LECTURE 22: AXONAL GUIDANCE REQUIRED READING: Kandel text, Chapter 54 ESTABLISHMENT OF NEURONAL...

LECTURE 22: AXONAL GUIDANCE

REQUIRED READING: Kandel text, Chapter 54

ESTABLISHMENT OF NEURONAL IDENTITY

AXONAL GUIDANCE TO TARGETS

NEUROTROPHIC SUPPORT FROM TARGET TISSUES

SYNAPTOGENESIS

HOW IS PROPER “WIRING” OF NERVOUS SYSTEM ACHIEVED?HOW IS PROPER “WIRING” OF NERVOUS SYSTEM ACHIEVED?

THEORY 1: AXON PROJECTIONS ARE IMPRECISE…PROPER CONNECTIONS ARE WORKEDTHEORY 1: AXON PROJECTIONS ARE IMPRECISE…PROPER CONNECTIONS ARE WORKED OUT BY ELECTRICAL ACTIVITIES DURING EARLY EXPERIENCEOUT BY ELECTRICAL ACTIVITIES DURING EARLY EXPERIENCE

THEORY 2: CHEMOSPECIFICITY MODEL….AXONS ARE DIRECTED TO THEIR PRECISE TARGETSTHEORY 2: CHEMOSPECIFICITY MODEL….AXONS ARE DIRECTED TO THEIR PRECISE TARGETS BY CHEMICAL SIGNALS THAT EXIST INDEPENDENT OF EXPERIENCEBY CHEMICAL SIGNALS THAT EXIST INDEPENDENT OF EXPERIENCE

Roger Sperry’s retinal axonRoger Sperry’s retinal axonregeneration experimentregeneration experiment

supported the supported the chemospecificity modelchemospecificity model

When optic nerve is cut, axonsWhen optic nerve is cut, axonsregenerate and restoreregenerate and restore

normal sight and behaviornormal sight and behavior

When optic nerve is cut When optic nerve is cut andandthe eyeball is rotated 180 degthe eyeball is rotated 180 degin socketin socket, axons regenerate, axons regenerate

and restore sight, but behaviorand restore sight, but behavioris maladaptiveis maladaptive

FROG THINKS “UP” IS “DOWN”FROG THINKS “UP” IS “DOWN”And “LEFT” is “RIGHT”And “LEFT” is “RIGHT”

TemporalRetina

NasalRetina

AXON GUIDANCE IS A MULTI-STEP PROCESSAXON GUIDANCE IS A MULTI-STEP PROCESS

1.1. Migration towards retinal exit point (axon convergence)Migration towards retinal exit point (axon convergence)2.2. Turning to exit retina and fasiculationTurning to exit retina and fasiculation3.3. Fasiculated extension towards chiasmFasiculated extension towards chiasm4.4. Ipsilateral vs. contralateral migration at optic chiasm (divergence point)Ipsilateral vs. contralateral migration at optic chiasm (divergence point)5.5. Fasiculated extension into brainFasiculated extension into brain6.6. Projection into lateral geniculate or into optic tectum (divergence point)Projection into lateral geniculate or into optic tectum (divergence point)7.7. Projection a certain distance along surface of tectum (divergence step)Projection a certain distance along surface of tectum (divergence step)8.8. Diving into the tectumDiving into the tectum9.9. Stopping at specific depths into tectum (divergence step)Stopping at specific depths into tectum (divergence step)10.10. SynaptogenesisSynaptogenesis

AXON DIVERGENCES AT OPTIC CHIASM ALLOW CONVERGENCE OFAXON DIVERGENCES AT OPTIC CHIASM ALLOW CONVERGENCE OFCONGRUENT INFORMATION FROM BOTH RETINASCONGRUENT INFORMATION FROM BOTH RETINAS

Nasal hemiretinal axons cross over at chiasmNasal hemiretinal axons cross over at chiasm(contralateral projection)(contralateral projection)

Temporal hemiretinal axons do not cross overTemporal hemiretinal axons do not cross over(ipsilateral projection)(ipsilateral projection)

ACTIN CYTOSKELETON DYNAMICS IN GROWTH CONEACTIN CYTOSKELETON DYNAMICS IN GROWTH CONEDURING ATTRACTION AND REPULSION:DURING ATTRACTION AND REPULSION:

GROWTH CONE STRUCTUREGROWTH CONE STRUCTURE

ACTIN FILAMENTSACTIN FILAMENTSIN FILOPODIAIN FILOPODIA

AND LAMELLIPODIAAND LAMELLIPODIA

MICROTUBULESMICROTUBULESALONG AXON ANDALONG AXON AND

IN CORE OFIN CORE OFGROWTH CONEGROWTH CONE


LAMELLIPODIA/FILOPODIA DRIVE AXON EXTENSIONLAMELLIPODIA/FILOPODIA DRIVE AXON EXTENSION


REPULSIVE CUES CAUSE GROWTH CONE COLLAPSEREPULSIVE CUES CAUSE GROWTH CONE COLLAPSE

GROWTH CONEGROWTH CONEBEFOREBEFORE

SEMAPHORINSEMAPHORINEXPOSUREEXPOSURE

SAME GROWTHSAME GROWTHCONE AFTERCONE AFTERSEMAPHORINSEMAPHORIN

EXPOSUREEXPOSURE

ACTIN CYTOSKELETON DYNAMICS: THE ROLE OF RHO-FAMILY G PROTEINS

Gs, Gi, Gq~45,000 MW HETEROTRIMERIC

H-Ras, K-Ras, N-Ras ~21,000 MW

RhoA, Rac, Cdc42 ~21,000 MW

RalA, RalB ~21,000 MW

RAS FAMILY

RHO FAMILY

RAL FAMILY

SMALL

GTPases

G : GTPG : GDP

GDP GTP

Pi

INA

CT

IVE

AC

TIV

E


Common structure of small G proteins allows design of constitutively active (CA) and dominant negative (DN) mutant proteins

CA-RhoA , CA-Rac , CA-Cdc42 expressed in fibroblasts induce specific architectural structures

FOCAL ADHESIONS&

STRESS FIBERS

LAMELLIPODIA FILOPODIA

EXTRACELLULAR AXON GUIDANCE MOLECULES ACT THROUGH RHO-FAMILY G PROTEINS TO MODIFY GROWTH CONE ARCHITECTURE AND STEER DIRECTION OF OUTGROWTH

ATTRACTANT GUIDANCE MOLECULES

LOCAL ACTIVATION OF RAC and CDC42

LOCAL EXTENSION OF FILOPODIA andSPREAD OF LAMELLIPEDIA

GROWTH CONE EXTENDS TOWARDATTRACTANT

REPULSIVE GUIDANCE MOLECULES

LOCAL ACTIVATION OF RHO

LOCAL ASSEMBLY OF ACTOMYOSINBRIDGES

GROWTH CONE CONTRACTS AWAYFROM REPELLANT

DIFFERENT TYPES OF AXON GUIDANCE CUESDIFFERENT TYPES OF AXON GUIDANCE CUES

TOPOGRAPHIC VISUAL MAP PROJECTED ONTO TECTUMTOPOGRAPHIC VISUAL MAP PROJECTED ONTO TECTUM

THE SURFACE OF TECTUM AS A TOPOLOGICALLY PRESERVED REPRESENTATIONTHE SURFACE OF TECTUM AS A TOPOLOGICALLY PRESERVED REPRESENTATIONOF THE VISUAL FIELDOF THE VISUAL FIELD

AXONS FROM MOST ANTERIOR OF RETINA PROJECT TO MOST POSTERIOR TECTUMAXONS FROM MOST ANTERIOR OF RETINA PROJECT TO MOST POSTERIOR TECTUMAND VISA VERSAAND VISA VERSA

WHAT IS NATURE OF THE GUIDANCE SYSTEM?WHAT IS NATURE OF THE GUIDANCE SYSTEM?

AN AXONAL REPELLENT PRODUCED BY TECTUMAN AXONAL REPELLENT PRODUCED BY TECTUM

POSTERIOR RETINA AXONSPOSTERIOR RETINA AXONSWON’T GROW ALONGWON’T GROW ALONG

POSTERIOR TECTAL MEMBRANES;POSTERIOR TECTAL MEMBRANES;THEY GROW ALONGTHEY GROW ALONG

ANTERIOR TECTAL MEMBRANESANTERIOR TECTAL MEMBRANESOR NO MEMBRANESOR NO MEMBRANES

ANTERIOR RETINA AXONSANTERIOR RETINA AXONSSHOW NO SUBSTRATESHOW NO SUBSTRATE

PREFERENCEPREFERENCE

CONCLUSION:CONCLUSION:POSTERIOR TECTUM PRODUCESPOSTERIOR TECTUM PRODUCESA CHEMOREPELLENT SPECIFICA CHEMOREPELLENT SPECIFIC

FOR POSTERIOR RETINA AXONSFOR POSTERIOR RETINA AXONS

THE CHEMOREPELLENT WASTHE CHEMOREPELLENT WASPURIFIED FROM TECTUMPURIFIED FROM TECTUMTISSUE HOMOGENATESTISSUE HOMOGENATES

REPELLENT FOUND TO BE PROTEINREPELLENT FOUND TO BE PROTEINNOW TERMED NOW TERMED EPHRINEPHRIN

EPHRINS AND THEIR RECEPTORSEPHRINS AND THEIR RECEPTORS

EPHRINS ARE CELL-SURFACEEPHRINS ARE CELL-SURFACEPROTEINS:PROTEINS:

““A-TYPE” ARE GPI-ANCHOREDA-TYPE” ARE GPI-ANCHORED““B-TYPE” ARE TRANSMEMBRANEB-TYPE” ARE TRANSMEMBRANE

EPHRIN RECEPTORS AREEPHRIN RECEPTORS ARERECEPTOR TYROSINE KINASESRECEPTOR TYROSINE KINASES

THE SIGNALING CAPACITIESTHE SIGNALING CAPACITIESOF EPHRIN RECEPTORSOF EPHRIN RECEPTORS

ARE VERY DIFFERENT FROMARE VERY DIFFERENT FROMOTHER RTKs,OTHER RTKs,SUCH AS THESUCH AS THE

NEUROTROPHIN RECEPTORSNEUROTROPHIN RECEPTORS

GRADIENT EXPRESSION OF EPHRINS ON TECTUMGRADIENT EXPRESSION OF EPHRINS ON TECTUMAND RECEPTORS ON RETINAL AXONSAND RECEPTORS ON RETINAL AXONS

EPHRIN EXPRESSION IS HIGHEST ON POSTERIOR TECTUMEPHRIN EXPRESSION IS HIGHEST ON POSTERIOR TECTUM

EPH EXPRESSION IS HIGHEST ON POSTERIOR RETINAL GANGLION CELLSEPH EXPRESSION IS HIGHEST ON POSTERIOR RETINAL GANGLION CELLSAND THEIR AXONS, WHICH STOP IN ANTERIOR TECTUMAND THEIR AXONS, WHICH STOP IN ANTERIOR TECTUM

LOW EPH EXPRESSION ON ANTERIOR GANGLION CELLS ALLOWSLOW EPH EXPRESSION ON ANTERIOR GANGLION CELLS ALLOWSTHEIR AXONS TO ADVANCE TO POSTERIOR TECTUMTHEIR AXONS TO ADVANCE TO POSTERIOR TECTUM

ECTOPIC EPH EXPRESSION DIVERTS POSTERIOR RETINAL AXONSECTOPIC EPH EXPRESSION DIVERTS POSTERIOR RETINAL AXONS

PATCHES OF ECTOPIC EPHRIN EXPRESSION WAS ACHIEVED BY INFECTINGPATCHES OF ECTOPIC EPHRIN EXPRESSION WAS ACHIEVED BY INFECTINGTECTUM WITH REPLICATION-COMPETENT VIRAL VECTOR EXPRESSING EPHTECTUM WITH REPLICATION-COMPETENT VIRAL VECTOR EXPRESSING EPH

POSTERIOR RETINAL AXONS WITH HIGH EPHRIN RECEPTOR LEVELSPOSTERIOR RETINAL AXONS WITH HIGH EPHRIN RECEPTOR LEVELSAVOID EPHRIN OVEREXPRESSION PATCHESAVOID EPHRIN OVEREXPRESSION PATCHES

ANTERIOR RETINAL AXONS ARE IMMUNE TO EPHRIN PATCHESANTERIOR RETINAL AXONS ARE IMMUNE TO EPHRIN PATCHES

(A MUTATION ELIMINATING EPHRIN EXPRESSION IN BRAIN ALLOWS(A MUTATION ELIMINATING EPHRIN EXPRESSION IN BRAIN ALLOWSPOSTERIOR RETINAL AXONS TO PROJECT THROUGHOUT TECTUM)POSTERIOR RETINAL AXONS TO PROJECT THROUGHOUT TECTUM)

EPHRIN INDUCES EPH RECEPTOR CLUSTERING AND ACTIVATES THE RECEPTORCYTOPLASMIC DOMAIN TO INITIATE SIGNALING LEADING TO REPULSION

SIGNALINGSIGNALING&&

REPULSIONREPULSION

SIGNALINGSIGNALING&&

REPULSIONREPULSION

EPH

EPHRINEPHRIN

TRKTRK-EPH

NGFNGF

CHEMOATTRACTANT PRODUCED BY FLOOR PLATE FOR COMMISURAL AXONS CHEMOATTRACTANT PRODUCED BY FLOOR PLATE FOR COMMISURAL AXONS

COMMISURAL AXONS PROJECT VENTRALLY TO FLOOR PLATECOMMISURAL AXONS PROJECT VENTRALLY TO FLOOR PLATE

FLOOR PLATE EXPLANT ATTRACT COMMISURAL AXONS IN EXPLANTFLOOR PLATE EXPLANT ATTRACT COMMISURAL AXONS IN EXPLANTOF DORSAL SPINAL CORDOF DORSAL SPINAL CORD

IDENTITY OF FLOOR PLATE CHEMOATTRACTANT AND ITS RECEPTORIDENTITY OF FLOOR PLATE CHEMOATTRACTANT AND ITS RECEPTOR

COMMISURAL AXONS IN NETRIN AND DCCCOMMISURAL AXONS IN NETRIN AND DCCMUTANT MICE FAIL TO PROJECT TO FLOOR PLATEMUTANT MICE FAIL TO PROJECT TO FLOOR PLATE

NETRIN/DCC INTERACTION CAN ALSO BE CHEMOREPULSIVENETRIN/DCC INTERACTION CAN ALSO BE CHEMOREPULSIVEROLE OF CYCLIC AMP?ROLE OF CYCLIC AMP?

UNC-5 STATUS DETERMINES WHETHER DCC-POSITIVEUNC-5 STATUS DETERMINES WHETHER DCC-POSITIVEAXON IS ATTRACTED OR REPELLED BY NETRINAXON IS ATTRACTED OR REPELLED BY NETRIN

TROCHLEAR MOTONEURONSTROCHLEAR MOTONEURONSNORMALLY REPELLED BY NETRIN,NORMALLY REPELLED BY NETRIN,BUT ARTIFICIAL ACTIVATION OFBUT ARTIFICIAL ACTIVATION OF

PKA IN CELLS CAUSESPKA IN CELLS CAUSESATTRACTION TO NETRIN SOURCEATTRACTION TO NETRIN SOURCE

GUIDANCEMOLECULES

GUIDANCERECEPTORS

CONSERVED FAMILIES OFGUIDANCE MOLECULES AND THEIR RECEPTORS

n

AXON NAVIGATION THROUGH MULTIPLE GUIDANCE REGIONS REQUIRESCHANGES IN GROWTH CONE SENSITIVITY TO INDIVIDUAL GUIDANCE CUES

SENSITIVITY TO ATTRACTANT IS LOST!!!

MECHANISMS FOR CHANGES IN SENSITIVITY TO AXON GUIDANCE CUESI. CHANGE IN INTRACELLULAR CYCLIC NUCLEOTIDE LEVELS

Netrin mRNANetrin mRNA(blue)(blue)

Retinal ganglionRetinal ganglionaxons (brown)axons (brown)

from Shewan et al., Nature Neuroscience 5:955 (2002)

Early in their outgrowth, retinal axonsEarly in their outgrowth, retinal axonstravel intotravel into netrin-expressing region netrin-expressing region

of optic stalkof optic stalk

Netrin is attractive guidance cueNetrin is attractive guidance cue

Axons express high cAMPAxons express high cAMP

Later in their outgrowth, retinal axonsLater in their outgrowth, retinal axonsavoidavoid netrin-expressing deep regions netrin-expressing deep regions

of thalamus and tectumof thalamus and tectum

Netrin is repulsive guidance cueNetrin is repulsive guidance cue

Axons express low cAMPAxons express low cAMP

MECHANISMS FOR CHANGES IN SENSITIVITY TO AXON GUIDANCE CUESII. SILENCING A GUIDANCE CUE

COMMISURAL AXONS ARE COMMISURAL AXONS ARE ATTRACTED TO MIDLINE FLOOR PLATE, ATTRACTED TO MIDLINE FLOOR PLATE,

CROSS ONCE,CROSS ONCE,AND NEVER CROSS AGAINAND NEVER CROSS AGAIN

Midline floor plate producesMidline floor plate producesNetrin attractant and Slit repellentNetrin attractant and Slit repellent

Initially, axons are attracted by netrin andInitially, axons are attracted by netrin andare insensitive to slit repulsionare insensitive to slit repulsion

Upon reaching floor plate, Slit does two things:Upon reaching floor plate, Slit does two things:1)1) SILENCES netrin attractionSILENCES netrin attraction

2)2) INDUCES Slit repulsionINDUCES Slit repulsion

SLIT ACTS LOCALLY TO MODIFYSLIT ACTS LOCALLY TO MODIFYTHE PROPERTIES OF THETHE PROPERTIES OF THE

GROWTH CONEGROWTH CONE

SLIT SILENCES NETRIN BY GENERATING ROBO/DCC RECEPTOR COMPLEXESAND SILENCING MUST PRECEDE SLIT-MEDIATED REPULSION

Slit induces DCC/Robo complexes,and the cytoplasmic domain of Robo

binds the cytoplasmic domain ofDCC to block attractive signaling

The formation of DCC/Robo complexesmay also promote exocytosis of

Robo-containing vesicles,thereby increasing surface Robo andallowing for Slit-mediated repulsion

Only after axon passes through floor plate,Only after axon passes through floor plate,it is no longer sensitive toit is no longer sensitive to

floor plate attractionfloor plate attraction

Only after axon passes through floor plate,Only after axon passes through floor plate,it becomes sensitive toit becomes sensitive to

Slit repulsionSlit repulsion

LECTURE 22: AXONAL GUIDANCE REQUIRED READING: Kandel text, Chapter 54 ESTABLISHMENT OF NEURONAL...

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