Introduction to Neurotrophin

1. Introduction to Neurotrophin • Include: • Nerve Growth Factor (NGF) • Brain-Derived Neurotrophic Factor (BDNF) • Neurotrophin T-3 and NT-4 • Receptors: • Low Affinity: p75 • High Affinity: TrkA, TrkB, and TrkC • In development: Regulates neuronal innervation to specific targets • In adults: • Regulates neuronal plasticity • Regulates the number of neuronal progenitor cells http://www.devbio.com/article.php?ch=13&id=143

2. Effects of Exercise Following Lateral Fluid Percussion Brain Injury in Rats Romana R. Hicks, et al. University of Kentuky Restorative Neurology and Neuroscience 12 (1998) 41-46

3. Background • After brain injurymany impairments • Exercise has shown to: • Maybe maintain memory: increases 5HT and noradrenaline • With PreTx of Exc: attenuates ischemic damage • Activate different molecular cascades: increase of EC lactate, increases antioxidant enzymes, and increases BDNF, NGF, and bFGF • BDNF: • Most prevelant in the brain • Amoung other rolesIs involved in activity-dependent plasticity • Enriched housing increases BDNF mRNA levels in rats

4. Hypothesis “…tested whether exercise following a lateral fluid percussion (FP) brain injury could increase BDNF mRNA expression in the hippocampus and attenuate the neuropathology and behavioral deficits that are associated with this model of experimental brain injury in rats.”

5. Materials and Methods • Surgery: • Sprague-Dawley rats, n=20 • Utilizing the stereotaxic and FP device placed a unilateral brain injury to the left parieto-occipital cortex

6. Treadmill Training: Began the day after FP injury (n=10) with 5min Time increased 5min per day until 60min reached Control (n=9): Handled 30-60sec daily Behavioral Tasks MWM: day15-17 post-op  after 12 trials, 90sec probe Inclined plane test Visual Limb Placing Vertical Righting Response Grip Test Materials and Methods, cont

7. Materials and Methods, cont In Situ Hybridization: Three sections from dorsal hippocampus were analyzed per animal CA3 • Histological Evaluation: Eosin stain • Cortical Volumes (left vs. right) • %lesion volume= (ipsi/contra)-100 • CA3 cell loss/injury scored on scale of 0-4

8. Results ? Histology * Morris Water Maze Battery of Behavioral Tasks

9. Results Basal Levels of BDNF?

10. Discussion • Exercise after FP injury does not: • Attenuate histological, cognitive, or neuromotor deficits (were there any deficits?) • Exercise after FP injury does: • Increase hippocampal BDNF mRNA compared to injured/no exercise group Even though BDNF has previously been shown to increase solely in response to injury, authors believe that the increase of BDNF mRNA in exercised animals occurred via a separate pathway….exercise induced pathway?

11. Discussion cont • Suggestions: • Unanswered questions of benefits vs. harm of exercise • Stress Response • Motor learning better than repetitive exercise Conclusion: “…experimental brain trauma are able to increase neurotrophin levels in the brain in response to exercise.”

12. ﮋ ﮋ questions?

13. Voluntary exercise increases axonal regeneration from sensory neurons Raffaella Molteni, Jun-Qi Zheng, et al. University of California, Los Angeles PNAS June 1, 2004 vol. 101 no. 22 8473-8478

14. Background • Neuronal Platicity • Development: amount of activity influences cortical circuitry • Adulthood: Activity-dependent plasticity is retained • Both require morphological changes of synaptic structures • Neurotrophin • Role in growth and differentiation of neurons • Regulators of synaptic plasticity • BDNF and NT-3 important in regulating the function of the muscle- dorsal root ganglion -spinal cord interface • Previously showed that exercise increases BDNF and NT-3 expression in spinal cord and skeletal muscle

15. Hypothesis “…asked howvoluntary exercise affects the structural plasticity of the DRG [dorsal root ganglion].” via a neurotrophin-dependent mechanism Isolated L4-5 Dorsal Root Ganglion

16. Materials and Methods • Exercise Conditioning • DRG Culture and Gene Expression Groups: Control(0 day) and Run (3 day, 7day) • Trk Inhibitor Groups: microbeads implantation 12h before exercise • Nerve Crush Groups: Run(7 day) and Control • DRG Culture • L4-5 DRG isolated • After 20-22h, fixed with 4%ParaImmuno • Longest process of >70 neurons for ea. condition was measured • DRB included in transcription-independent culture medium

17. Materials and Methods, cont • Nerve Crush • Unilateral sciatic crush: 7d-exercised or control • Three days after crush: nerve was transected • Transected end place in cuff containing FlouroGold • Animals sac two days later • Sciatic nerve and L4-5 DRG isolated, fixed, sectioned, and used for immunoflourescence • mRNA analysis • Quantitative real-time RT-PCR utilized • Analysis completed on total isolated mRNA

18. Manipulations • DRG culture- does exercise effect elongation of neurons in vitro? • DRG culture with RNA synthesis inhibitor- if exercise-elongation occur, is it also dependent on gene expression? • Nerve Crush- if elongation occurs in in-vitro, does exercise conditioning effect elongation in vivo? • Gene Expression- does exercise-conditioning effect gene expression? • Trk inhibitor- are exercise conditioning effects truly neurotrophin dependent?

19. 1. Results: Exc. Increases Growth In Vitro Sedentary Animal 7 day exercise 3 day exercise NOTICE ANYTHING?

20. 1. Results: Exc. Increases Growth In Vitro AXONAL OUTGROWTH INCREASED WITH LONGER PERIODS OF EXERCISE POSITIVE CORRELATION

21. 2. Results: Transcription-Independent Growth DRB: mRNA transcription inhibitor Gene expression is not required for rapid neurite out- growth not sig. significant

22. 3. Results: Regeneration In Vivo Sedentary Animal 7d-Exercise FlouroGold Positive Neurofiliment 50 um

23. 3. Results: Regeneration In Vivo %retrogradely-labeled neurons Number of Neurons

24. Uninjured Sciatic Nerve Exercised-Injured Nerve 3. Results: Regeneration In Vivo Sedentary-Injured Nerve 20 um

25. 4. Results: Exercise Influences Gene Expression BDNF regulates synthesis of Synapsin I to increase NT release Synapsin I: synaptic vesicle protein GAP43: Growth- Associated Protein

26. 5. Results: Inhibition of Trk Tyrosine Kinase Synapsin I mRNA levels Mean Neurite Length Activation of neurotrophin rec. via exercise regulates synapsin I expression GAP43 mRNA levels Lack of effect of GAP43 expression with inhibition of Trk rec indicates separate signaling pathways Decreased growth argues Neurotrophins are essential in regulating growth potential Grey Bars: Presence of Trk Inhibitor

27. Discussion • Does exercise effect elongation of neurons in vitro?Yes. With positive correlation. • If exercise-elongation occur, is it also dependent on gene expression?No. Rapid neurite outgrowth can occur without new mRNA. • If elongation occurs in in-vitro, does exercise conditioning effect elongation in vivo?Yes. In response to injury pretreatment, greater elongation occurs in exercised condition. • Does exercise-conditioning effect gene expression?Yes. Increased mRNA; shows direct functional effect of exercise. • Are exercise conditioning effects truly neurotrophin dependent?Yes. Activation of neurotrophin pathways are critical for growth potential.

28. Discussion cont • Elongation vs. Arborization • Exercise activates mRNA expression which encode proteins important for elongation of axons  “Priming Effect” • Inhibition of mRNA synthesis: • Supports above • Could mean that exercise activates protein translation pre- or post- synaptically? • Do not distinguish between activation OR alleviation of inhibition

29. Supports an exercise induced pathway? ﮋ ﮋ questions?