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M2 P7- Cellular Neurobiology and Development - 2009

 

Lundi 21/9

Mardi 22/9

Mercredi 23/9

Jeudi 24/9

Vendredi 25/9

9h-11h

Thierry

Galli,

Frédéric Saudou, Frederic.Saudou@curie.fr, Mécanismes moléculaires de la neurodégénation dans la maladie de Huntington / Molecular mechanisms of neurodegeneration in Huntington's disease

 

François Tronche, francois.tronche@gmail.co m, Approches de génétique moléculaire pour l'étude des fonctions cérébrales / Molecular Genetics for the study of brain functions

Jean-Christophe Poncer,

thierry@tgalli.net, Cours Optionnel d'Introduction:

Rappels de M1 / Optional Introductory Lecture from

M1§

Isabelle

isabelle.caille@snv.jussieu. fr, Traduction locale dans

les neurones / Neuronal local translation

Caillé,

poncer@fer-a-

moulin.inserm.fr, Plasticité synaptique dans les réseaux corticaux / Synaptic plasticity in cortical networks

11h15-

         

13h15

Evelyne Bloch-Gallego,

bloch-

Jamel

Chelly,

gallego@cochin.inserm.fr

,

chelly@cochin.inserm.fr ,

Facteurs de guidage et réorganisations intracellulaires durant la coissance axonale et la migration neuronale / Guiding factors and intracellular rearrangements during axonal outgrowth and neuronal migration

titre Causes génétiques et concepts neurobiologiques impliqués dans le déficit mental / Genetic causes and neurobiological concepts involved in mental deficiency (learning disability)

Isabelle

Caillé,

Nathalie Spassky, nathalie.spassky@upmc.fr. Cellules ciliées et neurogénèse/ Ciliated cells

and neuro enesis

Thierry

Galli,

isabelle.caille@snv.jussieu.f

thierry@tgalli.net, Biologie cellulaire du Neurone / Cell biology of the neuron

r, Les cellules gliales comme cellules souches

 

neurales / Glial cells as neural stem cells

g

14h15-

         

16h15

Alessandra Pierani, pierani@ijm.jussieu.fr,

Nathalie

Rouach,

Lydia Danglot, danglot@ijm.jussieu.fr, Développement et synaptogenèse de l'hippocampe / Development and synaptogenesis of the hippocampus

Thierry

Galli,

nathalie.rouach@college-de-

france.fr,

Astrocytes

et

thierry@tgalli.net, Trafic et différenciation neuronale /

membranaire

Régionalisation dorso- ventrale du tube neural:

domaines progéniteurs et développement des classes neuronales / Dorso-ventral regionalisation of the neural tube : progenitor domains and development of neuronal classes

Examen Final / Final Exam

plasticité

synaptique

/

Astrocytes

and

synaptic

 

plasticity

Membrane Traffic and neuronal differentiation

§ Ce cours a été demandé par les étudiants des années précédentes. Il est recommandé pour ceux qui n'ont pas suivi le cours de M1 d'I Caillé et T Galli. This lecture was requested by the students of last year. It is intended for the students who did not follow the M1 course by I Caillé and T Galli.

Connect…

• http://sites.google.com/site/insermu950/ home

• Frederic.Saudou@curie.fr, isabelle.caille@snv.jussieu.fr, evelyne.bloch- gallego@inserm.fr, pierani@ijm.jussieu.fr, francois.tronche@gmail.com, danglot@ijm.jussieu.fr, jean- christophe.poncer@inserm.fr, jamel.chelly@inserm.fr, nathalie.rouach@college-de-france.fr, nathalie.spassky@upmc.fr

Introduction

How the nervous system is organized

• Nerve cell types and roles

• Excitability and electrical signals

Graded and action potentials initiation and conduction

• Neurotransmitters and signal conduction cell to cell

• Modulation and integration of the signals

Organization of the Nervous System

• Rapid communication for homeostatic balance

• Emergent properties of intelligence & emotion

• Central Nervous system (CNS)

• Peripheral Nervous system (PNS)

Organization of the Nervous System

Or g anization of the Nervous S y stem Figure 8-1: Organization of the nervous system

Figure 8-1: Organization of the nervous system

A Typical Neuron Overview

• Dentrites

Cell Body

• Axon

• Terminal

nervous system A T yp ical Neuron Overview • Dentrites • Cell Body • A xon

Figure 8-2: Model neuron

Diverse Neuron Forms and Functions

Pseudounipolar

• Bipolar

• Anaxionic

• Multipolar–CNS

• Multipolar–efferent

Diverse Neuron Forms and Functions

Multi p ola r– efferent Diverse Neuron Forms and Functions Figure 8-3: Anatomic and f unctional

Figure 8-3: Anatomic and functional categories of neurons

Metabolism and Synthesis in a Neuron

• Cell body site of energy generation and synthesis

Axonal transport

– Vesicles –

• Fast axonal transport to terminal

• Retrograde to cell body

Electrical depolarizations

Metabolism and Synthesis in a Neuron

Electrical depolarizations Metabolism and Synthesis in a Neuron Figure 8-4: Axonal transpor t of membranous organelles

Figure 8-4: Axonal transport of membranous organelles

Glial Cell Functions

• Support neuron bodies, form myelin sheaths

• Barriers between compartments

Scavenger/defense & metabolic assistance

Neuron & friends

sheaths • B arr iers bet ween compar t men ts • Scavenger/defense & metabolic assistance

Glial Cell Functions

Glial Cell Functions Figure 8-5: Glial cells and their functions Electrical Signals: Ionic Concentrations and Potentials

Figure 8-5: Glial cells and their functions

Electrical Signals:

Ionic Concentrations and Potentials

Nernst & GHK Equations predict

Membrane potential

Cell concentration gradients

2+

[Na

[K + ] higher ICF

+

, Cl

-

& Ca

] higher in ECF

Depolarization causes electrical signal

Gated channels control permeability

Electrical Signals:

Ionic Concentrations and Potentials

Electrical Signals: Ionic Concentrations and Potentials Table 8-2: Ion Concentrations and Equilibrium Potentials

Table 8-2: Ion Concentrations and Equilibrium Potentials

Graded Potentials

• Incoming signals

Vary in strength

– Lose strength over distance

Are slower than action potentials (AP)

• Travels to trigger zone

Subthreshold

• Too weak

• No generation of AP

– Suprathreshold – generate AP

Graded Potentials

Graded Potentials Figure 8-7: Graded potentials decrease in strength as they spread out from t he

Figure 8-7: Graded potentials decrease in strength as they spread out from the point of origin

Trigger Zone: Cell Integration and Initiation of AP

• Excitatory signal: depolarizes, reduces threshold

Inhibitory signal: hyperpolarizes, increases threshold

Trigger Zone: Cell Integration and Initiation of AP

Trigger Zone: Cell Integration and In iti a ti on o f AP Figure 8-8a: Subthreshol

Figure 8-8a: Subthreshold and suprathreshold graded potentials in a neuron

Trigger Zone: Cell Integration and Initiation of AP

Trigger Zone: Cell Integration and In iti a ti on o f AP Figure 8-8b: Subthreshol

Figure 8-8b: Subthreshold and suprathreshold graded potentials in a neuron

Action Potential Stages: Overview

• "All or none"

• Signal does not diminish over distance

Action Potential Stages: Overview

none " • Signal does not diminish over distance Action Potential Stages: Overview Figure 8-9: The

Figure 8-9: The action potential

Membrane & Channel Changes during an Action Potential

• Initiation

Depolarization

• Signal peak

• Repolarization

Membrane & Channel Changes during an Action Potential

Membrane & Channel Changes during an Action Potential Figure 8-10: Model of t he voltage-gated channel

Figure 8-10: Model of the voltage-gated channel Na +

Introduction to Cell Biology of the Neuron

Introduction t o Cell Biology of the Neuron
Introduction t o Cell Biology of the Neuron
Introduction t o Cell Biology of the Neuron
Introduction t o Cell Biology of the Neuron
Introduction t o Cell Biology of the Neuron
Introduction t o Cell Biology of the Neuron
2 2 compartments: compartments : •• AAxxoonn •• SomatodendriticSomatodendritic AxonAxon (NF)(NF)

22 compartments:compartments:

•• AAxxoonn •• SomatodendriticSomatodendritic

AxonAxon (NF)(NF) SpinalSpinal cordcord neuronneuron
AxonAxon
(NF)(NF)
SpinalSpinal cordcord neuronneuron
SomaSoma ++ DendritesDendrites (MAP(MAP 2)2) HippocampalHippocampal neuronneuron
SomaSoma
++
DendritesDendrites
(MAP(MAP 2)2)
HippocampalHippocampal neuronneuron

Neuron Parts:

2)2) HippocampalHippocampal neuronneuron Neuron Parts: Maj or s it es to receive input Major sites for

Major sites to receive input

neuronneuron Neuron Parts: Maj or s it es to receive input Major sites for output The

Major

sites for

output

The question we are focusing on:

Neuron polarization: Development of axon & dendrites.

Neuronal p olarit y : domains NEURONS ARE POLARIZED L1-CAM LDLR

Neuronal polarity:

domains

NEURONS ARE POLARIZED

L1-CAM LDLR
L1-CAM
LDLR

Different axonal domains

Different axonal domains CamKII
CamKII
CamKII
Pioneering in vitro study by Ganry Banker i n 1980’s-1990’s Craig AM, Banker G. Annu

Pioneering in vitro study by Ganry Banker in 1980’s-1990’s

in vitro study by Ganry Banker i n 1980’s-1990’s Craig AM, Banker G. Annu Rev Neurosci.
in vitro study by Ganry Banker i n 1980’s-1990’s Craig AM, Banker G. Annu Rev Neurosci.
in vitro study by Ganry Banker i n 1980’s-1990’s Craig AM, Banker G. Annu Rev Neurosci.

Craig AM, Banker G. Annu Rev Neurosci.

1994;17:267-310.

Thus, the question of neuron polarization can be simplified as the selection of one neurite growth to become axon during the transition from stage 2 to 3 in the in vitro case of this specific type of neuron.

Neuronal Differentiation

Neuronal Differentiation Molecular markers for axon or dendrite Tau1 MAP2 Axon: Tau1 , GAP43 , synapsin,

Molecular markers for axon or dendrite

Differentiation Molecular markers for axon or dendrite Tau1 MAP2 Axon: Tau1 , GAP43 , synapsin, synaptotagmin
Differentiation Molecular markers for axon or dendrite Tau1 MAP2 Axon: Tau1 , GAP43 , synapsin, synaptotagmin

Tau1

MAP2

Axon: Tau1, GAP43, synapsin, synaptotagmin …

Dendrite: MAP2, Glycine receptor, GABAa receptor …

Hippocampal Neuron Polarization from Stage 2 to Stage 3

Hippocampal Neuron Polarization from Stage 2 to Stage 3 da Silva JS, Dotti CG. Nat Rev

da Silva JS, Dotti CG. Nat Rev Neurosci. 2002

Sep;3(9):694-704.

Silva JS, Dotti CG. Nat Rev Neurosci. 2002 Sep;3(9):694-704. Neuron growth cone Evident from intensive axon

Neuron growth cone

Evident from intensive axon guidance researches, neurite growth is tightly controlled by the structure in its tip ---growth cone. Maybe the question of polarization can be furthur simplified as the selection of growth cone for growth.

Neuronal differentiation

I. Membrane growth and polarized sorting

Precursor

I. Membrane growth and polarized sorting Precursor Immature Neuro n Mature Neuro n Dendrites: TfR and

Immature Neuron

Mature Neuron

Dendrites: TfR and Golgi Axon: Synaptotagmin
Dendrites: TfR and Golgi
Axon: Synaptotagmin

Neuronal

differentiation

II. Plasticity in membrane growth

Precursor

Immature Neuron

P-lysine
P-lysine

Mature Neuron

growth Precursor Immature Neuron P-lysine Mature Neuron L1 Neuronal differentiation III. Directed g row th From:
L1
L1

Neuronal

differentiation

III. Directed

growth

Neuron L1 Neuronal differentiation III. Directed g row th From: Hong-jun Song and Mu-ming Poo Nature
Neuron L1 Neuronal differentiation III. Directed g row th From: Hong-jun Song and Mu-ming Poo Nature
Neuron L1 Neuronal differentiation III. Directed g row th From: Hong-jun Song and Mu-ming Poo Nature
Neuron L1 Neuronal differentiation III. Directed g row th From: Hong-jun Song and Mu-ming Poo Nature

From: Hong-jun Song and Mu-ming Poo Nature Cell Biology 2001

1. Role of membrane trafficking in neuritogenesis

1. Role of membrane trafficking in neuritogenesis Phase contrast time-lapse recording of a neuron forming an

Phase contrast time-lapse recording of a neuron forming an axon. The video shows a rapid playback of a 16 hour recording with one image taken every 10 minutes.

a neuron forming an axon. The video shows a rapid playback of a 16 hour recording
a neuron forming an axon. The video shows a rapid playback of a 16 hour recording
a neuron forming an axon. The video shows a rapid playback of a 16 hour recording
a neuron forming an axon. The video shows a rapid playback of a 16 hour recording
a neuron forming an axon. The video shows a rapid playback of a 16 hour recording

Membrane traffic: basic mechanisms

Synaptobrevin 2 Regulation: Syntaxin 1 SNAP25 Rab GTPases Brunger, 1998
Synaptobrevin 2
Regulation:
Syntaxin 1 SNAP25
Rab
GTPases
Brunger, 1998

Cai et al, Dev Cell 2007

basic mechanisms Synaptobrevin 2 Regulation: Syntaxin 1 SNAP25 Rab GTPases Brunger, 1998 Cai et al, Dev
basic mechanisms Synaptobrevin 2 Regulation: Syntaxin 1 SNAP25 Rab GTPases Brunger, 1998 Cai et al, Dev

Neuron maturation and membrane trafficking

Neuron maturation and mem b rane tra ffi c ki ng Neurite: Kinesin-dependent transport of cytoskeleton

Neurite:

Kinesin-dependent transport of cytoskeleton components & regulators, and of vesicle-associated cargos

Arimura & Kaibuchi Nat Neurosci 2007

Growth cone:

Main site of membrane insertion

Craig 1995, & others

SNAREs at the plasma membrane

NO EFFECT ON NEURON MATURATION

SNAP-25

• Genetic invalidation in mouse

Washbourne Nat Neurosci 2002

Syntaxin 1

• Genetic invalidation in worm

Saifee Mol Biol Cell1998

• Genetic invalidation in mouse

Fujiwara J Neurosci 2006

• RNA interference

Darios & Davletov Nature 2006

EFFECT ON NEURON MATURATION

SNAP-23?

• Could compensate SNAP-25 absence

Washbourne Nat Neurosci 2002

S

i

yn ax n

t

3

• RNA interference

Darios & Davletov Nature 2006

On the vesicular side

On the vesicular side TeNT Syb 2 Contrast FM1-43 uptake Osen-Sand, J. Comp. Neurol. 1996 Synaptobrevin
TeNT Syb 2
TeNT
Syb 2
On the vesicular side TeNT Syb 2 Contrast FM1-43 uptake Osen-Sand, J. Comp. Neurol. 1996 Synaptobrevin
Contrast FM1-43 uptake Osen-Sand, J. Comp. Neurol. 1996
Contrast
FM1-43
uptake
Osen-Sand, J. Comp. Neurol. 1996

Synaptobrevin 2 KO mic

Schoch, Science 2001
Schoch, Science 2001

TeNT cleavage of Synaptobrevin 2

Neuritogenesis is TeNT resistant

Precursor Immature Neuron Mature Neuron Growth cone Tetanus Neurotoxin resistant Syb2 independent
Precursor
Immature Neuron
Mature Neuron
Growth cone
Tetanus
Neurotoxin
resistant
Syb2 independent

TI-VAMP in neuritogenesis

TI-VAMP in neuritogenesis Suivi e d e GFP - TIVAMP pendant la neuritogenèse TI-VAMP in growth

Suivie de GFP-TIVAMP pendant la neuritogenèse

TI-VAMP in growth cones

Ursula Schenk
Ursula Schenk

Presynaptic markers and growth cone proteins are enriched in GCP re arations

TI-VAMP Syb/VAMP2 (Coco et al., J.Neurosci.1999)
TI-VAMP
Syb/VAMP2
(Coco et al., J.Neurosci.1999)

TI-VAMP is essential for neurite outgrowth in neurons

TI-VAMP is essential for neurite outgrowth in neurons Alberts & al MBoC 2003 Flux of secretory

Alberts & al MBoC 2003

Flux of secretory vesicles in the growing neurite: a main player

al MBoC 2003 Flux of secretory vesicles in the growing neurite: a main player Krasimira Tsaneva-Atanasova

Krasimira Tsaneva-Atanasova David Holcman

TI -VAMP RFP Synaptobrevin 2 GFP What is needed to build a neurite? • Vesicles:

TI-VAMP RFP Synaptobrevin 2 GFP

What is needed to build a neurite?

• Vesicles: TI-VAMP mediated transport and regulators

• Vesicles: TI-VAMP mediated transport and regulators • Microtubules and regulators • Actin filaments and

• Microtubules and regulators

Vesicles: TI-VAMP mediated transport and regulators • Microtubules and regulators • Actin filaments and regulators
Vesicles: TI-VAMP mediated transport and regulators • Microtubules and regulators • Actin filaments and regulators

• Actin filaments and regulators

Involvement of syntaxin 3 in neurite outgrowth

Involvement of s y ntaxin 3 in neurite outgrowth Darios & Davletov, Nature 2006 Neurite outgrowth

Darios & Davletov, Nature 2006

Neurite outgrowth is impaired in SCG explants from Syt VII-/-mice

outgrowth is impaired in SCG explants from Syt VII-/-mice Arantes & al J. Neurosci . 2006

Arantes & al J. Neurosci. 2006

Copyright ©2006 Society for Neuroscience

Syt VII-/-mice Arantes & al J. Neurosci . 2006 Copyright ©2006 Society for Neuroscience Rao &

Rao & al JBC 2004

Role of exocytosis in neuronal morphogenesis

Basic molecular mechanism mediated by:

• TI-VAMP/VAMP7 as v-SNARE

• Syntaxin 3 as t-SNARE

• Synaptotagmin VII

TIVAMP
TIVAMP
• S yntaxin 3 as t- S NARE • Synaptotagmin VII TIVAMP TI-VAMP/VAMP7 Syntaxin3 Cargo: Neural

TI-VAMP/VAMP7

Syntaxin3

Cargo: Neural cell recognition molecule L1

-L1: member of the immunoglobulin superfamily of cell adhesion molecules

-involved in axonal growth and pathfinding

-mutation in man: MASA- syndrome (mental retardation, spasticity, hydrocephalus)

-KO-mice: malformation of the corticospinal tract

- KO -m i ce: ma lf ormat i on o f the corticospinal tract F.

F. Rathjen http://www.mdc-berlin.de/~devneuro/fgr-intr.htm

L1-CAM is a TI-VAMP’s Cargo

L1-CAM is a TI- V AMP’s Car go Alberts & al MBoC 2003 TI-VAMP: v-SNARE mediating
L1-CAM is a TI- V AMP’s Car go Alberts & al MBoC 2003 TI-VAMP: v-SNARE mediating

Alberts & al MBoC 2003

TI-VAMP: v-SNARE mediating neurite outgrowth

al MBoC 2003 TI-VAMP: v-SNARE mediating neurite outgrowth TI-VAMP Syb/VAMP2 (Coco et al., J.Neurosci.1999) Targeting
TI-VAMP Syb/VAMP2 (Coco et al., J.Neurosci.1999)
TI-VAMP
Syb/VAMP2
(Coco et al., J.Neurosci.1999)
Targeting AP-3 Auto-inhibition LONGIN SNARE TM TI-VAMP 1 120 180 220 SNARE TM Synaptobrevin 2
Targeting
AP-3
Auto-inhibition
LONGIN
SNARE
TM
TI-VAMP
1 120
180
220
SNARE
TM
Synaptobrevin 2

Neurite outgrowth

TeNT

Longin-TIVAMP

Longin-TIVAMP

ARNi

Conclusion: Integration of signaling, actin, and exocytosis in neurite outgrowth

-TI-VAMP is a vesicular SNARE that is necessary for neurite outgrowth -TI-VAMP transports the IgCAM L1. The TI-VAMP dependent membrane trafficking regulates the stability of L1-dependent adhesive contacts -L1 mediated adhesion induces a polarization of TI-VAMP vesicles to sites of contact -The exocytosis of TI-VAMP is positively controlled actin dynamics and cdc42

cdc42 cdc42 cdc42
cdc42
cdc42
cdc42

Philipp Alberts

2. The cytoskeleton and neuronal polarity

controlled actin dynam ics and cdc42 cdc42 cdc42 cdc42 Philipp Alberts 2. The cytoskeleton and neuronal

MICROTUBULES (organising centres and polarity)

MTOC

Centrosome

Centrioles Migrating cell +
Centrioles
Migrating cell
+
+ Basal body Cillia or Flagella +
+
Basal body
Cillia
or Flagella
+
+ - + Spindle poles
+
-
+
Spindle poles
Neurones +
Neurones
+

Mitotic spindle Dividing cell

body Cillia or Flagella + + - + Spindle poles Neurones + Mit o ti c

BBA, 1376:27 (1998)

Ce ll po l ar iza ti on requ i res cap t ure o

Cell polarization requires capture of microtubules at the leading edge

Ce ll po l ar iza ti on requ i res cap t ure o f

Role of microtubules and their regulators

Role of microtubules and their regulators Motors carrying different car goes in different directions fibroblast neuron
Role of microtubules and their regulators Motors carrying different car goes in different directions fibroblast neuron
Role of microtubules and their regulators Motors carrying different car goes in different directions fibroblast neuron

Motors

carrying

different

cargoes

in different

directions

fibroblast neuron
fibroblast
neuron

Anterograde and retrograde transport

Anterograde and retrograde transport Cargo structures Overall   Instantaneous Directionality Duty ratio

Cargo structures

Overall

 

Instantaneous

Directionality

Duty ratio

 

rate

rate (light

   

(pulse

microscopy)

labeling)

 

Golgi-derived

200–400

1–5 µm/s b

Anterograde

High

vesicles

mm/d a (2–

     

(fast anterograde)

5

µm/s)

Endocytic vesicles,

100–250

1–3 µm/s b

Retrograde

High

lysosomes,

mm/d a (1–

     

autophagosomes

3

µm/s)

(fast retrograde)

       

Mitochondria

<70

 

0.3–0.7 µm/s d

Bidirectional

Intermediate

 

mm/d c

     

(<0.8

µm/s)

   

e

     

Microfilaments,

2–8 mm/d

Unknown

Unknown

Unknown

cytosolic protein

(0.02–0.09

     

complexes (slow

µm/s)

 

component b)

 

Microtubules,

0.2–1

 

0.3–1 µm/s f

Bidirectional

Low

neurofilaments (slow component a)

mm/d e

       

(0.002–

 

0.01 µm/s)

Role of kinesins

(slow component a) mm/d e         (0.002–   0.01 µm/s) Role of kinesins
(slow component a) mm/d e         (0.002–   0.01 µm/s) Role of kinesins
(slow component a) mm/d e         (0.002–   0.01 µm/s) Role of kinesins
(slow component a) mm/d e         (0.002–   0.01 µm/s) Role of kinesins

Centrosome localization determines neuronal polarity

Centrosome localization determines neuronal polarity Froylan Calderon de Anda, Giulia Pollarolo, Jorge Santos Da Silva ,

Froylan Calderon de Anda, Giulia Pollarolo, Jorge Santos Da Silva, Paola G. Camoletto, Fabian Feiguin & Carlos G. Dotti Nature (2005)

Centrosome localization determines neuronal polarity

(2005) Centrosome localization determines neuronal polarity Froylan Calderon de Anda, Giulia Pollarolo, Jorge Santos Da

Froylan Calderon de Anda, Giulia Pollarolo, Jorge Santos Da Silva, Paola G. Camoletto, Fabian Feiguin & Carlos G. Dotti Nature (2005)

Microtubule stabilization by CRMP2

In cultured hippocampal neurons, one axon and several dendrites differentiate from a common immature process. Here we found that CRMP-2/TOAD-64/Ulip2/DRP-2 (refs. 2-4) level was higher in growing axons of cultured hippocampal neurons, that overexpression of CRMP-2 in the cells led to the formation of supernumerary axons and that expression of truncated CRMP-2 mutants suppressed the formation of primary axon in a dominant- negative manner. Thus, CRMP-2 seems to be critical in axon induction in hippocampal neurons, thereby establishing and maintaining neuronal polarity.

thereby establishing and maintaining neuronal polarity. Red: actin Green: microtubule Inagaki et al., Nat Neurosci.
thereby establishing and maintaining neuronal polarity. Red: actin Green: microtubule Inagaki et al., Nat Neurosci.

Red: actin

Green: microtubule

maintaining neuronal polarity. Red: actin Green: microtubule Inagaki et al., Nat Neurosci. 2001 Aug;4(8):781-2.

Inagaki et al., Nat Neurosci. 2001 Aug;4(8):781-2.

Conversion of preexisting dendrite to axon by GSK-3 inhibition

Inagaki et al., Nat Neurosci. 2001 Aug;4(8):781-2. Conversion of p reexistin g dendrite to axon by

Model

Model MICROFILAMENTS ACTIN STRUCTURES IN CELLS : MICROVILLI STRESS FIBRES FOCAL ADHESIONS LAMELLIPODIA FILOPODIA (or

MICROFILAMENTS

ACTIN STRUCTURES IN CELLS:

MICROVILLI
MICROVILLI

STRESS FIBRES FOCAL ADHESIONS

LAMELLIPODIA FILOPODIA (or MICROSPIKES

CONTRACTILE RING (cell division)

HPC neuron,

24h

H P C n e u r o n , 2 4 h DNA- blue; µtubules-

DNA- blue; µtubules- green; actin- red

HPC neuron, 3 weeks

H P C n e u r o n , 2 4 h DNA- blue; µtubules-
H P C n e u r o n , 2 4 h DNA- blue; µtubules-
?
?

Actin instability in growth cone

Local perfusion of cytochalasin D onto a growth cone induces it to grow as an axon, indicating that actin destabilization is sufficient for axon formation. These data strengthen the proposed hypothesis that polarized actin-filament instability determines initial neuronal polarization

instability det ermines initial neuronal polarization Red: actin Green: microtubule Bradke F, Dotti CG. Science.
instability det ermines initial neuronal polarization Red: actin Green: microtubule Bradke F, Dotti CG. Science.
instability det ermines initial neuronal polarization Red: actin Green: microtubule Bradke F, Dotti CG. Science.

Red: actin

Green: microtubule

Bradke F, Dotti CG. Science. 1999 Mar 19;283(5409):1931-4.

-The Role of Local Actin Instability in Axon Formation. Frank Bradke and Carlos G. Dotti. (1999) Science 283: 1931-1934

-Establishment of neuronal polarity: lessons from cultured hippocampal neurons, Frank Bradke and Carlos G Dotti (2000). Curr Opin. Neurobiol. 10:

574-581

Actin instability
Actin instability
hippocampal neurons , Frank Bradke and Carlos G Dotti (2000). Curr Opin. Neurobiol . 10: 574
hippocampal neurons , Frank Bradke and Carlos G Dotti (2000). Curr Opin. Neurobiol . 10: 574

Role of the actin cytoskeleton

Role of the actin cytoskeleton The sequential act i v i ty o f t h
Role of the actin cytoskeleton The sequential act i v i ty o f t h
Role of the actin cytoskeleton The sequential act i v i ty o f t h

The sequential activity of the GTPases Rap1B and Cdc42 determines neuronal polarity.

Phalloidine Cdc 42 Rap1b Phalloidine
Phalloidine
Cdc 42
Rap1b
Phalloidine

Schwamborn JC, Puschel AW, Nat Neurosci. (2004) .

The sequential activity of the GTPases Rap1B and Cdc42 determines neuronal polarity. Nat Neurosci. 2004 Schwamborn JC,

Puschel

AW

The establishment of a polarized morphology is an essential step in the differentiation of neurons with a single axon and multiple dendrites. In cultured rat hippocampal neurons, one of several initially indistinguishable neurites is selected to become the axon. Both phosphatidylinositol 3,4,5-trisphosphate and the evolutionarily conserved Par complex (comprising Par3, Par6 and an atypical PKC (aPKC) such as PKClambda or PKCzeta) are involved in axon specification. However, the initial signals that establish cellular asymmetry and the pathways that subsequently translate it into structural changes remain to be elucidated. Here we show that localization of the GTPase Rap1B to the tip of a single neurite is a decisive step in determining which neurite becomes the axon. Using GTPase mutants and RNA interference, we found that Rap1B is necessary and sufficient to initiate the development of axons upstream of Cdc42 and the Par complex.

of axons upst ream o f Cdc42 and the Par complex. The sequential activit y of

The sequential activity of the GTPases Rap1B and Cdc42 determines neuronal polarity. Nat Neurosci. 2004 Schwamborn JC, Puschel AW.

y of the GTPases Rap1B and Cdc42 determines neurona l polar it y. Nat Neurosci. 2004

Neuronal

Polarity:

PI3Kinase

etc…

Neuronal Polarity : PI3Kinase etc… Rho GTPases

Rho GTPases

Neuronal Polarity : PI3Kinase etc… Rho GTPases

4. Inside- out OR Outside-in

?

4 . Inside - out OR Outside - in ? Polarity
4 . Inside - out OR Outside - in ? Polarity
4 . Inside - out OR Outside - in ? Polarity

Polarity

4 . Inside - out OR Outside - in ? Polarity

Inside-Out

Selective sorting of proteins

Inside-Out:

sorting

signals

Inside-Out Selective sorting of proteins Inside-Out: sorting si gna ls
Inside-Out Selective sorting of proteins Inside-Out: sorting si gna ls
Inside-Out Selective sorting of proteins Inside-Out: sorting si gna ls
Inside-Out Selective sorting of proteins Inside-Out: sorting si gna ls

Inside-Out : signals and sorters

Inside-Out : signals and sorters Example of signal: the axonal initial seg ment (AIS) of Dar

Example of signal: the axonal initial segment (AIS) of Dargent & coll.

Inside-Out : signals and sorters Example of signal: the axonal initial seg ment (AIS) of Dar
Inside-Out : signals and sorters Example of signal: the axonal initial seg ment (AIS) of Dar

Selective sorting or selective retrieval?

Selective sorting or selective retrieval? Transcytosis of NgCAM in neurons
Selective sorting or selective retrieval? Transcytosis of NgCAM in neurons

Transcytosis of NgCAM in neurons

Selective sorting or selective retrieval? Transcytosis of NgCAM in neurons

Raft: axonal signals?

Raft: axonal si g nals? Missorting of the axonal Thy-1 but not of a dendritic me

Missorting of the axonal Thy-1 but not of a dendritic membrane protein occurred in sphingolipid-deprived cells. These results indicate that neurons sort a subset of axolemmal proteins by a mechanism that requires the formation of protein-lipid rafts. The involvement of rafts in axonal membrane sorting may explain the neurological deficits observed in patients with certain types of Niemann-Pick disease.

in patients with certain types of Niemann-Pick disease. L e esma, d M ar a i
L e esma, d M ar a i D o ores et a . (
L
e esma,
d
M
ar a
i
D
o ores et a . (
l
l
1998 P
)
roc.
N
at .
l
A
ca
d
.
S
c .
i USA 95 3966 3971
,
-
Copyright ©1998 by the National Academy of Sciences

Outside-In

Cell polarity is regulated by signaling molecules that localize to the leading edge

1. Membrane receptors (GPCRs, RTKs) detect an asymmetric signal from outside the cell

2. Receptors activate Ras-like small G proteins (Rho proteins)

3. Rho proteins induce cytoskeletal changes at the leading (Rac, Cdc42) and trailing (Rho) edges of the cell

IGF1R? IGF-1 receptor is essential for the establishment of hippocampal neuronal polarity Lucas Sosa, Sebastian

IGF1R?

IGF1R? IGF-1 receptor is essential for the establishment of hippocampal neuronal polarity Lucas Sosa, Sebastian Dupraz,
IGF1R? IGF-1 receptor is essential for the establishment of hippocampal neuronal polarity Lucas Sosa, Sebastian Dupraz,

IGF-1 receptor is essential for the establishment of hippocampal neuronal polarity Lucas Sosa, Sebastian Dupraz, Lisandro Laurino, Flavia Bollati, Mariano Bisbal, Alfredo Cáceres, Karl H Pfenninger & Santiago Quiroga Nature Neuroscience 2006

Rho GTPases

Bollati, Mariano Bisbal, Alfredo Cáceres, Karl H Pfenninger & Santiago Quiroga Nature Neuroscience 2006 Rho GTPases
Signalling & outgrowth
Signalling & outgrowth

Signalling & outgrowth

Signalling & outgrowth

Growth, Guidance, Synapse… The END

Growth, Guidance, Synapse… The END Synaptic transmission: communication between neurons
Synaptic transmission: communication between neurons
Synaptic transmission: communication between
neurons

Two principal kinds of synapses: electrical and chemical

Two principal kinds of synapses: electrical and chemical Chemical synapses: the predominant means of communication between

Chemical synapses: the predominant means of communication between neurons

kinds of synapses: electrical and chemical Chemical synapses: the predominant means of communication between neurons
Presynaptic Active Zone

Presynaptic Active Zone

Presynaptic Active Zone
Presynaptic Active Zone
An early experiment to support the neurotransmitter hypoth Criteria that define a neurotransmitter: 1. Must

An early experiment to support the neurotransmitter hypoth

Criteria that define a neurotransmitter:

1. Must be present at presynaptic terminal
2. Must be released by depolarization, Ca ++ -dependent
3. Specific receptors must be present

ynap tic terminal 2. Must be released by depolarization, Ca + + -dependent 3. Specific receptors
ynap tic terminal 2. Must be released by depolarization, Ca + + -dependent 3. Specific receptors

Neurotransmitters may be either small molecules or peptide

Mechanisms and sites of synthesis are different

P ept id es, or p
P
ept
id
es, or
p

Small molecule transmitters are synthesized at terminals, packaged into small clear-core vesicles (often referred to as s na tic vesicles

y

neuropeptides are synthesized in the endoplasmic reticulum and transported to the synapse, sometimes they

are processed along the way. Neuropeptides are packaged in large dense-core vesicles

Neuropeptides are packaged in large dense-core vesicles Neurotransmitter is released in discrete packages, or quanta

Neurotransmitter is released in discrete packages, or quanta

Failure analysis reveals that neurons release many quanta of neurotransmitter when stimulated, that all contribute to the response

individual q
individual
q

Quantal content:

Quantal size:

The number of quanta released by stimulation of the neuron

How size of the

uanta

Quanta correspond to release of individual synaptic vesicles

EM images and biochemistry suggest that a MEPP could be caused by a single vesicle

EM studies revealed correlation between fusion of vesicles with plasma membrane and size of postsynaptic response

EM studies revealed correlation between fusion of vesicles with plasma membrane and size of postsynaptic response
EM studies revealed correlation between fusion of vesicles with plasma membrane and size of postsynaptic response
EM studies revealed correlation between fusion of vesicles with plasma membrane and size of postsynaptic response

4-AP was used to vary the efficiency of release

4- AP was u sed to var y the efficiency of release Calcium influx is necessary

Calcium influx is necessary for neurotransmitter release

var y the efficiency of release Calcium influx is necessary for neurotransmitter re lease Voltage-gated calcium

Voltage-gated

calcium

channels

Calcium influx is sufficient for neurotransmitter release
Calcium influx is sufficient for neurotransmitter release

Synaptic release II

The synaptic vesicle release cycle

1. Tools and Pools

2. Molecular biology and biochemistry of vesicle release:

1. Docking

2. Priming

3. Fusion

3. Recovery and recycling of synaptic vesicles

The synaptic vesicle cycle

The synaptic vesicle cycle How do we study vesicle dynamics? Morphological techniques Electron microscopy to obtain

How do we study vesicle dynamics?

Morphological techniques

Electron microscopy to obtain static pictures of vesicle distribution; TIRFM (total internal reflection fluorescence microscopy) to visualize movement of vesicles close to the membrane

Physiological studies

Chromaffin cells Neuroendocrine cells derived from adrenal medulla with large dense-core vesicles. Can measure membrane fusion (capacitance measurements), or direct release of catecholamine transmitters using carbon fiber electrodes (amperometry)

Neurons Measure release of neurotransmitter from a presynaptic cell by quantifying the response of a postsynaptic cell

Genetics

Delete or overexpress proteins in mice, worms, or flies, and analyze phenotype using the above techniques

Synaptic vesicle release consists of three principal steps:

1. Docking

Docked vesicles lie close to plasma membrane (within 30 nm)

1. Priming

Primed vesicles can be induced to fuse with the plasma membrane by sustained depolarization, high K + , elevated Ca ++ , hypertonic sucrose treatment

2. Fusion

Vesicles fuse with the plasma membrane to release transmitter. Physiologically this occurs near calcium channels, but can be induced experimentally over larger area (see priming). The active zoneis the site of physiological release, and can sometimes be recognized as an electron- dense structure.

Neurotransmitter Release

ys i o l og i ca l release, and can sometimes be recognized as an
ys i o l og i ca l release, and can sometimes be recognized as an
ys i o l og i ca l release, and can sometimes be recognized as an
ys i o l og i ca l release, and can sometimes be recognized as an

Vesicle release requires many proteins on vesicle and plasma membrane

Vesicle release requires many proteins on vesicle and p lasma membrane SNARE s: t arge t
Vesicle release requires many proteins on vesicle and p lasma membrane SNARE s: t arge t
Vesicle release requires many proteins on vesicle and p lasma membrane SNARE s: t arge t
Vesicle release requires many proteins on vesicle and p lasma membrane SNARE s: t arge t

SNAREs: targets of clostridial NTs

Vesicle release requires many proteins on vesicle and p lasma membrane SNARE s: t arge t
Vesicle release requires many proteins on vesicle and p lasma membrane SNARE s: t arge t
Vesicle release requires many proteins on vesicle and p lasma membrane SNARE s: t arge t
Vesicle release requires many proteins on vesicle and p lasma membrane SNARE s: t arge t
SNAREs: tar g ets of clostridial neurotoxins
SNAREs: tar g ets of clostridial neurotoxins
SNAREs: tar g ets of clostridial neurotoxins

SNAREs: targets of clostridial neurotoxins

SNAREs: tar g ets of clostridial neurotoxins
SNAREs: tar g ets of clostridial neurotoxins
SNAREs: tar g ets of clostridial neurotoxins
SNAREs: tar g ets of clostridial neurotoxins

Priming

Vesicles in the reserve pool undergo priming to enter the readily- releasable pool

At a molecular level, priming corresponds to the assembly of the SNARE complex

releasable pool At a molecular level, priming corresponds to the assembly of the SNARE complex The

The SNARE complex

releasable pool At a molecular level, priming corresponds to the assembly of the SNARE complex The
Inhibitory domain, folds back on itself “open” syntaxin doesn ’ t fold properly Synaptotagmin functions

Inhibitory domain, folds back on itself “open” syntaxin doesnt fold properly

Synaptotagmin functions as a calcium sensor, promoting vesicle fusion

“open” syntaxin doesn ’ t fold properly Synaptotagmin functions as a calcium sensor, p romotin g

Calcium & exocytosis

Calcium & exocytosis Regulation by calcium: through synap totagmin?

Regulation by calcium: through synaptotagmin?

Calcium & exocytosis Regulation by calcium: through synap totagmin?
Calcium & exocytosis Regulation by calcium: through synap totagmin?

Mutants of syt

Mutants of syt SNARE et synaptotagmine

SNARE et synaptotagmine

Mutants of syt SNARE et synaptotagmine

Syt accelerates membrane fusion in vitro

Syt accelerates membrane fusion in vitro AnnuaAnnua ll RevRev ii ewsews
Syt accelerates membrane fusion in vitro AnnuaAnnua ll RevRev ii ewsews
Syt accelerates membrane fusion in vitro AnnuaAnnua ll RevRev ii ewsews

AnnuaAnnuall RevReviiewsews

Syt acts through SNAREs and lipids

Syt acts through SNAREs and lipids Regulation by complexin & sy na p tota g min
Syt acts through SNAREs and lipids Regulation by complexin & sy na p tota g min

Regulation by complexin & synaptotagmin

Syt acts through SNAREs and lipids Regulation by complexin & sy na p tota g min
A complexin-tagmin cycle?

A complexin-tagmin cycle?

A complexin-tagmin cycle?
A complexin-tagmin cycle?
Regulation, regulation • Much more is known: Munc - 13 Munc - 18 • Much

Regulation, regulation

Much more is known:

Regulation, regulation • Much more is known: Munc - 13 Munc - 18 • Much more

Munc-13

Munc-18

Regulation, regulation • Much more is known: Munc - 13 Munc - 18 • Much more

Much more to come:

?????

Synaptic vesicles exist in multiple pools within the nerve terminal

(reserve pool)
(reserve pool)

(Release stimulated by flash-photolysis of caged calcium)

Becherer, U, Rettig, J. Cell Tissue Res (2006) 326:393

Morphologically, vesicles are classified as docked or undocked. Docked vesicles are further subdivided into primed and unprimed pools depending on whether they are competent to fuse when cells are treated with high K + , elevated Ca ++ , sustained depolarization, or hypertonic sucrose treatment.

In CNS neurons, vesicles are divided into

Reserve pool (80-95%)

Recycling pool (5-20%)

Readily-releasable pool (0.1-2%; 5-10 synapses per active zone)

Rizzoli, Betz (2005). Nature Reviews Neuroscience 6:57-69)

A small fraction of vesicles (the recycling pool) replenishes the RRP upon mild stimulation. Strong stimulation causes the reserve pool to mobilize and be released

Docking:

UNC-18 (or munc-18) is necessary for vesicle docking

(Weimer et al. 2003, Nature Neuroscience 6:1023)

1. unc-18 mutant C. elegans have neurotransmitter release defect

2. unc-18 mutant C. elegans have reduction of docked vesicles

Unc-18 mutants are defective for evoked and spontaneous release

mutant C. elegans have reduction of docked vesicles Unc-18 mutants are defective for evoked and spontaneous

Unc-18 mutants are defective for calcium-independent release

Unc-18 mutants are defective for calcium-independent release primed vesicles occasionally fuse in the absence of calcium;

primed vesicles occasionally fuse in the absence of calcium; a calcium-independent fusion defect suggests a lack of primed vesicles

UNC-18 (munc18) is required for docking:

unc-18 mutants have fewer docked vesicles

suggests a lack of primed vesicles UNC-18 (munc18) is required for docking: unc- 18 mutants have
suggests a lack of primed vesicles UNC-18 (munc18) is required for docking: unc- 18 mutants have

Summary:

Unc-18 mutants are unable to dock vesicles efficiently. Impaired docking leads to fewer primed vesicles; fewer primed vesicles leads to reduced overall neurotransmitter release.

docking leads to fewer primed vesicles; fewer primed vesicles leads to reduced overall neurotransmitter release .

Synaptic vesicles recycle post-fusion

Synaptic vesicles recycle post-fusion Modern methods to track recycling membrane

Modern methods to track recycling membrane

Synaptic vesicles recycle post-fusion Modern methods to track recycling membrane

Endocytosis retrieves synaptic vesicle membrane and protein from the plasma membrane following fusion

The ATP-ase NSF disassembles the SNARE complex
The ATP-ase NSF disassembles the
SNARE complex