Effects of CV4 on Rodent Model of Alzheimer’s Disease
Blaise Costa PhDAssociate Professor of Pharmacology
Edward Via College of Osteopathic Medicine –VA Campus
Adjunct faculty: Dept. of Biochemistry,
School of Neuroscience & College of Veterinary Medicine, Virginia Tech
American Academy of Osteopathy 2019 Convocation
March 13-17, Orlando, FL
Outline
Cranial Osteopathic Manipulation (COM) on Animal Models of AD
Effect of CV4 Treatment
Spatial Learning and Memory Assay
Immunoassays
Aβ Plaque Imaging
Alzheimer’s Disease (AD) and Glutamate receptors
Summary and Closing Remarks
Alzheimer’s Disease
Cellular Events
Molecular Events
Glutamate
By NMDA ReceptorImages courtesy of AD education and referral center National Institute on Aging
NMDA Subtype of Glutamate Receptors
Glutamate
Costa et al., JPET 2010
Clinical Use of NMDA Receptor Blocker
Rx for Alzheimer's disease (AD): Memantine (Namenda -Allergan)
• NMDA receptor blocker
• For use in moderate to severe AD
• Oral solid formulations
Adverse effects
• Stevens-Johnson syndrome
• Epidermal necrolysis
• Suicidal ideation
Etiology of Alzheimer’s Disease
• Age dependent reduction in fluid circulation
• Increased accumulation of metabolic wastes
• Neuroinflammation
• Release of proinflammatory mediators
• Hyperactivation of ion channels (NMDA receptors)
Lymphatic vessels
CSFISF
Louveau etal., 2015 ; Aspelund et al., 2015
Alzheimer’s Disease
An Osteopathic approach for AD
• Cranial Osteopathic Manipulation (COM)
o In practice for decades
o Mechanism of action largely unknown
Outline
Cranial Osteopathic Manipulation (COM) on Animal Models of AD
Effect of COM (CV4) Treatment
Spatial Learning and Memory Assay
Immunoassays
Aβ Plaque Imaging
Summary and Closing Remarks
Alzheimer’s Disease (AD) and Glutamate receptors
Cranial Osteopathic Manipulation (COM) on Animal Models of AD
Aim.2Cranial osteopathic manipulation (COM)
on TgF344 or Aged rat model of AD.
Assessment of Spatial Learning & Memory by Morris water Maze
Identify changes in brain gene, protein and metabolite levels
Experimental Design
Demonstration of Aβ clearance in liveanimals by PET imaging
Aim.1Aim.3
18months old rats treated for 7days
Hope Tobey, DO
Anesthesia & COM MWM
4.00pm7.00am
7days
Per Gunnar Brolinson, DO Bradley Klein, PhD
COM improves Spatial Learning and Memory
d a y 1 d a y 2 d a y 3 d a y 4 d a y 5 d a y 6 d a y 7 d a y 8
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COM improves Spatial Learning and Memory
COM improves spatial learning and memory in naturally aged rat model of AD. Representative video shows the movement of UT (left, swim starts at 13th
sec.) and COM (right, swim starts at 13th sec.) treated eighteen months old naturally aged rats. Four quadrants, platform location (circle at northwest quadrant)
are marked in the video.
Untreated COM
Outline
Alzheimer’s Disease (AD)
Cranial Osteopathic Manipulation (COM) on Animal Models of AD
Effect of COM (CV4) Treatment
Spatial Learning and Memory Assay
Immunoassays
Aβ Plaque Imaging
Research Background
Summary and Closing Remarks
COM Reduces Aβ Protein
Experiment:• Positron Emission Tomography (PET) Imaging• Florbetapir F18 (AmyVid - Lilly)• 300-400µCi -tracer IV injection• PET scan after 45min
Observation:• ↓ Florbetapir F18 signal • Midbrain• Ventral Tegmental Area (VTA)• Hippocampus
Inference:• COM increases the clearance of Aβ proteins
18 month Old Wild Type Rats
UVA Radiology and Medical Imaging
COM Reduces Aβ Protein
Experiment:• Positron Emission Tomography (PET) Imaging• Florbetapir F18 (AmyVid - Lilly)• 300-400µCi -tracer IV injection• PET scan after 45min
Observation:• ↓ Florbetapir F18 signal
Inference:• COM increases the clearance of Aβ proteins
4 month old TgF344-AD Rats
TgF344-AD rat contains mutations (K595N, M596L in APP) and PS1 with deletion of exon 9
COM Reduces Aβ Protein in Transgenic AD Rats
11-16 month old TgF344-AD Rats
Outline
Alzheimer’s Disease (AD)
Cranial Osteopathic Manipulation (COM) on Animal Models of AD
Effect of COM (CV4) Treatment
Spatial Learning and Memory Assay
Immunoassays
Aβ Plaque Imaging
Research Background
Summary and Closing Remarks
Amyloid Precursor Protein (APP)
Cleavage sites for α- β- and γ-secretase on APP
AmyloidogenicNon-Amyloidogenic
Bergström et al., 2016
COM Alters Aβ Levels
Immuno assays
Tyler Lucas
COM Alters Aβ Levels
In association with astrocyte activation and upregulation of aquaporin and lymphatic vessels
Aa
Louveau etal., 2017
COM Alters Astrocytes (GFAP) Activation
Increase in expression of GFAP at dentate gyrus in COM group animals
Mike Mykins
COM Alters VEGF-C levels in TgF344-AD rats
11-16 month old TgF344-AD Rats
Immuno assays UT
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COM Numerically Reduces Aβ42 in TgF344-AD rats
11-16 month old TgF344-AD Rats
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Glutamate and GABA in Synaptic Transmission
Excitatory Inhibitory
Signaling Mechanisms
Vesicular glutamate transporter-1 (vGlut1)Glutamate decarboxylate-67 (GAD67)
COM Modulates Synaptic Transmission
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GAD67+ VGluT1+DAPIGAD67 VGluT1 DAPIa
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Hippocampal CA1 region
COM Modulates Synaptic Transmission
GAD67+ VGluT1 DAPIGAD67 VGluT1 DAPIU
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b.
COM treatment increases the GAD67 expression in the prefrontal cortex (PFC) of aged rats. a, Images represent the UT and COMtreated rat brain sagittal sections immunostained for GAD67 (red), VGluT1 (green) and DAPI(blue). b, Puncta density analysisreveal significant increase in (UT, puncta density 100.0±7.4% vs COM, 279.8±22.4%, p<0.01) PFC region. Scale bar: 20uM. UT,untreated; COM, cranial osteopathic manipulated.
Proteomic Assay
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P-value Vs Ion Intensity Ratio
Immuno assays
1 D4A435 Intercellular adhesion molecule 5 1.31
2 Q63564 1.08
3 P31596-2 0.61
4 P32851 0.76
5 P21707 0.68
6 P97686-2 0.67
7 P09606 -0.92
8 P07936 Neuromodulin (Axonal membrane protein GAP-43) -0.94
9 P02688 -1.22
10 P19527 -1.65Neurofilament light polypeptide (NF-L)
Neuronal cell adhesion molecule (Nr-CAM)
Synaptotagmin-1 (Synaptotagmin I) (SytI) (p65)
Sl
#
Protein
IDProtein Name
log2
(Treated/
Contol)
Myelin basic protein (MBP)
Synaptic vesicle glycoprotein 2B
Excitatory amino acid transporter 2 (GLT-1)
Syntaxin-1A (Neuron-specific antigen HPC-1)
Glutamine synthetase
Summary
Effect of COM (CV4) Treatment
Spatial Learning and Memory Assay
ImmunoassaysAβ Plaque Imaging
Possible Mechanism of Action
Clinical Application
Future Directions
• Increase the sample size and repeat the assays
• Quantitative COM
• Ultrasound in clinical trials
Ben-Tzvi et al., 2016
Acknowledgements
Principle Investigator:• Blaise Costa
Co-Investigators: • Hope Tobey (Pediatrician -VCOM ) • Gunnar Brolinson (Vice Provost for Research - VCOM)• Bradley Klein (Associate Professor of Neurobiology, Virginia Tech)
Collaborators: • Rich Helm (VT Biochemistry)• Stuart Berr (UVA)• Soumen Paul (UVA)
Lab members:(Present) • Tyler Lucas • Bryanna Vacca• Tullia Johnston
(Past) • Douglas Bledsoe • Caroline Campbell• Mike Mykins
Costa Lab, Spring 2018