Download Early Neural Patterning •Neural induction

Early Neural Patterning
•Neural induction
-Neural induction = differentiation of ectoderm to epidermis and neural tissue (between ectoderm and dorsal
mesoderm)
-Lip of blastula = line that separates dorsal mesoderm and endoderm
-During gastrulation, endoderm rises up and fills up the centre of embryo; endoderm invaginates from the lip of
blastula (forms a pore) and drags the dorsal mesoderm up with it. The dorsal mesoderm becomes a long, thin strip
lying immediately ventral and parallel to prospective neural tissue
-End of gastrulation: endoderm fills up the centre; the derivative of the dorsal mesoderm forms the notochord;
neural plate starts to differentiate on the dorsal surface of embryo (bigger rostral than caudal); neural folds flank the
peripheries of neural plate proper
•Neurulation = formation of the neural tube
-Evolutionary value (fragile, non-regenerative nervous system gets tucked inside, away from the surface)
-At the caudal cervical/ rostral thoracic region
-Midline has neural plate flanked by neural folds/crests which are flanked by epidermal ectoderm; notochord is
ventral to the neural plate at the midline
-Neural folds/crests get tucked up and act as hinges to fold downwards while the midline buckle upwards to form
the neural groove
-Neural folds/crests (lateral borders) continue to move towards each other and fuse at the midline → neural groove
becomes one continuous, hollow neural tube
-Neural crests migrate inside dorsal and lateral to the neutral tube proper as neural crest cells/ somites
-Neural crest cells migrate out from the dorsal neural tube and give rise to sensory neurons (DRG),
autonomic ganglia, enteric neurons, Schwann cells, melanocytes and chromaffin cells
-Epidermal tissue fuses at the midline → con nuous layer of epidermal ectoderm on top of the neural tube
-Notochord lies ventral to the neural tube
-19 days of gestation: neural plate begins to fold at the caudal cervical/rostral thoracic level first and forms the
neural tube (22 days)
-Process continues both rostrally and caudally
-24-26 days: Rostral/cranial neural tube closure - Failure=Anencephaly (no development of brain)
-26-28 days: Caudal neural tube closure - Failure=Spina bifida
•Differentiation of the neural tube
-Optic cup = C-shaped bulges emanating from lateral borders of diencephalon = contain retina progenitors
-Retinal ganglion progenitor cells are only cells that exit the retina; their axons exit retina to grow back along the
optic stalk and form synapses in the diencephalon (LGN) and mesencephalon (SC)
-Olfactory nerve = out-pocketing of telencephalon
-CN1 and CN2 are not nerves but CNS tracts
-Cephalic flexure = between forebrain and midbrain; persists in adulthood to create the bend in neuraxis
-Cervical flexure = between rhombencephalon and spinal cord
-Pontine flexure = between rostral and caudal hindbrain; transient
Primary vesicles
Secondary vesicles
Telencephalon
Prosencephalon
Diencephalon
Mesencephalon
Rhombencephalon
Caudal neural tube
Mesencephalon
Metencephalon
Myelencephalon
Caudal neural tube
Adult derivatives
Cerebral cortex
Hippocampus
Amygdala
Striatum (basal ganglia)
Olfactory bulb
Thalamus
Hypothalamus
Epithalamus
Subthalamus
Retina
Midbrain
Pons
Cerebellum
Medulla
Spinal cord
Ventricle
Lateral
3rd
Cerebral aqueduct
4th
4th
Central canal
•The Organiser experiment – Spemann and Mangold (1924)
-In albino amphibian embryos, every single cell is non-pigmented (tagging of cell possible to trace lineage)
-Noted tissue immediately adjacent to dorsal lip of blastula is important → dorsal mesoderm
-Transplant donor tissue to where ventral mesoderm is in the recipient embryo
-Recipient has 2 patches of dorsal mesoderm (ventral and dorsal locations)
-Embryo develops to have secondary embryonic axis (neuraxis) where the abdomen should be
-Only the notochord was directly from the donor; the neural tube and somites were from the recipient
-Dorsal mesoderm tissue doesn’t become the secondary embryonic axis but has the capacity to induce embryonic
axis in the surrounding tissue
-Dorsal mesoderm is the Organiser - can independently organise an entire secondary embryonic axis via signalling
molecules (which can diffuse out and bring changes in the environment)
•Mechanism of neural induction
-Signalling molecules called bone morphogenic proteins (BMPs) are normally expressed in ectoderm
-BMPs diffuse out and signal nearby cells to induce epidermis
-Inhibition of BMP signalling induces the formation of the neural plate
=Inhibition of endogenously present proteins to form the nervous system from skin
•Axis organisation
-Developing nervous system is organised along 3 axes:
1. Neuraxis/ Rostrocaudal axis/ Anteroposterior axis
2. Mediolateral axis
3. Ventricular-pial or deep-superficial axis
1. Neuraxis/ Rostrocaudal axis/ Anteroposterior axis
-Corresponds with main body axis
-Establishment is concurrent with the neural induction
-Dorsal mesoderm expresses and releases BMP inhibitors (noggin, chordin, follistatin)
-Neural plate forms
-Somites adjacent to caudal portion of neural plate express and release posteriorising agents (retinoic acid, FGF)
-A gradient of exposure to posteriorising agents forms
-Results in crude rostrocaudal axis
-Neural identity (caudal and rostral neural plate) is defined then refined
-Neuraxis is then subdivided into smaller and smaller subunits or segments called neuromeres
▪Rhombomeres
-Segmentation/ neuromeric structure is most obvious in the hindbrain and disappears as development proceeds
-8 rhombomeres (bulges in rhombencephalon)
-Segmental organisation has a close relationship with structures that mediate feeding and respiration by controlling
structures around the face and neck (highly conserved across species)
-Trigeminal, facial and glossopharyngeal cranial motor nerves form individual branchial arches (b1, b2 and b3)
-2-segment repeat pattern: adjacent pairs of rhombomeres contains cell bodies of neurons which innervate each
branchial arch
-The motor nerve exits only via the even numbered rhombomere
-E.g. cell bodies giving rise to CN V are in r2 and r3 but CN V exits only by r2
-This pattern is maintained for sensory organisation
-Somites that lie alongside the spinal cord and caudal hindbrain express retinoic acid (RA)
-Retinoic acid is a biologically active derivative of vitamin A (potent at changing neural identity)
-Gradient of RA is set up across rhombomeres
-RA receptors are ligand-specific transcription factors which majorly targets the Hox family
-Hox family = class of transcription factors that regulate development in all organisms = a group of homeobox genes:
encode proteins that contain a highly conserved DNA binding domain called a homeodomain (conserved across
almost all species from yeasts to mammals)
-RA can drive the expression of Hox genes in a concentration dependent manner
-Depending on the concentration of RA, different sets of Hox gene are turned on (higher dose, more genes)
-Hox gene expression then encodes the identity of each rhombomere
-Each rhombomere has a specific pattern of Hox expression (RA gradient is transformed into neural identity)
-Excess/deficiency of RA causes a shift in Hox gene expression and which then changes the identity of each
rhombomere → changes the development of fundamental motor neurons of face and neck
-E.g. HoxB1 is only expressed in r4 – if this gene becomes mutated, no facial nerve can form and 2 trigeminal nerves
form instead
▪Patterning of the midbrain
-Midbrain is rostral to the upper limit of rhombencephalon at which Hox gene expression (and segmentation)
abruptly stops
-Isthmus (neural junction between midbrain and hindbrain) releases signalling molecules/ morphogens such as
fibroblast growth factor 8 (FGF8) and Wnt-1 which organises the cell pattern in midbrain
-Morphogens diffuse and spread out to set up a concentration gradient and activate transcription factors
-FGF8 acts through homeodomain proteins such as Engrailed to regulate rostrocaudal identity in the tectum
-E.g. retina maps into the superior colliculus (dorsal part of the tectum) in a specific manner – helps to mediate visual
ability
2. Mediolateral axis
-Closure of the neural tube deflects this axis to become the dorsoventral axis
-Important for brainstem and spinal cord patterning
▪Ventral signalling
-Notochord = derivative of dorsal mesoderm = ventral to the midline of the neural plate
-Notochord expresses signalling molecule/ morphogen sonic hedgehog (Shh)
-Shh diffuse out and act on the cells in the middle of the developing neural tube
-The concentration of Shh that a ventral neuron encounters determines its identity
-Highest dose of Shh at the ventral midline causes the differentiation of the specialised type of glial cells = floor plate
cells (FPC)
-FPCs themselves begin to produce Shh (2 sources)
-Shh acts on cells near the midline and induce them to form motor neuron (MN) precursors (by the time neural tube
closes)
-MN precursors differentiate into motor neurons which begin to extend axons towards their targets
▪Experiment 1
-Normal: 2 motor neuron pools symmetrical on either side and FPC in the centre
-Graft extra notochord on the lateral side of the developing neural tube leads to induction of secondary motor
neuron pool more dorsally
-Removal of notochord leads to absence of floor plate and motor neuron
→ Notochord (Shh) is both necessary and sufficient for the induc on of motor neuron iden es in the ventral part of
the spinal cord
▪Experiment 2
-There is correlation between the identity of motor neurons in the ventral spinal cord and the dosage of Shh
-Highest dose leads to the production of V3 cells (most ventral set of neurons in the spinal cord)
-Slightly less dose leads to the production of classic motor neuron pool
→ The dosage of Shh directly determines the iden ty of motor neurons
▪Experiment 3
-Sagittal section of developing embryo
-In situ hybridisation stains for mRNA (location of gene expression)
-High level of Shh at the notochord and in FPCs inducing motor neurons in the ventral neural tube/ spinal cord
-Detailed look at motor neurons in developing embryo reveals different pools of motor neurons with different
functions happening depending on the level (despite same signalling molecule Shh)
-E.g. serotonergic neurons (raphe nuclei) and dopaminergic neurons (substantia nigra)
-Dorsoventral signalling acts within the context of the rostrocaudal signalling
-Rostrocaudal signalling precedes dorsoventral signalling and sets up some determination/ identities
→ Shh induces different types of ventral neurons at different rostrocaudal levels
▪Dorsal signalling
-BMP signalling in the epidermal ectoderm (adjacent to the lateral part of the developing neural plate) induces
differentiation of the parts of the dorsal spinal cord
-BMPs induce Roof Plate Cells to form at the dorsal midline and to begin to express BMPs
-The more ventral population gets progressively smaller dose of BMP and forms the sensory interneuron precursors
-BMP signalling induces neural crest cells (those receiving high dose in neural folds) to differentiate into DRG and
sensory interneurons precursors to form sensory interneurons
▪Summary of dorsoventral patterning signals
-Dorsoventral signalling is mediated by morphogens expressed in adjacent non-neural structures acting in
concentration-dependent manner
3. Ventricular-pial or deep-superficial axis
-Most prominent in cortex, cerebellum and retina
Neuromuscular Junction Development: 1. Postsynaptic agrin/ MuSK pathway
•Introduction
-NMJ = large relay synapse between the motor axon terminal and the motor endplate in the middle of a skeletal
muscle fibre
-When an AP depolarises the motor axon, the nerve terminal releases ACh via exocytosis and ACh binds to and
activates AChR on the postsynaptic membrane
-AChR activation generates the depolarising endplate potential (EPP) which then triggers AP that spreads
depolarisation along the muscle fibre
-AChRs are packed with high density in the mature postsynaptic membrane of the NMJ (~104 per μm2) → CLUSTERS
-High AChR packing density makes the membrane highly sensitive to ACh → STRONG endplate poten al
-When rapidly dividing muscle precursor cells (myoblasts) reach a certain density their membranes fuse together to
form differentiated post-mitotic myotubes
-Myotubes are immature muscle fibres containing many nuclei
-AChR clusters form on the myotubes cell
•Timeline
-At embryonic day 14, the growth cone of motor neuron is finding its way to developing muscle mass
-Within 4 hours, the ACh receptors (which are initially spread all over the muscle mass) reorganise themselves to
form clusters underneath the axon process
-At postnatal day 1, the motor endplate becomes innervated by several (up to 10) different axons
-At postnatal day 14, the motor endplate forms a mature pattern, innervated by SINGLE axon
•Methods used to study development
1) Targeted gene inactivation/gene knockout in the germline of the mouse
-Engineer mutations and put into cloned gene
-Mutated gene is expressed while normal gene is prevented from being expressed
-Introduce the mutation into the sequence of the chromosomal gene in embryonic stem cells (homologous
recombination)
-Implant early embryos populated by the mutant stem cells into uterus of a host mouse
-Breed the mutant progeny to generate a knockout mouse line
2) Immunofluorescence microscopy
-Diaphragm muscle is used as the sample as it is flat with the intramuscular nerve running perpendicular to the
muscle fibres (runs diagonally left to right)
-All the NMJs line up beside nerve in the middle of the diaphragm muscle
a) Visualising motor nerves
-Thin slices or whole mounts of muscle tissue are incubated with a primary antibody that binds specifically to a
synaptic vesicle protein (synaptophysin or synapsin)
-The sample is washed to remove unbound antibody
-The sample is incubated with a fluorophore-labelled secondary antibody that binds specifically to primary antibody
and emits fluorescent light
→An bodies against synapsin, synaptophysin, SV2 and Bassoon assesses presynap c differen a on
b) Visualising postsynaptic AChR
-The sample is incubated with fluorophore-labelled α-bungarotoxin (an 8kD polypeptide toxin from snake venom)
that binds tightly to the AChR to block the channel opening
→α-BGT measures postsynaptic differentiation
-Merging the fluorescence image: motor nerves (green), AChRs (red) and single NMJ/ synaptic alignment (yelloworange spot)
•2 views of how a synapse might be established
1) Neurocentric view theory: When the ingrowing axon growth cone contacts the muscle cell, it passes a signal to
induce synaptic differentiation (receptor clustering) at that contact site → the nerve controls the synapse forma on
and determines the location of synapse on target cell
2) Pre-formed target theory: The muscle fibre pre-determines the synaptic site, which then becomes innervated by
the nerve
•Neurocentric hypothesis experiments
1) Neural tube excision
Method
-Lumbar/sacral section of the neural tube was excised from a chick embryo
-Hind limb of the chick embryo developed without any motor innervation
Results
-1a: control; neurofilament showing the axon growing in and branching out to form synapses
-1b: clusters of AChR where the synapses formed
Conclusions
-1c: in aneural muscle, no axons and no receptor clusters were seen
→Receptor clusters are only seen when the muscle is innervated by axons (not in aneural muscle)
-Nerve induces the receptor clusters which form synapses (nerocentric hypothesis)
2) Nerve-muscle co-culture
Method
-Place explant of frog embryo spinal cord (with motor neurons) in a culture dish together with
cultured muscle cell of frog embryo
-After several days, the culture as stained for AChRs with fluorescent-α-bungarotoxin
Results
Conclusions
-The motor neurons in the explant grew out their axons over the surface of the muscle cell
-AChR clusters on the surface of the muscle cell where there was contact with the axons from the
motor neurons (in vitro NMJs)
-Spontaneous receptor clusters (hot spots) were also present in parts of the cell not related to any
nerve
-After 2 days, the spontaneous receptor clusters disappeared
Nerve induces AChRs to reorganise themselves underneath the nerve process to form a synapse
3) Sniffer electrodes
Method
-Patch electrode (working like a pipette) sucked up a portion of surface membrane with AChR clusters
so that it is held on the end of the path electrode → bio-sniffer that can detect low concentrations of
ACh in real time
-Placed a current electrode into the cell body of a developing neuron culture and placed the patch
electrode on the growing end of the growth cone
-Stimulated the cell body with a chain of impulses to send action potential down the developing axon
Results
-Patch electrode detected signals from AChR opening
-ACh was exocytosed from the growth cone to open the AChR
Conclusions -The growth cone, even before it makes contact with muscle cell, is capable of activity-dependent
release of ACh
-The growth cone could possibly be releasing other molecules
-Presynaptic axon terminal is partially differentiated and induces changes (AChR clustering) in the
postsynaptic neuron
•Synaptic basement membrane
= Location of proteins involved in postsynaptic differentiation
-Specialised synaptic cleft basement membrane contains acetylcholinesterase (AChE)
1) Denervation and regeneration
Method
Results
Conclusions
1. Cut the nerve, which leads to degeneration and phagocytosis of the nerve terminal
2. Kill the muscle fibre by freeze damage (liquid nitrogen probe at the end of muscle fibre): muscle
cell degenerates and is phagocytosed leaving behind the basement membrane
3. Muscle precursor cells repopulate the basement membrane sheath
-New ACh receptor clusters appear in the new plasma membrane exactly where they had been in
the previous muscle fibre (even without the nerve innervation)
-The synaptic basement membrane contains some type of signalling molecules that is retained in
the membrane to induce receptor clustering in the postsynaptic membrane
Q) Describe a key experiment that showed that a component of the synaptic the basement membrane contains the
information needed to organise AChRs in the postsynaptic membrane.
•Proteins of postsynaptic differentiation
1) Neural agrin
= Initially believed to be a natural inducer of AChR clusters (postsynaptic differentiation) but discovered to be a
stabiliser of AChR clusters
-Agrin mRNA expressed by motor neurons of skeletal muscle
-Contains multiple protein domains that allow agrin to attach to extracellular proteins
-Motor neurons make a special form of the agrin mRNA that encodes 4 additional amino acid resides at the carboxyl
Z-splicing site → Z-splice insert allows neural agrin to bind and activate the LRP4-MuSK complex
-Agrin protein transported anterogradely down motor axons to nerve terminals
1) Bioassay
Method
-Torpedo has modified muscle cells with layers of AChR clusters that generate inward inplate
potential current to produce high voltage
-Dissect out torpedo muscle cell basement membrane and grind them up
-Chromatography: fractionate proteins according to size, charge etc
-Bioassay
1. Add the test substance to a culture of myotubes
2. Stain AChRs with fluorescent-α-bungarotoxin
3. Count the number of AChR clusters under the microscope (inductive capacity)
-Select the best fraction (maximum number of AChR clusters per cultured muscle cell) which would
have enriched source of inducers
-Column chromatography for purification
-Isolate a single molecular component (which could induce maximum number of AChR clusters)
Results
Conclusions
-Identified neural agrin
-Cloned neural agrin – C terminal can induce receptor clustering and dystroglycan and laminin
binding sites help it bind to the basement membrane
-Recombinant neural agrin is potent at causing AChR clustering in muscle cell cultures (only nm is
needed)
2) Agrin knockout (1996)
Method
Generated agrin knockout (deficient) mice
Results
-Agrin-/- embryos were paralyzed and died at birth (P0)
-AChRs were expressed on muscle fibres but only a few AChR clusters formed
-The receptor clusters were not well aligned with axon endings
Conclusions
-Agrin is important for organising and aligning receptor clusters underneath the nerve terminals
(the location of clustering)
-But agrin is not necessary for clustering the receptors
▪Competition with ARIA (AChR Inducing Activity)
Agrin
-Isolated from basement membrane of Torpedo electric
organ by UJ McMahon
-Induces AChR cluster formation on myotubes
ARIA
-Isolated from chicken brain by Gerry Fischbach
-Increased expression of AChRs on myotubes
-Turned out to be a form of Neuregulin I which works
via ErbB receptors
-Real role still not well understood
2) Rapsyn ( 43kD protein)
= Machinery holding AChRs together in clusters
-Receptor associated protein of the synapse
-~45 kD cytoplasmic protein that co-purifies with the AChR
-Scaffolding protein containing multiple domains which can bind to various other proteins (e.g. AChR, dystroglycan)
-Helps to immobilize AChRs within the 2D plane of the membrane
-Peripheral membrane protein (could be removed from membrane by adding alkaline solution)
-Gene for similar 43kD/rapsyn protein cloned from mouse and human
-Myristate group at the N terminus attaches to the inner face of the plasma membrane
-Protein interaction domains which can bind to calpain, rapsyn
-AChR
-β-dystroglycan
1) Electron microscopy and 3D reconstruction
Method
-Performed electron microscopy of torpedo electric organ
-Reconstructed 3D image of the postsynaptic membrane
-Co-purified rapsyn from Torpedo postsynaptic membranes bound to AChRs
Results
Conclusions
-Single receptor cluster has a central pore and there are bridges present between the receptors
-Tall 6 AChR forming a cluster with bridges on the cytoplasmic side between adjacent receptors
-Rapsyn is the bridge protein holding the AChR clustered together
2) Transfection into heterologous cells
Method
-Take the gene for AChR subunits with or without rapsyn and transfect (insert into mammalian
cells) them into cultured myotubes
-After waiting overnight, stain for AChRs with fluorescent α-bungarotoxin
-Put under fluorescent microscope
Results
a) AChR alone: AChRs spread diffusely over the surface of the cell
b) AChR + rapsyn: AChRs reorganised into clusters
Conclusions
Rapsyn clusters AChR when overexpressed in heterologous cells
3) Rapsyn knockout (1995)
Method
Generated rapsyn knockout (deficient) mice
Results
-Rapsyn-/- embryos were paralysed and died just before birth (E19) – no functional NMJ
-AChRs were expressed on muscle fibres but not in clusters
Conclusions
Rapsyn is necessary for postsynaptic AChR clustering
3) MuSK (Muscle specific kinase)
= Central coordinator of NMJ formation
-Transmembrane receptor tyrosine kinase with multiple protein interaction sites in both extracellular and
intracellular regions
-Extracellular region is large and can bind to LRP4
-The assembly of neural agrin-LRP4 with MuSK activates MuSK
-2 MuSK molecules come together as a dimer, the intracellular catalytic domains phosphorylate each other and this
switches MuSK to kinase-active mode
1) MuSK knockout
Method
-Generated MuSK knockout (deficient) mice
-Stained for presynaptic nerve termianal (vesicles) with antibodies against synaptophysin
-Stained for AChRs on the postsynaptic muscle membrane with α-BGT
-Stained for AChE activity
Results
Conclusions
-MuSK knockout mice were stillborn while control mice could wriggle around
-In MuSK+/+ embryo, there were receptor clusters where the nerves innervate
-In MuSK-/- embryo, there were no receptor clusters (D) and the axons continued growing without
forming synapses immediately in the muscle
-In control mice, AChE is localised to the centrally located endplate band but accumulations of AChE
are absent in muscle from mutant mice
-MuSK is necessary for sending a recognition signal to the nerve to stop growing and undergo
presynaptic differentiation
-MuSK is a potential receptor target for nerve-derived agrin
2) AChR quantitation
Method
-Myotubes derived from control and MuSK-/- mice were cultured and treated with varying
concentrations of agrin
-Stained for AChRs with fluorescent α-BGT
-Subjected to AChR cluster quantitation
Results
Conclusions
-The control mice myotubes had formed receptor clusters in agrin concentration-dependent way
-MuSK-/- mice myotubes didn’t form any receptor clusters in response to neural agrin
MuSK is the postsynaptic membrane receptor that responds to agrin to induce AChR clustering
▪Candidate agrin co-receptors: dystroglycan vs. LRP4
-No experiment could show that neural agrin could actually bind directly to MuSK
-1996: MASC (myotubes associated specificity component) postulated to describe a hypothetical co-receptor
-Agrin could bind to dystroglycan and LRP4
-Dystroglycan = Transmembrane protein in muscle fibres that helps to link extracellular basement
membrane proteins (e.g. laminins and agrin) to intracellular proteins (e.g. dystrophin, rapsyn)
-2008: LRP4 discovered to be the true MASC
4) LRP4 (Low density lipoprotein Receptor-related Protein-4)
= Co-receptor with MuSK for neural agrin
-Transmembrane protein with large extracellular region that has binding sites for neural agrin and MuSK
-Aids the activation of MuSK by agrin
1) LRP4 knockout (1995)
Method
Generated LRP4 knockout (deficient) mice and stain the embryo diaphragm muscle
Results
Conclusions
-Control mouse embryo: nerve axons go through the muscle fibre and form synapses immediately
(AChR clusters seen)
-LRP4-/- embryo: nerve axons come out of the trunk but they continue to grow out along the muscle
fibres without forming syanpses immediately – no AChR clusters form
-Results similar to MuSK-/- knockout mice
LRP4 is another necessary postsynaptic membrane receptor that responds to agrin to induce AChR
clustering
2) Western blot
Method
-Myotubes derived from control and LRP4-/- mice were cultured and treated with agrin
-Restored the expression of wild-type LRP4 by transfection with retroviral vector
Results
-MuSK protein expression was found to be normal in LRP4-/- as well as control myotubes
-Agrin stimulated MuSK tyrosine phosphorylation (blotted anti-pTyr) in wild-type but not in LRP4-/myotubes
-Restoring LRP4 expression in LRP4-/- myotubes restores agrin-stimulated MuSK phosphorylation
and AChR clustering
-LRP4 also co-immunoprecipitated with MuSK (not shown)
Conclusions
-LRP4 is not required for the expression of MuSK on the cell surface
-LRP4 is necessary for agrin to activate MuSK and induce receptor clustering
-LRP4 can cause MuSK to become phosphorylated (once MuSK is activated, it dimerises and each
unit has a kinase domain that phosphorylates the other unit) by forming a tetramer
Q) What kind of proteins are: neural agrin, MuSK, LRP-4 and rapsyn and their proposed functional relationships?
Q) Explain the key experimental evidence that supports the proposed roles of neural agrin, MuSK and rapsyn.
•MuSK signalling protein complex
• The agrin/MuSK paradigm of the 1990s
-Ingrowing motor nerve terminal secrete neural agrin
-Neural agrin binds to LRP4
-LRP4 helps to assemble a MuSK dimer and forms a tetramer → Ac ve MuSK complex
-DOK7 binds and stabilises the MuSK complex
-MuSK phosphorylates and activates non-receptor tyrosine kinases: Abl and Src kinases → tyrosine kinase cascade
-Src binds to the cytoplasmic domain of AChR
-Src and Abl together phosphorylate the AChR β subunit
-Phosphorylation of the AChR β-subunit recruits rapsyn to AChR
-Rapsyn cross-links adjacent AChRs and stabilise and retain them in clusters
-MuSK activation can also increase transcription of AChRs, rapsyn and MuSK to build the synapse
→Agrin/MuSK signalling works to stabilize and grow the specialised postsynap c membrane of the NMJ
•Fall of the agrin/MuSK paradigm (2001)
1) Agrin and motor neuron knockout mice
Method
-Generated mouse embryos lacking both agrin and motor neurons
-Myotubes derived from control and agrin-/- and HB9-/- mice were cultured and stained
Results
Conclusions
-Control: Axons grew through the myotubes and AChR clusters formed
-Agrin-/- and HB9-/- myotubes: AChR cluster still appeared in the central part of the muscle fibres
where the NMJs normally form on day 14/15 when the nerve first normally gets there
-The receptor clusters were transient, dispersing and disappearing within a day or two
-AChR clusters form spontaneously where they should form without the nerve (agrin) but require
agrin to retain the clustering and stabilising
-Agrin does not induce receptor clustering and synapse formation
-Agrin is necessary to stabilise and reinforce the developing receptor clusters
2) Cell culture
Method
Results
Aneural myotubes were placed on a very adhesive substratum
Conclusions
Aneural myotubes formed large (NMJ-llike) AChR clusters through adhesive contacts
-Muscle cell is capable of organising AChR clusters (which can then be innervated by the nerve)
-Implies a role for adhesive extracellular matrix proteins
•Basis for a revised hypothesis
-Correctly identified and cloned agrin, rapsyn, MuSK and LRP4 as components of synaptic formation but
misinterpreted their roles
-Neural agrin from nerve can activate MuSK
-LRP4 & MuSK are essential for AChR clustering
-Muscle cells need neither nerve nor neural agrin to create a ‘postsynaptic like’ AChR cluster
-Neural agrin’s role is to stabilize the new synaptic AChR cluster by enhancing MuSK activation
-Muscle fibres contain many nuclei but a small group of muscle nuclei are concentrated beneath the motor endplate
-These soleplate nuclei express higher levels of MuSK and other genes that encode postsynaptic proteins
-Modified Hypothesis: Local expression of MuSK is sufficient to generate postsynaptic differentiation
-Why you get the spontaneous formation of postsynaptic receptors even in the absence of nerves is that local nuclei
make more MUSK and that gets put into the membrane and that makes this area become specialised
1) In situ hybridisation
Method
-Generated HB9-/- knockout (motor neuron deficient) mice
-Probed for the expression of synapse-specific genes (mRNA that encode AChR and MuSK) in
diaphragm muscle
Results
Conclusions
-Wild type and HB9-/- mice exhibited similar results
-mRNAs encoding MuSK and AChR selectively expressed in the soleplate nuclei, even in muscles of
aneural mouse embryos
-More MuSK was made underneath the nerve (wild type)
-MuSK alone is capable of forming receptor clusters if it is sufficiently expressed
-Agrin may be increasing the activity of MuSK
2) Transgenic mice with ectopic MuSK expression
Method
-Create transgenic mice that express MuSK ectopically throughout the muscle fibre using a promoter
-Ectopic expression = expression of a gene at an inappropriate point of development when it is not
normally expressed (experimentally determines the function of the gene)
1. Generate HB9-/- mice carrying either the MuSK-L or MuSK-H transgene and stain AChRs with α-BGT
2. Stain whole mounts of diaphragm muscles of wild-type mice carrying either MuSK-L or MuSK-H
transgene with antibodies against neurofilament and synaptophysin to visualise axons and nerve
terminals and with α-BGT to mark postsynaptic AChRs
Results
-Endogenous MuSK is expression in the central region of muscle whereas exogenous MuSK-H and
MuSK-L were expressed throughout muscle (ectopic regions)
1. High ectopic MuSK expression induced ectopic AChR clusters and disrupts AChR pre-patterning
-High MuSK expression induced ectopic AChR clustering even in the absence of motor nerve
innervation
-AChR clusters were concentrated in the central region of muscle from wild-type mice while AChR
clusters were expressed throughout muscle of MuSK-H mice (even on the peripheral ends of the
muscle fibres where they wouldn’t normally form)
-Ectopic AChR clusters are not induced in MuSK-L mice
2. High ectopic MuSK expression promoted motor axon outgrowth
-In wild-type mice, motor axons branched and terminated adjacent to the main trunk and AChRs were
clustered selectively at synaptic sites (similar for MuSK-L mice)
-In mice carrying MuSK-H transgene, the main nerve trunk was positioned correctly but motor axons
failed to stop and instead grew throughout the muscle
-Motor axons extended to innervate pre-patterned ectopic AChR and formed synapses
Conclusions -Induction of ectopic AChR clusters is dependent on the level of MuSK expression
-High ectopic MuSK expression is sufficient to perturb muscle pre-patterning and to induce ectopic
AChR clustering in muscle lacking motor axons
-High ectopic MuSK expression is sufficient to promote motor axon growth and disruption of muscle
pre-patterning does not perturb motor axon growth
-Muscle prepatterning restricts motor axons to this pre-specialized region of the muscle
→Pre-formed target theory: nerve grows towards a preformed target
•The role of agrin/MuSK system is to prevent the destruction of postsynaptic cluster of AChRs (inhibition of
negative pathway)
Q) What influence do MuSK expression levels have upon the location of AChR cluster on embryonic muscle fibres.
Describe an experiment to support your contention.
Q) Does the ingrowing motor axon induce the formation of the postsynaptic AChR cluster in embryonic muscle?
Outline the key evidence for and against this proposition.
•MuSK pathway disruption as the cause of myasthenia gravis
▪Myasthenia gravis
-Develop autoimmune antibodies that attack proteins at the NMJ:
-By binding the extracellular domains of the AChRs (destabilisation and complement-mediated damage)
-By binding to MuSK/ LRP4
-Congenital myasthenia gravis is often caused by mutations in rapsyn, DOK7 and MuSK -Loss of postsynaptic AChRs, failure of neuromuscular transmission, fatiguing muscle weakness
1) IgG injection
Method
Anti-MuSK-positive IgG from MG patient were intraperitoneally injected daily into mice for 14 days
Results
-After 10 days, the mice began to lose weight and developed weakness
-They showed all signs of myasthenia gravis
Conclusions
-IgG-treated mice lost AChRs in the postsynaptic membrane (reduced receptor area)
-mEPP (miniature end plate potential =small depolarisations in the postsynaptic membrane caused
by spontaneous leakage of ACh to NMJ ) amplitude decreased
-EPP (endplate potential) amplitude decreased below the AP threshold
MuSK damage/loss causes destabilisation and loss of AChR and causes NMJ to decline in function
▪MuSK signalling deficiency timeline
-2002: Sequencing of rapsyn, LRP4, MuSK, DOK7 genes from some patients with congenital myasthenia reveals
mutations that cause failure of AChR clustering
-2001: MuSK antibodies discovered in blood of some myasthenia gravis patients
-2008: Immunoglobulin G (IgG) from these patients shown to cause myasthenia gravis when injected into mice
-2013: Anti-MuSK patient antibodies found to block MuSK activation
-2015: ‘Gene therapy’ shown to relieve anti-MuSK and congenital myasthenia in mice
Q) Outline how developmental studies have advanced our understanding of a disease of the NMJ.
Neural Networks Associated with GM Loss Patterns
▪Major brain areas involved in PTSD marked in red: mPFC, amygdala, hippocampus
•Schematic of the inter and intra-connectivity of the major regions involved in fear conditioning and extinction with
the input and output of the circuitry
-Input from thalamus to LA
-Intralamina brings in signal from UCS (pain sensation from electric shock)
-MGN brings in auditory signal of CS (bell)
-Output to PAG (dorsal and ventral) and hypothalamus
•Neurons and their synapses subserving the fear conditioning and extinction circuitry
-White = excitatory neuron; Red = inhibitory neuron
-Thalamus → LA (lateral amygdala)
1) LA → Fear neuron (F) in BA (basal amygdala)
-When US comes in, the fear neuron is excited directly from intralamina
-Fear neuron has direct output to CEA (central amygdala)
-CEA neuron has output to PAG and hypothalamus (control HPA axis, cortisol release, motor performance)
2) LA → ITCd (Dorsal inhibitory intercalated region of amygdala)
-ITCd neuron projects ventrally to inhibit another inhibitory neuron, ITCv (ventral ITC)
-ITCv normally inhibits the CEA output neuron
-If ITCv is active, it inhibits output to and activation of PAG and hypothalamus and prevents fear acquisition
-If ITCv is inhibited, there is no inhibition of output → fear neuron will produce fear response via PAG and
hypothalamus without being regulated by inhibitory pathway
→ITCv pathway must be activated to alleviate fear
Q) How can we activate the inhibitory pathway when the fear neuron is activated?
3) LA → Extinction neuron (E) in BA
-In extinction, the extinction neuron gets input not only from the thalamus/LA but also from the infralimbic mPFC
-E neuron’s excitatory output is to ITCv neuron
-Driving input from mPFC turns on ITCv inhibitory pathway (via E neuron) and turns off output to PAG and
hypothalamus → extinction
-In order to treat PTSD, we must potentiate:
-ITCv inhibitory system
-Extinction neuron (to overwhelm any other circuit and turn off output circuitry)
•Model of amygdala network engaged in extinction (with each number denoting each neural population)
-During conditioning, thalamic input to (1) in LA which projects to fear neuron (2)
-Fear neuron’s main pathway is to output neuron in CEA (3)
-At the same time (during fear acquisition), there is a signal from LA to inhibitory ITCd neuron (9) which projects to
another inhibitory ITCv neuron (7) which normally turns off output neuron
-In extinction, IL mPFC neuron projects into LA, BLA and ITC neurons
-mPFC excites the extinction neuron and turns off output via ITCv
-mPFC also sends a direct input to ITCv neuron to make it hyperexcitable so it will easily do its inhibitory action on
the output neuron (3)
-Once the extinction neuron is excited (by mPFC), it also projects to PV+ inhibitory neuron (8) which inhibits the fear
neuron (2) and effectively turns off the fear acquisition pathway
-The inhibitory pathway prevails and turns off output to PAG and hypothalamus
-Extinction neuron (6) is fed by inhibitory neuron (5) which is getting input from LA neuron (4)
-CCK+ inhibitory neuron (5) can turn off extinction neuron and is deleterious
-Discovery: inhibitory neuron (5) turns itself off
-When extinction neuron is highly excited by mPFC drive, it releases an endocannabinoid (anadamide) which diffuses
in a retrograde manner to the nerve terminal of the inhibitory neuron (5) and turns it off
-The endocannabinoid regulation allows extinction neuron to flourish and inhibit output
-The release of ECB in amygdala greatly increases during extinction and the blockade of ECB release prevents
extinction (high level of acquisition remains)
Synapse Loss/ Regression Underlies GM Loss
•Berkley vs. Cajal’s drawings of dendritic spines
-Collateral axons = a branch of the main axon that usually projects and feeds back toward the cell soma
-Collateral axons forming synapses on the spines of dendrites of another neuron:
•No loss of neurons, astrocytes or oligodendrocytes were detected in stressed animals
A) Nissl-stained section through the ACC showing Neurons (red), Astrocytes (blue) and Oligodendrocytes (green)
B-E) Average number of different cell types before and after restraint stress in ACC (B), CA1 hippocampus (C),
amygdala (D) and RSG (E)
-Insignificant differences in density of cell types
→NO change in the cellular components under conditions of stress and trauma
*RSG (retrosplenial granular area) is used as a positive control as it is not part of the fear/stress/trauma circuitry
•Changes in the number of neurons, astrocytes and oligodendrocytes following stress
-No statistical significance reached
•Golgi-stained neurons in brain regions
•Changes in number of dendritic spines
A) Photomicrograph of spines on 4th order dendrites within the ACC of control (above) and stressed (below) mice,
showing reduced spine density in stressed mice
B-E) Density of dendritic spines per 10 μm length of dendrites for different order dendrites (2 to 5) in the ACC (B),
CA1 hippocampus (C), amygdala (D) and RSG (E)
F-I) Percentage of changes in the number of spines per 10 μm for the different order dendrites in the animals
following stress for ACC (F), CA1 (G), amygdala (H) and RSG (I)
-The order refers to how far the dendrites are from the cell body of neuron (5 being the furthest)
-There was no significant difference in dendrite diameters between control and stressed animals
-There was no spine loss for the different order dendrites in the amygdala and RSG (both exhibit no GM loss
following stress)
-Significant decreases occur in stressed animals in the number of synaptic spines along dendrites of neurons in the
ACC and CA1 (both show significant GMV loss using MRI)
-As the order increases, the number of spines declines more
•Quantitative changes in the density of synapses along dendrites of individual neurons
-Only ACC and hippocampus reached statistical significance (especially as order increases)
-Amygdala: non-specific changes with high p values
•Percentage loss of synaptic spines for higher-order dendrites (orders 3-5)
-Loss of spines occurs because synapses which define them are gone
-Boutons sitting on spines come off and spines retract
-As a consequence of the spine loss, dendrites collapse and are severely curtailed from the pyramidal neurons in
mPFC and hippocampus
-Dendrites that support the spines decrease length
•Thin tortuous and beaded dendrites in the LGN of cat after MD
-Remove the synapse from the dendrite by cutting the optic nerve
-As a consequence of the loss of synapse, the spines disappear and dendrites pull in and turn into a mess
-When you totally deafferent a neuron, dendrites pull in and the cell body collapses
-Loss of synapses → Loss of dendri c spines → Loss of dendrites (but NOT loss of cell body/ NO cell death)
-In stress and trauma, loss of neurons or glia is not observed but only loss of spines and dendrites
•Changes in length of apical dendrites of neurons
A-B) Photomicrograph of Golgi-stained pyramidal CA1 neuron of a non-stressed mouse (A) and stressed mouse (B),
showing reduced cumulative dendritic length in stressed mouse
C-D) Changes in the average of cumulative dendritic length per neuron in ACC, CA1, amygdala and RSG comparing
control (blue) and stressed rats (red) + Percentage changes
E-F) Changes in the average cumulative volume of dendrites per neuron in ACC, CA1, amygdala and RSG +
Percentage changes
-No significant change in dendritic length or volume in the amygdala or RSG (both showed no change in GMV loss)
-Significant decreases occur in stressed animals in the cumulative length of apical dendrites of neurons = sum of all
different order dendrites in the ACC and CA1
-When the dendrites did collapse, the % change in dendritic volume didn’t seem to quantitatively match the GM
volume loss (dendrites take up half the volume of soma?)
•Changes in GM volume due to changes in volume of dendrites following stress vs. Changes in GM volume
determined with MRI (Calculate the extent to which dendrites made a volumetric difference to the volume of GM)
•Volume of GM taken up by different volumes of its cellular constituents and their processes
-Dendrites, axon collaterals and extracellular
space (containing CSF) dominate
-Surprisingly, synapses take up 6%
-1011 neurons in the GM of cortex and
each neuron can have up to 50 000 synapses =
~1015 synapses in GM
-GM loss happens because of the loss of
dendrites; exact measured loss of dendrites is the
measured loss of GM (as calculated using MRI)
•GM volume change vs. Cumulative dendritic volume change/% change in volume of dendrites
-In stressed animals, there is a linear relationship between the loss of GMV determined with MRI and the cumulative
loss of dendritic volume
-Loss of dendrites accounts for the loss of grey matter
Alzheimer’s Disease
•Introduction
Symptoms
Loss of learning and memory retention
Brain region
Entorhinal cortex
Hippocampus
Temporal lobe
Inability to recognize familiar places, objects, and people
Deterioration of reading, writing and linguistic skills
Parietal lobe
Increasing inability to properly locate objects within 3D space
The gradual loss of inhibition and impulsive behaviour regulation
Frontal lobe
-Main correlate to cognitive decline is the loss of synapses, and later, neurons
1) Inherited AD (only ~1% of all AD cases)
-Early onset of ~40 years
-Caused by dominantly inherited mutations in amyloid precursor protein (APP) or presenilin (γ secretase) genes
-Familial AD involves the same brain regions with faster disease progression
-Familial AD mutations have 100% penetrance (propportion of carriers showing the characteristic phenotype)
2) Late-onset AD (>90% of all AD cases)
-Affects ~20% of all people aged over 85 years
-Genetic and environmental risk factors but no single cause
-At least 70% of AD has no known causes
-Major risk factor is old age itself
▪Risk factors/associations:
-Diabetes Mellitus (Type 2 Diabetes)
-Obesity
-Hypertension
-Depression
-Smoking
-Physical inactivity
-Cognitive inactivity or low educational attainment
-Up to a maximum of 30-50% cases associated with a combination of these factors
-However, due to the overlapping nature of these risks (e.g. obesity + diabetes, smoking + hypertension), the
proportion of cases associated with these factors is less than 30-50%
-Cause or effect is not known (e.g. is depression a causal risk or a consequence of AD?)
▪Risk genes
-Innate immune system, phagocytosis, debris clearance, endocytosis, lipid transport, apoptosis:
-ApoE (apolipoprotein E)
-Clusterin (apolipoprotein J)
-TREM2 (triggering receptor expressed on myeloid cells)
-CD33 (myeloid cell surface antigen)
-ABCA7 (ATP-binding cassette A7: transporter on macrophages)
-CR1 (complement component 3b/4b receptor 1)
-PICALM (phosphatidylinositol binding clathrin assembly protein/lymphoidmyeloid leukemia)
-BIN1 (bridging interactor 1 – involved in clathrin-mediated endocytosis)
▪ApoE
-Found in lipoprotein particles
-Produced by astrocytes
-Transports cholesterol to neurons (via low density lipoprotein receptors, ApoE receptor)
-Encoded by polymorphic alleles - ε2, ε3 and ε4
-These allelic forms differ from each other by only one or two amino acids at positions 112 and 158 but this alters
ApoE structure and function
-Presence of the ε4 allele is a major AD risk factor (may account for 40% of AD)
-Increased AD frequency
-Decreased mean age of clinical onset
-APOE4 probably has both “Aβ-dependent” and “Aβ-independent” roles in AD pathogenesis
-Gain of toxic function: ↑ Brain atrophy, neuronal toxicity, Aβ aggregation, tangle formation, aberrant brain activity
-Loss of neuroprotective function: ↓ Synaptic function/pruning, glucose metabolism, neurogenesis, Aβ clearance,
vascular function, mitochondrial function, lipid and cholesterol metabolism
•Current therapeutics
-Acetylcholine esterase inhibitors (acetylcholine is depleted in AD brain)
-NMDA-type glutamate receptor antagonists (to prevent aberrant neural excitability)
-Effect of these drugs is weak to moderate and transient
-They help maintain the activity of the residual neurons but do not stop the ongoing neuronal cell death and loss of
synapses
-99.6% of all new AD treatments tested from 2002-2012 have failed in clinical trials
•Pathology
1) Extracellular amyloid-beta plaques
-Aβ40 and Aβ42 (products of APP metabolism) form plaques outside neurons in the parenchyma
2) Intraneuronal filamentous tangles of phosphor-Tau proteins
-Tau protein is mutated, detached from microtubules, hyperphosphorylated and assembled into filaments
-The aggregates form the neurofibrillary tangles (neurons filled up with aggregates of Tau protein)
-Tau dysfunction causes disease in the absence of amyloid pathology (e.g. FTLD)
→The Amyloid Cascade Hypothesis: Aβ plaques form first and lead to downstream cascade of events that cause Tau
aggregation
▪Amyloid plaques
-Amyloid plaques contain extracellular fibrils of aggregated Aβ peptides and dystrophic neuronal processes that
accumulate phosphorylated Tau proteins (neuritic plaques)
-APP precursor protein (type 1 transmembrane protein) found all over the body
-Cleavage of APP by BACE (β secretase) releases a large ectodomain (sAPPβ) into the interstitial compartment,
leaving behind a longer C terminal fragment (C99) embedded in membrane
-C99 gets cleaved in the membrane by γ-secretase complex (containing presenilin 1 or 2 = catalytic site)
-This generates Aβ peptides in various isoforms (mainly Aβ40, Aβ42)
-Aβ is secreted and aggregates into fibrils that make up amyloid plaques in AD (not cleared, degraded by
extracellular proteases)
▪APP and presenilin mutations
-Cause familial early-onset AD
-APP mutations (~30 known) and PSN1 or PSN2 mutations (>80 known)
-γ secretase cleavage occurs at a progressive manner
-First cleavage at ε-site then consecutive cleavages that trim down the peptide
-Aβ peptide released contains either 40 or 42 amino acids
-Mutations in presenilin increase:
1) Overall production of Aβ peptides (more efficient cleavage towards amyloidogenic pathway)
2) Relative generation of longer Aβ peptides (Aβ42)
-Aβ42 has a higher propensity to aggregate (compared to shorter Aβ40)
-Aβ42 is the predominant isoform found in amyloid plaques
▪Neuritic plaques contain dystrophic neurites filled with abnormal tau protein filaments (as well as Aβ)
▪Neurofibrillary tangles and threads: Tau is mislocalized from axons to dendrites/cell bodies and forms aggregates
-Plaques tend to be in layers 2/3
-NFT cluster in various other lamina of cortex
▪Tau protein function
-The domains of Tau bind different types of molecules suggesting roles (other than formation of toxic aggregates) in
signalling pathways (N-terminal) and cytoskeletal organisation (binding to actin filaments)
-Phosphorylation at different sites may further regulate/reduce different functions (e.g. Phospho-Tau has lower
affinity for microtubules → Free, mislocalised Tau dissociated from microtubules → Tau aggregates)
▪AD tau filaments are hyperphosphorylated
-Immunolabelling with Phospho-Tau antibody (AT8) reveals hyperphosphorylated Tau filaments
-Electrophoresis of Tau filaments and normal Tau with alkaline phosphatase (ALP) reveals
-Normal Tau and filamentous Tau move faster on gel with ALP (phosphorylated and hence cleaved)
-Filamentous Tau moves slower on gel without ALP (not cleaved into smaller pieces)
▪Aβ and tau protein aggregates are similar in structure
-Neurofibrillary tangles and threads are comprised of intracellular “Paired Helical Filaments” (PHFs) of Tau protein
-These structures are similar to Aβ aggregates (except Tau is much larger)
-They both have underlying β-sheet conformation
▪Nucleation-Elongation model for Tau and Aβ fibrils/filaments assembly
-Tau and Aβ form aggregates via a process of nucleation and elongation
-In a slow nucleation phase (RDS), the protein forms a dimer which further aggregate to form an oligomer
-This causes the lag time (green curve)
-Once the nucleus/oligomer forms, this initiates a fast elongation of filaments
-Exponential increase of the aggregation
-If you add a seed (= large or higher order aggregate = nucleus/oligomer or fragmented mature fibrils), this skips the
lag time and speeds up the elongation process
-Oligomers may be more important than deposits of large aggregates (drug targeting)
-This may underlie how AD pathology spreads from one cell to another (passing on seeds to accelerate aggregation)
-Lag phase is dependent on the concentration of monomer
-Increased concentration of proteins = higher propensity to form aggregates
•Pathology and disease
-The extent of tau pathology correlates with disease progression
-Density of NFTs and NTs, tangle counts, level of deposition of Tau
-Decline in cognitive status and neuronal function
-The extent of amyloid plaque deposition does NOT correlate well with disease progression
-In old age, people have high amyloid load but some remain cognitively normal
-Amyloid plaques form first and saturate out early in the disease
process, before the onset of symptoms
-In the preclinical stage, the amyloid plaques are maxed out →
cognitively normal
-Tau NFTs and NTs are observed in small amounts (entorhinal
cortex, hippocampus)
-The growth/spread/progression of tau pathology follows the
course of AD from mild to severe