Janani Iyer

Bhattacharya Lab

NASA Ames Research Center

Investigating neuro-consequences of

spaceflight using Drosophila melanogaster

A .MES FIESE C ,E::: NATFICH ER

techshot /

Physiological response to spaceflight

Microgravity

(Lack of gravity)Radiation

Social Isolation

Cardiovascular

deconditioning

Fluidic shift

Bone loss

Muscle atrophy

Maladaptive immune system

Central Nervous system

Genetic changes - Telomeres

slow response

• ~75% of human disease-causing genes have a functional homolog in the fruit fly• Model for human innate immunity, circadian rhythms,

development, neurobehavior etc.

• Amenable to experimental manipulation• Readily available genetic tools and fly lines• Well-annotated genome • Fast generation time• Low gene redundancy

• Small, convenient for ISS experimentation (requires little power, mass, volume) to get statistically significant sample sizes

Drosophila melanogaster (fruit fly) – the model organism

2018

In Brief: Electron microscopy imaging of the entire adult fruit fly brain at synapse resolution reveals circuitry spanning multiple regions and connectivity between known and previously unknown cell types.

Shi et al., 2017; Kaiser M 2015; Heuvel et al., 2016

Drosophila melanogaster Homo sapiens

135000 neurons 100 billion neurons

Neural connectivity is strikingly similar across species

Cell A Complete Electron Microscopy Volume of the Brain of Adult Drosophila melanogaster

Graphical Abstract

< MB-C1

OtherKCs

--- • Bouton

Kenyon Cell (KC)

Bouton

Authors

Zhihao Zheng, J. Scott Lauritzen, Eric Perlman, ... , Stephan Saalfeld,

Richard D. Fetter, Davi D. Bock

4

Multi-use Variable-g Platform (MVP) Validation

Credits: Techshot Inc.

• Optimized for life/physical science research in microgravity

• 2 independent centrifuges (each can spin up to 2g)

• Controls temperature, relative humidity

• Cycles fresh cabin air into habitat

• Telemetry/real-time video & other data; ground commanding

• Reusable/reconfigurable MVP facility

• Up to 12 simultaneous experiment modules

MVP-Fly-01 experiment on SpaceX CRS-14:

Launched on: 4/2/18

Returned on: 5/5/18 (33 days)

Ground control dates: 5/27/18 – 6/29/18

techshot -----====== /

• 1,160 total Micro-g adult flies: 506 males, 654 females

• 1,076 total Space 1g adult flies: 517 males, 559 females

(+ thousands more larvae and eggs in each case)

• 883 total asynchronous ground flies: 362 males, 521 females

LARGE SAMPLE NUMBERS RETRIEVED POST-FLIGHT

A) Behavior: Contraction and crawling

B) Synaptic architecture using NMJ

Glutamatergic synapses

C) Brain morphology

• Neuron

• Glia

• DA neurons

• Apoptosis

• Proliferation

A) Behavior: In-flight locomotion assay

C) Brain morphology

• Neuron

• Glia

• DA neurons

• Apoptosis

• Oxidative stress

markers

D) Genomic and proteomic analysis D) Genomics and Proteomics analysis

Perez Torres et al., (2011). Revista Clinica.

B) Behavior: Climbing ability

• Multi-generation studies

Measuring neurobehavioral health of the flies

Janani Iyer,Siddhita Mhatre, Amber Paul and Jhony Zavaleta

Larva

Drosophila life cycle

Larva )/

I

Larva

Song et al., 2007

Beh

avioral an

d b

rain m

orp

ho

logy d

efects in sp

aceflight

larvae suggest n

euro

logical im

pairm

entsJananiIy

er, S

iddhita

Mhatre

, Jhony

Zavale

ta

Earth

Space

0

10

20

30

40

50

Larv

al c

on

tractio

n s

ex c

om

bin

ed

Body wall contractions per minute

P v

alu

e

Exa

ct o

r ap

pro

xim

ate

P v

alu

e?

P v

alu

e s

um

ma

ry

<0

.00

01

Exa

ct

****

Earth

Space

0 5

10

15

Dis

tan

ce c

raw

led

sex c

om

bin

ed

Distance crawled in 3 min (cm)

P v

alu

e

Exa

ct o

r app

roxim

ate

P v

alu

e?

P v

alu

e s

um

ma

ry

<0

.000

1

Exa

ct

****

Gro

un

d 1

gS

pace μg

pH

3

Pro

lifera

tion

mark

er

CA

B

Earth

Space

0

20

40

60

80

100

Larv

al P

H3_sex c

om

bin

ed

Normalized to area x104

P v

alu

e

Exa

ct o

r ap

pro

xim

ate

P v

alu

e?

P v

alu

e s

um

ma

ry

0.0

485

Exa

ct

*

Earth

Space

0

5000

10000

15000

20000

Larv

al E

LA

V_sex c

om

bin

ed

ELAV fluorescence

normalized to brain area

P v

alu

e

Exa

ct o

r ap

pro

xim

ate

P v

alu

e?

P v

alu

e s

um

ma

ry

0.0

106

Exa

ct

*

Earth

Space

0

50

100

150

Larv

al D

CP

1_sex c

om

bin

ed

# DCP1 +ve cells

P v

alu

e

Exa

ct o

r ap

pro

xim

ate

P v

alu

e?

P v

alu

e s

um

ma

ry

0.0

006

Exa

ct

***

Earth

Space

0

20

40

60

80

Larv

al T

H_sex c

om

bin

ed

# Dopaminergic neurons

P v

alu

e

Exa

ct o

r ap

pro

xim

ate

P v

alu

e?

P v

alu

e s

um

ma

ry

0.0

033

Exa

ct

**

***

***

**

***

**

Ne

uro

na

l ma

rker

DC

on

tractio

nD

ista

nce

cra

wle

d

EF

DA

ne

uro

n m

ark

er

Ap

op

totic

ma

rker

Larva

l bra

in

i i

ii

Direction of Movement

<IIIDIDD <IIIrrIID> Eill> (2:§TII:t>

~

~

<IIIII:ITID

PHALLOIDINHRP

A1

A2

A3

A4

A5

A6

A7

A3

Muscle 6/7

PHALLOIDINHRP

Synaptic abnormalities in spaceflight larvae

Janani Iyer,Siddhita Mhatre, Jhony Zavaleta

A

Sp

ac

e μ

g

Gro

und 1g

Spac

e µg

0.0000

0.0005

0.0010

0.0015

Bo

uto

n# / M

uscle

are

a

*

B Total Boutons

Gro

un

d 1

g

20mm

p<0.05*

HRP

Gro

und 1g

Spac

e µg

0

10

20

30

40

% S

ate

llit

e B

ou

ton

s

*

p<0.05*

C Satellite Boutons

Synaptic abnormalities in spaceflight larvae

Janani Iyer, Siddhita Mhatre

Decreased total

number of boutons

and active zones

while

increased number

of satellite boutons

in

spaceflight larvae

suggests decreased

synaptic

transmission

Gro

und 1g

Spac

e µg

0

20

40

60

80

Brp count /Area of NMJ_A3_Female_AGC to ug

Acti

ve Z

on

e / N

MJ a

rea

x10

2

*

Active zones

Gro

un

d 1

gS

pa

ce

μg

BRP

20mm

D

E Muscle areaF

Gro

und 1g

Spac

e ug

0

20000

40000

60000

80000

100000

Mu

scle

are

a

n.s.

.---------- : ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' L ________________ _

r---- --::::i :._ _____ _

Climbing abilityA

Neuronal loss in spaceflight condition: Adult flies

Decreased neuronal area and dendritic field, and reduced dopaminergic neurons in space reared flies suggests neuronal loss

E Dopaminergic neuronsFGround 1g Space 1g Space μg

Janani Iyer, Siddhita Mhatre

Gro

und 1g

Spac

e 1g

Spac

e ug

0

10

20

30

40

50

60

70

80

90

100

%C

lim

bed

in

18s

*

*

B C D

ELAV

Neuronal field area Dendritic area

Gro

und 1g

Spac

e 1g

Spac

e ug

0.000

0.005

0.010

0.015

0.020

0.025

ELAV_DC_agc, 1g, ug sex combined

Den

dri

tic f

ield

/bra

in

****

*

TH

ca cr::;--o• ... 0 :::::, ,... G) >< c-'C ! -~ cu - cu cu E e o 0 "' z

*

Neuronal loss in spaceflight condition: Adult flies

Increased apoptotic cells and decreased neuronal area and dendritic field suggests neuronal loss

Janani Iyer, Siddhita Mhatre

A Apoptotic cellsBGround 1g Space 1g Space μg

CC3

Gro

und 1g

Spac

e 1g

Spac

e ug

0.000

0.001

0.002

0.003

CC3_agc, 1g, ug sex combined

CC

3 p

osit

ve p

un

cta

n

orm

alized

to

bra

in a

rea

******

• Validation spaceflight mission for Drosophila MVP hardware → Successful

• Novel study assessing spaceflight effect on nervous system

• Behavioral deficits in larvae and adults correlates with observed morphological

defects

• Adult fly brain results suggest gravity as a major factor in neuronal deficits in

spaceflight

• Results from this mission would help in shaping hypothesis for future missions

and ground based studies

• On going: Ground acclimatization and multi-generational studies along with

genomic and proteomic analysis on adult flies

This study will help elucidate the underlying anatomical, functional and molecular changes

in the nervous system resulting from spaceflight, which in future will help identify putative

gene pathways for countermeasure studies

Summary and Future Directions

Principal Investigator:

Sharmila Bhattacharya

Scientists:

Janani IyerRachel R. Gilbert

Amber M. Paul

Egle Cekanaviciute

Christina Cheung

Medaya Torres

Students :

Ivy Fernandes

Jhony Zavaleta

COMMERCIAL PARTNER: TECHSHOT, INC.

ISS Research Integration Office for support and funding

KSC/ARC project management and staff for flight support

THANK YOU!!