Lecture 4: Neurotrophics + BDNF Autoregulation + Synaptic Health + Neurotrophic Pathways + Injections

Wiley Experiment

1. remove limb bud from chick embryo -> reduced sensory/motor neurons in SC; 2) extra limb bud to embryo -> increased sensory/motor neurons in SC

Victor Hypothesis

extra limb -> differentiates progenitor cell in SC -> mature neurons in SC

Updated Hypothesis

survival issue, target-derived factor kept differentiated cells alive; limb provides factors

Neurotrophic Hypothesis

GFs necessary to keep neurons alive; innervation targets secrete survival factors to balance target organ size + innervating neurons; peripheral + CNS neurons

NGF Purification

growth assay in ciliary ganglia of eye, added ground limb tissue -> neurite growth

Neurotrophins

precursors (250AA) -> 100-120 AA; NGF, BDNF, NT-3/NT-⅘ (PCR-based cloning)

NGF

prototypical nerve growth factor; cell survival, neurite outgrowth; not in CNS, mainly in PNS

BDNF

brain-derived neurotrophic factor; 50% homology with NGF, purified from pigs

Trk-r

tropomyosin-related kinase, receptor for neurotrophins

TrkA

NGF

TrkB

BDNF + NT-4/5 + NT-3

TrkC

NT-3

p75LNTR

low affinity, homologous to TNFr, all pro-NTs bind but large 250AA form; inhibitory -> apoptosis

BDNF Autoregulation

1. ER -> golgi -> proconvertase cleavage -> released; 2) furin cleaves pro-BDNF; 3) pro-BDNF -> plasmin -> trkB; 4) pro-BDNF -> p75NTR -> safeguard removal -> release -> normal

BDNF Autoregulation Key Factors

proconvertase (in), furin (in), plasmin (out); convert pro-BDNF -> BDNF

Synapses/BDNF

stronger presynaptic input -> high release (BDNF) -> post-synaptic growth + AMPAR + nNOS

BDNF Polymorphism

Met66 (bad) version + Val66 (good) version

Decrease BDNF

decrease release -> reduction in episodic memory + increase in anxiety/depression

Block BDNF Signalling

anti-trkB antibodies = reduces synapses + exercise-mediated memory increase + synaptic proteins -> NCAM-180, N-cadherin, AMPA receptor (glutamate); important in hippocampus

BDNF/Schizo Results

reduction in hippocampal memory in controls (Val); schizo (Met) always have bad memory

Synaptic Health

MRI/MRS, NAA (N-acetyl-aspartate) marker; lower in Val/Met, Met/Met

MRS

magnetic resonance spectroscopy; measures protons in biomaterials; NAA + creatinine; higher = better

MRS Function

measure health status of individual brain regions/neurons; NAA

Creatinine Levels

used because have a peak that is very constant in the brain

Val66Met Implications

result in behavioural changes -> eating disorders + seizure activity; bigger brain -> Val

Downstream Effectors

synapsin 1 promoter (neuron-specific) -> gene V12-Ha-ras -> reporter gene; increase ras = increase length/width of brain

V12-Ha-ras

downstream molecule that gets turned on after BDNF binding

Neurotrophic Hypothesis 2

absence of neurotrophins -> neurons die; cells normally die but pathway is inhibited

Neurotrophin GF Pathway

GF -> Trk -> ras -> PI3K -> Akt -> anti-apoptotic factors -> survival

Neurotrophin Deficiency Pathway

no GF -> MEKK1 -> ASK1 -> MKK4/7 -> JNK -> c-jun -> pro-apoptotic

Introduce Genes -> Cell

1. inject into nucleus (sliced neurons), 2) gene gun biolistics (slices/tissues), 3) transfection -> CaPO4/lipofectin (dissociated cell line cultures), 4) viruses (HSV/AAV)

Secretion Measuring Process

two vectors; 1) BDNF - GFP, 2) DsRed; inject genes into neuron

MAP2

marker for mature neurons

Val66 Secretion Experiment

inject DsRed into neuron (fills entire neuron, shape) -> inject BDNF-GFP (see yellow) -> see green in new neuron (released BDNF in connected neighbour) -> stain all neurons with MAP2; does cell 1 release BDNF to cell 2? Yes -> Valine 66

Met66 Secretion

less or no BDNF release into cell 2; BDNF produced but not released effectively -> decrease in synaptic plasticity, decrease ras pathway in neighbouring cells; would not see an green in cell 2

Val/Met Differences

val secreted easily, nourishes neighbours, increase in exercise; Met not released