effects of cv4 on rodent model of alzheimer’s...

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

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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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Hippocampal CA1 region

COM Modulates Synaptic Transmission

GAD67+ VGluT1 DAPIGAD67 VGluT1 DAPIU

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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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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