The effect of the Val66Met BDNF polymorphism on axon regeneration after peripheral nerve injury Open Access

Abstract

Although peripheral nerves can spontaneously regenerate after injury, outcome is generally very poor. Activity-dependent treatments to enhance peripheral nerve regeneration after injury have shown great promise, and clinical trials implementing them have begun. Success of these treatments requires activity-dependent release of brain derived neurotrophic factor (BDNF). A single nucleotide polymorphism (SNP) in the BDNF gene known as Val66Met found in nearly one third of the human population results in defective activity-dependent BDNF secretion and could impact the effectiveness of these therapies. We used a mouse model of this SNP to test the efficacy of treadmill exercise in enhancing axon regeneration in animals both heterozygous (V/M) and homozygous (M/M) for the SNP. Axon regeneration was studied four weeks after complete transection and repair of the sciatic nerve, using both electrophysiological and histological outcome measures. Regeneration was enhanced significantly without treatment in V/M mice, compared to wild type (V/V) controls. Unlike V/V mice, treatment of both V/M and M/M mice with treadmill exercise did not result in enhanced regeneration. These results were recapitulated in vitro using dissociated neurons containing the light-sensitive cation channel, channelrhodopsin. Three days after plating, neurites of neurons from V/M and M/M mice were longer than those of V/V neurons. In neurons from V/V mice, but not those from V/M or M/M animals, longer neurites were found after optical stimulation. Taken together, Met carriers possess an intrinsically greater capacity to regenerate axons in peripheral nerves, but this cannot be enhanced further by activity-dependent treatments. This enhanced axon regeneration can be blocked by application of a trkB antagonist, ANA-12. In culture, application of ANA-12 to adult dorsal root ganglion (DRG) neurons blocks enhanced neurite outgrowth in cells from M/M mice. Application of the fusion protein trkB-Fc which sequesters trkB ligands also results in decreased neurite outgrowth in all genotypes. Inhibiting the pan-neurotrophin receptor, p75NTR, resulted in a modest improvement in V/V axon regeneration, and no effect in Met-carriers. Thus, the enhanced axon regeneration in Met-carriers is trkB-dependent.

Table of Contents

Chapter 1: Introduction to the role of BDNF in peripheral nerve injury and regeneration ... 1

Peripheral Nerve Structure .................................................................................................. 3

Peripheral Nerve Injury ........................................................................................................ 4

Brain Derived Neurotrophic Factor ................................................................................... 11

Regulation of BDNF Transcripts......................................................................................... 11

BDNF Trafficking and Secretion ......................................................................................... 14

BDNF Receptors ................................................................................................................ 15

Role of BDNF in Peripheral Nerve Injury ........................................................................... 18

TrkB in Peripheral Nerve Injury .......................................................................................... 19

p75NTR in Peripheral Nerve Injury ....................................................................................... 20

Activity-Dependent Treatments Enhance Regeneration .................................................. 22

Electrical Stimulation .......................................................................................................... 23

Exercise Treatment ............................................................................................................ 26

Optogenetic Stimulation ..................................................................................................... 29

Mechanisms ........................................................................................................................ 30

Neurotrophins .................................................................................................................... 30

Neuronal Activity ................................................................................................................ 32

Androgens ......................................................................................................................... 33

Synaptic Rearrangements ................................................................................................. 34

BDNF Val66Met Polymorphism ......................................................................................... 35

Dissertation Overview ........................................................................................................ 38

References .......................................................................................................................... 40

Chapter 2: The Val66Met BDNF polymorphism and peripheral nerve injury: Enhanced regeneration in Met-carriers that is not further improved with activity-dependent treatment ..62

Abstract ............................................................................................................................... 63

Introduction ........................................................................................................................ 64

Methods .............................................................................................................................. 66

Animals and Surgical Treatments ...................................................................................... 66

Treadmill Training .............................................................................................................. 66

Motor Unit Number Estimation (MUNE) ............................................................................. 67

Retrograde Labeling of Motoneurons and Muscle Afferent Neurons .................................. 68

Motor Endplate Reinnervation ............................................................................................ 69 

Dorsal Root Ganglion Cell Culture ..................................................................................... 69

Experimental Design and Statistics .................................................................................... 71

Sample Sizes and Statistical Analysis by Experiment ........................................................ 71

Results ................................................................................................................................ 73

Functional Recovery is Enhanced in untreated V/M and M/M mice .................................... 73

Motor Axon Regeneration Is Enhanced in Untreated But Not Treadmill Trained Mice Heterozygous for BDNFMet ...... 74

Muscle Fiber Reinnervation Is Enhanced in Untreated But Not Treadmill Trained Mice Heterozygous for BDNFMet .....75

Treadmill Training Does Not Enhance DRG Regeneration................................................. 76

Neurite Outgrowth Is Enhanced in V/M and M/M Neurons ................................................. 77

Optical Stimulation Enhances Neurite Elongation in V/V Neurons In Vitro ......................... 78

There Is No Difference in Basal Release of BDNF between Genotypes ............................. 79

Discussion .......................................................................................................................... 80

Conclusion .......................................................................................................................... 84

Figures ................................................................................................................................ 85

References .......................................................................................................................... 95

Chapter 3: Enhanced regeneration in Met-carriers of BDNF Val66Met polymorphism is trkB-dependent ..... 100

Abstract ............................................................................................................................. 101

Introduction ...................................................................................................................... 102

Methods ............................................................................................................................ 104

Animals and Surgical Treatments .................................................................................... 104

Motor Unit Number Estimation (MUNE) ........................................................................... 104

Retrograde Labeling of Motoneurons ............................................................................... 105

Motor Endplate Reinnervation .......................................................................................... 106

Dorsal Root Ganglion Cell Culture ................................................................................... 107

Experimental Design and Statistics .................................................................................. 107

Results .............................................................................................................................. 109

trkB Blockade Inhibited Functional Recover in Heterozygous Met-Carriers ...................... 109

trkB Blockade Inhibited Motor Axon Regeneration in V/M Mice ........................................ 110

trkB Blockade Increased Motor Endplate Recoccupation ................................................ 110

trkB Inhibition Results in Decreased Longest Neurite Length in DRG Neurons from M/M Mice ......111

Discussion ........................................................................................................................113

Conclusion ......................................................................................................................... 116 

Figures .............................................................................................................................. 117

References ........................................................................................................................ 124

Chapter 4: General Discussion and Future Directions ....................................................... 127

Discussion ......................................................................................................................... 128

There Is More trkB Expression in Met-Carriers ................................................................ 130

There Are Higher Levels of trkB Ligand Secretion in Met-Carriers ................................... 131

Ligand-Independent Activation of trkB ............................................................................. 133

Personalized Medicine ..................................................................................................... 137

Val66Met and Neuronal Insult ........................................................................................... 137

Future Directions ............................................................................................................... 140

Conclusion ......................................................................................................................... 140

References ........................................................................................................................ 142 

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Neuroscience Biology, Physiology
Keyword	DRG neuron activity-dependent treatment Val66Met optogenetics peripheral nerve injury BDNF exercise motoneuron
Committee Chair / Thesis Advisor	English, Arthur, Emory University
Committee Members	Alvarez, Francisco, Emory University Gourley, Shannon, Emory University Hochman, Shawn, Emory University Feng, Yue, Emory University Jones, Kathryn, Indiana University

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