as HEK1 or Cos7. This will reveal if the lynx1 protein interacts with other GPI-anchored proteins (36). b) We will next explore the nature of the membrane environment around nAChR receptor clusters, in terms of the disposition of lynx1FP and ly6-HFP molecules. This will involve identifying nAChR cluster-rich areas at the cell surface and addressing if these regions are enriched in nanoclusters

of lynx1FP or other GPI-anchored proteins. Potential problems, aim 2, experiment #2: a) It is possible that attaching a FP to a 6kD protein will alter its

function. To ascertain that the lynx1FP protein retains its function we will utilize it in some of the assays outlined in Figures 1 -3 to determine this specifically. We have long-standing experience in generating functional FP-tagged proteins utilizing knowledge of the structure of the molecule(54). b) It is also conceivable that the nanocluster enriched domains will not be obviously overlapped with the receptor aggregates: this may require a sophisticated statistical analysis. For this purpose we will look at the joint-probability distribution of the presence of nanoclusters enriched regions with the occurrence e of receptor clusters. We will also explore a statistical method to explore if the receptors themselves exhibit any difference in receptor organization due to the presence of the lynx1 protein (Figure 9).

Aim 2, experiment #3: We will test whether expression of lynx1 alters the endocytosis of nAChRs, using the several fluorescently labeled receptors aswell as lynx1FP protein generated in this study, and assays developed by the Bangalore group. Initially we will utilize the muscle receptor expressed in C2C12 cells where we are able to monitor receptor phosphorylation and actin recruitment to understand how lynx-nAChR interaction may change endocytosis characteristics of this receptor. This will be done in combination with actin perturbing drugs (Latrunculun) and RNA interference of molecules (such as Rac1/src protein) known to play a role in endocytosis of muscle nAChR. If the presence of the lynx1 protein alters the endocytic route, we will explore if the pathway reverts to any other route. Once again we will utilize standard assays to assess perturbations of the activity of key components of these altered routes (57). We will then explore the role of lynx1 in the endocytosis of multiple subtypes of labeled nAChR.

Figure 9: A. If two different GPI-APs (gray and black circles) occupy the same cluster (left), increasing expression of one GPI-AP (black circles) leads to decreased homo-FRET increasing emission anisotropy of the fluorescent GPI-AP species being monitored. Alternatively, if different GPI-AP species are present in separate clusters (right), there is no change in the emission. B. Data from an experiment monitoring the anisotropy of GFP-GPI in cell membranes that express increasing ratios of GPI-anchored (filled circles) or transmembrane anchored (open circles) folate receptors to GFP-GPI (31).

Figure 10: Alteration of membrane organization of nAChR after treatment with actin perturbing agents Actin depolymerization (LatA treatment) leads to a reduction in the number of spots of AChR at the cell surface, but an increase in their overall brightness upon antibody ligation. Thus, antibody-induced receptor clusters grow larger in the actin-depleted membrane, although the diffraction limit vitiates determining the actual cluster size even in images collected in a TIRF microscope. Bangalore data.

Untreated LatA

intensity and no. of spots normalized to control

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

intensity of spots no. of spots

Latrunculin A



Vertebrate Animals: mice (protocol 1386-10G) Numbers (Table 1)

Table 1. Mouse strains and numbers used over the course of this 5-yr project. All mice are pregnant females from isogenic colonies.

Strain Aim Backcross to C57Bl/6 # used Present location Comments

WT C57Bl/6 2a, 2b 100 Caltech Control Lynx1-KO 2b > N10 50 Caltech Role of lynx1 α5-KO 2a > N10 50 Caltech Role of α5 subunit

Justifications Mice have been chosen for several reasons. a. Because they are mammals they are more closely related to humans than non-mammalian

vertebrates or than invertebrates. b. They are considerably less expensive than primates and are not an endangered species; indeed all

strains we use are bred specifically for laboratory research. c. Many genetic and molecular techniques are available for mice and are directly applicable to the

experiments we plan. Animal Care Veterinary care is provided through the Caltech Office of Laboratory Animal Resources. Two board

certified laboratory animal veterinarians are assisted in providing veterinary care by three licensed veterinary technicians and a large number of animal care technical staff who are certified by the American Association of Laboratory Animal Science. Animals are observed daily including weekends and holidays and veterinary care is provided as needed on a daily basis to ensure the health and welfare of the animals.

Minor surgical procedures are performed using isoflurane gas anesthesia or ketamine/xylazine injectable anesthesia. Analgesics such as ketoprofen and buprenorphine are given at the time of surgery and then as needed to ensure the comfort of the animals.

The majority of animals will be euthanized by exposure to CO2 following the Caltech IACUC guidelines for use of the euthanasia agent. Cervical dislocation is performed in a small number of animals due to avoid changes in blood gases or pH which could compromise the integrity of cells or tissue obtained from the animal. This procedure is performed by well trained experienced individuals only after approval by the Caltech IACUC. Vertebrate Animals: Xenopus for oocyte harvest (protocol 1309-09)

Justification of animal use and numbers Oocytes from Xenopus laevis will be used in these studies to classify ion channels and receptors based

on optical and electrophysiological measurements after injection of RNA into the oocytes. Xenopus oocytes are used for expressing signaling proteins and are highly appropriate cells, and therefore relevant, for identifying similar for identical processes that occur in CNS neurons.

The anatomy and physiology of the Xenopus laevis oocyte is uniquely suited to the procedures to be performed. It is a commonly used cell for electrophysiological and optical recordings. The large size of each oocyte (~ 1.0 mm) greatly facilitates handling and manipulation. This makes them attractive as models for electrophysiology and optics. Oocytes are precursors to mature egg cells and are stored in the ovarian lobes of the adult female frog. They're classified in stages (from stage I to stage VI) depending their developmental state. Stage V and VI oocytes are used for the proposed experiments and are harvested by survival surgery or after euthanization of the frog.

On average the 25-member Lester-Dougherty lab group requires 3400 oocytes per week to perform in vitro experiments on 1700 viable oocytes (the remaining oocytes are not viable and cannot be used for experiments). On average, 1-2 frogs per surgery day, two surgeries per week, are used to obtain these 1700 viable oocytes. In a year (50 weeks) this equates to 100 surgeries. On average, frogs have three surgeries, once every four months. We require a minimum of roughly 50 frogs in an eight month period or at a minimum, 64 frogs per year. To assure an adequate supply, we are requesting 75 (20% more) per year. NS-11756 will use only 10% of these oocytes.

Animal care Oocytes are removed from the frogs surgically by a small incision made on the ventral surface of the

abdomen. Frogs are anesthetized prior to and during surgery. After surgery, the frogs are sutured, individually housed and treated with antibiotics for up to two weeks. Depending on the quality of the oocytes, frogs may

Vertebrate Animals Page 62


undergo multiple survival surgeries with a maximum of four surgeries. Frogs are rested between surgeries with an interval of approximately 4 months between each surgery. After the final surgery, euthanasia is performed by immersion in MS 222 or injection of MS 222 directly into the dorsal lymph sac. Carcasses are placed in a storage freezer.

Surgical oocyte harvest is necessary to isolate the correct developmental stage of the oocyte for injection with RNA as opposed to hormone priming and harvesting oocytes once they have been laid. The oocytes from the latter harvesting protocol are too late in development for the purposed studies.

The post-operative frogs are housed singly and monitored daily for at least 10 days after surgery. The frogs are checked for skin sloughing, discoloration, dehiscence, and irritation around the suture site. Should there be evidence of skin irritation, the frog is treated according to the veterinarian’s recommendations. Individual frogs are euthanized if they exhibit extremely poor oocyte quality or have undergone 4 oocyte retrieval surgeries. While we do not expect signs of ill health in these animals, if signs of poor health are noted, the veterinary staff will be notified and the animal will be treated per veterinary recommendations.

Vertebrate Animals Page 63


Multiple PI Leadership statement: Caltech Julie Miwa directs Aims of this project conducted at Caltech. She is responsible for communications with the NIH and for drafting most of the papers. Henry Lester provides additional oversight for the biophysical and fluorescence aspects of Caltech experiments in this project. Julie Miwa is not a tenure-track faculty member at Caltech. The Caltech Provost has directed the Director of Sponsored Research to sign this application, which includes the commitments described in the Facilities and Resources and Equipment pages, for the two-year period of this grant. In the longer term, we hope that this project will transition to an R01—what then? Henry Lester plans to recommend Julie Miwa for promotion to Senior Research Associate, which carries five-year rolling tenure assurance. In short, we have foreseen, and accounted for, the appropriate questions of independence associated with Julie Miwa’s status as PI. Like all Caltech employees, both Julie Miwa and Henry Lester executed patent agreements with Caltech. These agreements cover intellectual property generated by their research. Any scientific or strategic disagreements will be mediated by the Advisory Committee. If either PI moves to a new institution, attempts will be made to transfer the relevant portion of the grant to the new institution. In the event that a PI cannot carry out his/her duties, the other PI will assume responsibility for the entire project. The signatures by Caltech’s Office of Sponsored Research provide this assurance, even considering Julie Miwa’s status as a non-tenure track faculty member.

Multiple PI Leadership Plan Page 64


1. Kass, R. S. (2005) J Clin Invest 115, 1986-9. 2. Fernandes, C. C., Berg, D. K. & Gomez-Varela, D. (2010) J Neurosci 30, 8841-51. 3. Allen, J. A., Halverson-Tamboli, R. A. & Rasenick, M. M. (2007) Nat Rev Neurosci 8, 128-40. 4. Taly, A., Corringer, P. J., Guedin, D., Lestage, P. & Changeux, J. P. (2009) Nat Rev Drug Discov 8,

733-50. 5. Picciotto, M. R. & Zoli, M. (2008) Front Biosci 13, 492-504. 6. Kawamata, J. & Shimohama, S. (2002) J Alzheimers Dis 4, 71-6. 7. Barrantes, F. J., Borroni, V. & Valles, S. (2010) FEBS Lett 584, 1856-63. 8. Ritz, B., Ascherio, A., Checkoway, H., Marder, K. S., Nelson, L. M., Rocca, W. A., Ross, G. W.,

Strickland, D., Van Den Eeden, S. K. & Gorell, J. (2007) Arch Neurol 64, 990-7. 9. Tanner, C. M., Goldman, S. M., Aston, D. A., Ottman, R., Ellenberg, J., Mayeux, R. & Langston, J. W.

(2002) Neurology 58, 581-8. 10. Wirdefeldt, K., Gatz, M., Pawitan, Y. & Pedersen, N. L. (2005) Ann Neurol 57, 27-33. 11. Hernan, M. A., Takkouche, B., Caamano-Isorna, F. & Gestal-Otero, J. J. (2002) Ann Neurol 52, 276-84. 12. Picciotto, M. R., Addy, N. A., Mineur, Y. S. & Brunzell, D. H. (2008) Prog Neurobiol 84, 329-42. 13. Haydar, S. N. & Dunlop, J. (2010) Curr Top Med Chem 10, 144-52. 14. Court, J., Spurden, D., Lloyd, S., McKeith, I., Ballard, C., Cairns, N., Kerwin, R., Perry, R. & Perry, E.

(1999) J Neurochem 73, 1590-7. 15. Leonard, S., Gault, J., Hopkins, J., Logel, J., Vianzon, R., Short, M., Drebing, C., Berger, R., Venn, D.,

Sirota, P., Zerbe, G., Olincy, A., Ross, R. G., Adler, L. E. & Freedman, R. (2002) Arch Gen Psychiatry 59, 1085-96.

16. Gonzales, D., Rennard, S. I., Nides, M., Oncken, C., Azoulay, S., Billing, C. B., Watsky, E. J., Gong, J., Williams, K. E. & Reeves, K. R. (2006) JAMA 296, 47-55.

17. Klaassen, A., Glykys, J., Maguire, J., Labarca, C., Mody, I. & Boulter, J. (2006) Proc Natl Acad Sci U S A 103, 19152-7.

18. Teper, Y., Whyte, D., Cahir, E., Lester, H. A., Grady, S. R., Marks, M. J., Cohen, B. N., Fonck, C., McClure-Begley, T., McIntosh, J. M., Labarca, C., Lawrence, A., Chen, F., Gantois, I., Davies, P. J., Petrou, S., Murphy, M., Waddington, J., Horne, M. K., Berkovic, S. F. & Drago, J. (2007) J Neurosci 27, 10128-42.

19. Xu, J., Cohen, B. N., Zhu, Y., Dziewczapolski, G., Panda, S., Lester, H. A., Heinemann, S. F. & Contractor, A. (2010) Mol Psychiatry.

20. Croxen, R., Newland, C., Beeson, D., Oosterhuis, H., Chauplannaz, G., Vincent, A. & Newsom-Davis, J. (1997) Hum Mol Genet 6, 767-74.

21. Ryan, R. E., Ross, S. A., Drago, J. & Loiacono, R. E. (2001) Br J Pharmacol 132, 1650-6. 22. Quik, M., O'Neill, M. & Perez, X. A. (2007) Trends Pharmacol Sci 28, 229-35. 23. Dorsey, E. R., Constantinescu, R., Thompson, J. P., Biglan, K. M., Holloway, R. G., Kieburtz, K.,

Marshall, F. J., Ravina, B. M., Schifitto, G., Siderowf, A. & Tanner, C. M. (2007) Neurology 68, 384-6. 24. Adler, L. E., Hoffer, L. D., Wiser, A. & Freedman, R. (1993) Am J Psychiatry 150, 1856-61. 25. Royal_College_of_Physicians (2007) Harm reduction in nicotine addiction: helping people who can’t

quit. A report by the Tobacco Advisory Group of the Royal College of Physicians. (RCP, London). 26. Papke, R. L. & Thinschmidt, J. S. (1998) Neurosci Lett 256, 163-6. 27. Chimienti, F., Hogg, R. C., Plantard, L., Lehmann, C., Brakch, N., Fischer, J., Huber, M., Bertrand, D. &

Hohl, D. (2003) Hum Mol Genet 12, 3017-24. 28. Tsetlin, V., Utkin, Y. & Kasheverov, I. (2009) Biochem Pharmacol 78, 720-31. 29. Miwa, J. M., Ibanez-Tallon, I., Crabtree, G. W., Sanchez, R., Sali, A., Role, L. W. & Heintz, N. (1999)

Neuron 23, 105-14. 30. Hruska, M., Keefe, J., Wert, D., Tekinay, A. B., Hulce, J. J., Ibanez-Tallon, I. & Nishi, R. (2009) J

Neurosci 29, 14847-54. 31. Dellisanti, C. D., Yao, Y., Stroud, J. C., Wang, Z. Z. & Chen, L. (2007) Nat Neurosci 10, 953-962. 32. Harel, M., Kasher, R., Nicolas, A., Guss, J. M., Balass, M., Fridkin, M., Smit, A. B., Brejc, K., Sixma, T.

K., Katchalski-Katzir, E., Sussman, J. L. & Fuchs, S. (2001) Neuron 32, 265-75. 33. Lyukmanova, E. N., Shenkarev, Z. O., Shulepko, M. A., Mineev, K. S., D'Hoedt, D., Kasheverov, I. E.,

Filkin, S. Y., Krivolapova, A. P., Janickova, H., Dolezal, V., Dolgikh, D. A., Arseniev, A. S., Bertrand, D., Tsetlin, V. I. & Kirpichnikov, M. P. (2011) J Biol Chem.

34. Ibanez-Tallon, I., Miwa, J. M., Wang, H. L., Adams, N. C., Crabtree, G. W., Sine, S. M. & Heintz, N. (2002) Neuron 33, 893-903.

References Cited Page 65


35. Kumari, S., Borroni, V., Chaudhry, A., Chanda, B., Massol, R., Mayor, S. & Barrantes, F. J. (2008) J Cell Biol 181, 1179-93.

36. Sharma, P., Varma, R., Sarasij, R. C., Ira, Gousset, K., Krishnamoorthy, G., Rao, M. & Mayor, S. (2004) Cell 116, 577-89.

37. van Zanten, T. S., Cambi, A., Koopman, M., Joosten, B., Figdor, C. G. & Garcia-Parajo, M. F. (2009) Proc Natl Acad Sci U S A 106, 18557-62.

38. Bonnon, C., Wendeler, M. W., Paccaud, J. P. & Hauri, H. P. (2010) J Cell Sci 123, 1705-15. 39. Miwa, J. M., Stevens, T. R., King, S. L., Caldarone, B. J., Ibanez-Tallon, I., Xiao, C., Fitzsimonds, R. M.,

Pavlides, C., Lester, H. A., Picciotto, M. R. & Heintz, N. (2006) Neuron 51, 587-600. 40. Morishita, H., Miwa, J., Heintz, N. & Hensch, T. (2010) Science 330, 1238-1240. 41. Miwa, J., Freedman, R. & Lester , H. A. (2011) Neuron invited review being refereed. 42. Nashmi, R., Dickinson, M. E., McKinney, S., Jareb, M., Labarca, C., Fraser, S. E. & Lester, H. A. (2003)

J Neurosci 23, 11554-67. 43. Nashmi, R., Xiao, C., Deshpande, P., McKinney, S., Grady, S. R., Whiteaker, P., Huang, Q., McClure-

Begley, T., Lindstrom, J. M., Labarca, C., Collins, A. C., Marks, M. J. & Lester, H. A. (2007) J Neurosci 27, 8202-18.

44. Drenan, R. M., Nashmi, R., Imoukhuede, P. I., Just, H., McKinney, S. & Lester, H. A. (2008) Mol Pharmacol 73, 27-41.

45. Oancea, E., Wolfe, J. T. & Clapham, D. E. (2006) Circ Res 98, 245-53. 46. Fish, K. N. (2009) Curr Protoc Cytom Chapter 12, Unit12 18. 47. Khiroug, S. S., Pryazhnikov, E., Coleman, S. K., Jeromin, A., Keinanen, K. & Khiroug, L. (2009) BMC

Neurosci 10, 141. 48. Srinivasan, R., Pantoja, R., Moss, F. J., Mackey, E. D. W., Son, C., Miwa, J. & Lester , H. A. (2011) J.

Gen .Physiol. 137, 59-79. 49. Wade, L. A. (2010) in Division of Biology (California Insitute of Technology, Pasadena CA), Vol. Ph. D. 50. Moss, F. J., Imoukhuede, P. I., Scott, K., Hu, J., Jankowsky, J. L., Quick, M. W. & Lester, H. A. (2009) J

Gen Physiol 134, 489-521. 51. Pierleoni, A., Martelli, P. L. & Casadio, R. (2008) BMC Bioinformatics 9, 392. 52. van 't Hoff, M., Reuter, M., Dryden, D. T. & Oheim, M. (2009) Phys Chem Chem Phys 11, 7713-20. 53. Goswami, D., Gowrishankar, K., Bilgrami, S., Ghosh, S., Raghupathy, R., Chadda, R., Vishwakarma,

R., Rao, M. & Mayor, S. (2008) Cell 135, 1085-97. 54. Vyas, N., Goswami, D., Manonmani, A., Sharma, P., Ranganath, H. A., VijayRaghavan, K.,

Shashidhara, L. S., Sowdhamini, R. & Mayor, S. (2008) Cell 133, 1214-27. 55. Varma, R. a. M., S. (2006) in Methods Express Series (Scion Publishing. 56. Son, C. D., Moss, F. J., Cohen, B. N. & Lester, H. A. (2009) Mol Pharmacol 75, 1137-48. 57. Sabharanjak, S., Sharma, P., Parton, R. G. & Mayor, S. (2002) Dev Cell 2, 411-23.

References Cited Page 66


Resource sharing plan We send all plasmids to Addgene if we deem them useful. Upon first request for other plasmids, we send them to Addgene as well.

Resource Sharing Plan Page 67


PHS 398 Checklist


1. Application Type:From SF 424 (R&R) Cover Page. The responses provided on the R&R cover page are repeated here for your reference, as you answer the questions that are specific to the PHS398.

* Type of Application:

Federal Identifier:

2. Change of Investigator / Change of Institution Questions

Change of principal investigator / program director

Name of former principal investigator / program director:

Change of Grantee Institution

* Name of former institution:

3. Inventions and Patents (For renewal applications only)

* Inventions and Patents:

If the answer is "Yes" then please answer the following:

* Previously Reported:

Prefix:* First Name:Middle Name:

* Last Name:

Suffix:

New Resubmission Renewal Continuation Revision

NoYes

NoYes

Checklist Page 68


4. * Program Income

If you checked "yes" above (indicating that program income is anticipated), then use the format below to reflect the amount and source(s). Otherwise, leave this section blank.

Is program income anticipated during the periods for which the grant support is requested?

*Budget Period *Anticipated Amount ($) *Source(s)

Yes No

5. * Disclosure Permission Statement

If this application does not result in an award, is the Government permitted to disclose the title of your proposed project, and the name, address, telephone number and e-mail address of the official signing for the applicant organization, to organizations that may be interested in contacting you for further information (e.g., possible collaborations, investment)?

NoYes

Checklist Page 69


Subject: Fwd: Validations Complete/Check Assembled Application, Grants.gov Tracking Number GRANT10832625, PI Miwa, Julie - Warnings OnlyFrom: Satyajit Mayor <[email protected]>Date: Wed, 23 Mar 2011 08:27:25 +0530To: "Dr. Savita Ayyar" <[email protected]>

Dear Savita:This is just FYI:BestJitu

-------- Original Message --------Subject:Validations Complete/Check Assembled Application, Grants.gov Tracking Number GRANT10832625, PI Miwa, Julie - Warnings Only

Date:Tue, 22 Mar 2011 19:00:19 -0400From:<[email protected]>

Reply-To:<[email protected]>To:<[email protected]>

NIH has received the electronic application titled "Lynx in organization and dynamics of nicotinic acetylcholine receptor complexes" that was submitted through Grants.

Please review the assembled application within the next two business days (i.e., Monday-Friday; excluding federal holidays). If you wish to go forward with the warnin

If the SO does not explicitly stop the application within two business days, it will automatically move to the referral process. Once the application has moved to refNote that if the application correctly reflects your submission and you choose to stop the application from moving to referral, any subsequent application submissions If you have any questions about this email, please contact the eRA Helpdesk via Web Support at http://ithelpdesk.nih.gov/eRA/.

Fwd: Validations Complete/Check Assembled Application, G...

1 of 1 24/03/11 10:31 AM

national centre for biological sciences tata institute of...

Documents