Stress neuronal et activité électrique dans la sclérose latérale amyotrophique, maladie dégénérative du motoneurone

TC4 2020-F. Scamps, Institut des Neurosciences Montpellier, équipe Motoneurone, frederique.scamps@inserm.fr

(From Taylor, Brown, Cleveland, Nature 2016)

The Components of the Nervous System Impacted in ALS

Progressive Muscular Atrophy

Bulbar ALS

ALS: Fatal Motoneuron Degenerative Disease (death median 3 years)

10-20% familialPrimary Lateral Sclerosis

Lungs

10-20% familial

Modèle murin SOD1G93A

Presymptomatique(UPR, stress.UPS) Asymptomatique

(ER stress)

symptomatique

Un

ité Mo

trice

MOTONEURON SUB-TYPES and ALS

Sprint

Footing

Posture

Spinal cord

ALS

Sele

ctive m

oto

ne

uro

n d

eath

Sele

ctive m

uscle

de

ne

rvation

Slow muscle(soleus, oxidativeType I fiber)

Fast fatigable(tibialis anterior,glycolitic

Type IIB fiber)

Fast fatigue resistant(gastrocnemius, type IIA fiber)

symptomatiquepresymptomatique

Modèle murin SOD1G93A

IDENFICATION OF CELLULAR EVENTS DURING PRESYMPTOMATIC STAGE

Early cellular events in ALS:

Neuromuscular Junction

Motor Unit

Neuromuscular Junction

Early loss of synaptic vesicles (P38-P46) before denervation (P50)

From Pun et al. Nat.Neuro 2006

Neuromuscular junction

(P46)Muscle strength 100%)

SV2 NF160

Pre-synaptic Post-synaptic

Subtype-selective endoplasmic reticulum stress responses

ALS RESISTANT

soleus

ALS VULNERABLE

gastrocnemius

Early cellular events in ALS:

Motoneurone

Retrograde tracing

Although translation for all proteins begins in the cytoplasm, some are moved into the ER in order to be folded and sorted for different destinations

Rough Endoplasmic Reticulum

Smooth Endoplasmic Reticulum

Plays an important role in cholesterol and phospholipid biosynthesis.

Is an important site for the storage and release of calcium in the cell.

Endoplasmic Reticulum

Rough ER stress occurs when the capacity of the ER to fold proteins becomes saturated. ER stress may be caused by factors that impair protein glycosylation or disulfide bond formation, or by overexpression of or mutations in proteins entering the secretory pathway.

Smooth ER stress induced by pharmacologic disruption of ER calcium homeostasis activates UPR signaling pathways. Typically, theseagents cause ER stress by depleting the ER luminal calcium pool, suchas occurs when thapsigargin binds the smooth ER calcium ATPase and inhibits calcium uptake from the cytosol .

ER Stress

Conditions such as high protein demand, viral infection, mutant protein expression, hypoxia, energy deprivation, or exposure to excessive oxidative stress can trigger UPR or ER stress

Ubiquitination, BiP (binding immunoglobulin protein or HSPA5): dégradation des protéines

1. Endoplasmic reticulum/ER stress in ALS

Saxena et al Nat Neurosci 2009

From P12 (early) spinal cord of ALS mice:

. Upregulation of genes involved in stress-related pathways (Bip, ERO1alpha). Lost at P30

2. Unfolding Protein Response (UPR) promotes cellular survival in response to stress

3. UPR can initiate apoptosis under conditions of chronic stress

ER stress activates three main pathways of the UPR, mediated by the ER transmembraneproteins inositol‐requiring kinase 1 (IRE1), protein kinase RNA‐activated (PKR)‐like ER kinase (PERK), and activating transcription factor 6 (ATF6)

The 3 arms of the UPR (Unfolding Protein Responseis an ER stress response)

From Walker & Atkin, 2011

ERAD: ER-associated degradation

CHOP: pro-apoptotic transcription factor

UPS: Ubiquitin proteasome system

1. Increase folding capacity2. Inhibit general protein translation (reduced ER load)

eIF2alpha: eukariotic translation initiation factor 2

From P30 spinal cord from ALS mice:

. Upregulation of UPR-related genes& . Downregulation of ubiquitinproteasome system (UPS)-relatedgenes

Saxena et al Nat Neurosci 2009

Adaptive cell responses-neuroprotection

ALS mouse SOD1G93A Forced expression of ATF3 in motor neurons of transgenic SOD1G93A ALS mice

(Seijffers et al. PNAS 2013)

delays neuromuscular junction denervation by inducing axonal sprouting and enhancing motor neuron viability.

ATF4 silencing depresses MN survival

Increasing EIF2α phosphorylation confers MN protection

(Kiskinis et al. Cell Stem Cells, 2014)

iPSC from ALS patients

Adaptive cell responses-neuroprotection

•The ER is a membrane-enclosed organelle present in all eukaryotic cells that

serves to fold proteins destined for secretion or membrane insertion, synthesizes

lipids and sterols, and stores calcium.

•The UPR consists of molecular signal transduction pathways that detect

disturbances in the ER, such as misfolded proteins, and that determines whether a

cell survives or dies in response to the stress.

•UPR protective signaling results from the enhancement of ER protein-folding

capacity, degradation of misfolded ER proteins, and attenuation of translation.

UPR proapoptotic signaling is thought to involve the production of the CHOP

transcription factor, activation of the ASK1 and JNK kinases, and prolonged

inhibition of protein synthesis.

•Loss of UPR protective signaling may underlie the cell death in neurodegeneration

that causes ER stress.

The aggregating proteins in neurodegenerative disease do typically not accumulate in the ER and many of them do not enter the ER at any stage in their life cycle.

These findings raise the question of the source of ER stress that activates UPR signaling in this disease.

The precise upstream cause of UPR induction in both mutant SOD1‐linked and other forms of ALS remains to be ascertained, and whether ER stress is an upstream cause of disease processes remains unknown.

ER stress, vulnerability and electrical activity

Electrical Activity : Molecular Basis for Disease Selectivity ?

Functional basis for ALS ?

Phenotypic traits genetically correlated to ALS:

Education, Physical activity, Smoking,

Frequency of tenseness/restlesness

(Shared polygenic risk and causal inferences in ALS: The International ALS Genomics Consortium. Annals of Neurology 2019)

iPSC from SOD1A4V, C9orf72 and Fus patientsare hyperexcitable compared with control

(Wainger et al, Cell reports 2014)

Retigabine reduces motoneuron excitability and increases survival

(Wainger et al. 2014)

Retigabine: K+ channel activator*induces membrane hyperpolarization

*reduces electrical activity

Salubrinal , specific inhibitor of phosphatase that dephosphorylates pEIF2α, increases MN survival

(Kiskinis et al 2014, Cell Stem Cell)

Inherent ER stress in iPSC motoneurons (sXBP1) is alleviated by blocking actions potentials with TTX. Increasing electrical activity, increases XBP1.Vice versa, activation of stress response pathways with salubrinalreduces spontaneous activity in motoneurons.

Model: C9ORF72 iPSC (Selvaraj et al. Nat Comm 2018)

Hyperexcitability due to AMPAR induced excitotoxicity

GluA1 GluA2 GluA3 GluA4

C9-ALScontrol

post-mortem spinal cord of C9-ALS patient

Excitotoxicity increases ER stress,,,,

Expanded GGGGCC hexanucleotide repeat in non-coding region of C9ORF72 ALS & Frontotemporal dementia

Hypothesis of Enhanced MN vulnerability to glutamate-mediated excitotoxicityleading to intracellular Ca2+ overload

Con

C9

C9Δ

No change in MN intrinsic excitability

Novel therapies in development that inhibit motor

neuron hyperexcitability in amyotrophic lateral

Sclerosis

Yu-ichi Noto, Kazumoto Shibuya, Steve Vucic & Matthew C. Kiernan

Expert Review of Neurotherapeutics 2016

From Saxena & Caroni Neuron 2011

Electrical properties and motoneuron subtypes

MOTONEURON SUB-TYPES and ALS

Sprint

Footing

Posture

Spinal cord

ALS

Sele

ctive m

oto

ne

uro

n d

eath

Sele

ctive m

uscle

de

ne

rvation

Slow muscle(soleus, oxidativeType I fiber)

Fast fatigable(tibialis anteriorType IIB fiber)

Fast fatigue resistant(gastrocnemius, type IIA fiber)

MOTONEURON ELECTRICAL ACTIVITY: Excitability Threshold

P7Slice recordings

Putative S Putative FR Putative FF

SprintFootingPosture

Low Rheobase(muscular tone)

High Rheobase(exercise)

0

500

1000

1500

RB

(pA

)

S FR FF

Medium Rheobase(endurance)

α FR α FF

I = 800 pA type 3 I = 1800 pA

RB: Intrinsic properties

I(pA): Excitatory Synaptic inputsionotropic

Neuromodulation: metabotropic

I(pA )

Fre

quency

(Hz)

0 500 1000 1500 20000

10

20

30

40

50

Posture

Footing

Sprint

RB =

∆Vm

threshold current

MOTONEURON ELECTRICAL ACTIVITY: Firing Properties

Muscarinic Modulation of Firing Frequency

Sprint

Footing

Posture

f-I slo

pe incre

ase (

%)

0

50

100

150

ns

****

**

+ oxotremorine

Cholinergic C-bouton synapses (VAChT):

MOTONEURON ELECTRICAL ACTIVITY: Neuromodulation

VAChT

+ oxotremorine

* Oxotremorine enhances BiP signals in MN, enhanced UPR in FF MN, no effect on misfSOD1 accumulation

* Methoctramine reduces ER stress, delayed UPR, increases and spreads of misfSOD1 accumulation in MN

Dissociation between the accumulation of misfSOD1 and ER stress in FALS MNs(similar conclusion with iPSC which have ER stress and no SOD1 aggregation (Kiskinis et al.

and in agreement with cytoplasmic SOD1 protein, effect of soluble mutant SOD1?)

Saxena et al. Neuron 2013: Neuroprotection through excitability and mTOR required in ALS motoneurons….

Associated with FF motoneurons Excitability

TMEM16F, Ca-activated Cl current, is facing C-bouton

0

400

800

1200

ns

S

RB

(p

A)

Tmem

16f-/-

WT

ns

Tmem

16f-/-

WT

*FF

Tmem

16f-/-

WT

Contributes to rheobase

Soulard et al. Cell Rep 2020

0

5

10

15

20

25

**

ns

Runnin

g tim

e (

min

)

ns

Locomotor Behavior

endurance

CONTRIBUTION OF TMEM16F TO ALS PATHOGENESIS

SymptomaticPré-symptomaticBirth Onset Death

P0 105 150

SOD1G93A

80 120 160

50

0

119 d**

Male

Age (postnatal, d)

100

105 d

Pro

babili

ty o

f onset

Age (postnatal, d)

120 160 2000

20

40

60

80

100

166 d

145 d

ns

Pro

babili

ty o

f surv

ival

Delayed Onset Improved Strength

Tmem16f-/-

X

Survival

ATF3 ChAT

ventral horn tibialis anterior

BTX

NF/SV2

0

5

10

15

AT

F3

+ / C

hA

T+

neuro

ns (

%) **

Innerv

ate

d N

MJ (

%)

0

20

40

60

80

100

*

Decreased Stress Marker Expression Preserved Innervation

FF motoneurons Excitability

is a Contributing Factor

to Motoneuron Stress

& ALS Progression

From Soulard et al. Cell Rep 2020

100 pA25 pAstep current step current

S-motoneuron FF-motoneuron

1

23

RB

RB

tip electrode

Disease-VulnerableDisease-Resistant

Large Scale Analysis of Vulnerability

Mechanisms involved in ALS pathogenesis.

Many different processes selectively affect motor neurons, which are influenced by surrounding presynaptic neurons, astrocytes and microglia, in disease. These processes include: induction of ER stress; aggregation of misfolded proteins and formation of intracellular inclusions; macroautophagy; oxidative and nitrosative stress; excitotoxicity mediated by over‐stimulation of postsynaptic glutamate receptors; redistribution of TDP‐43 and FUS from the nucleus to the cytoplasm; fragmentation of the Golgi apparatus; dysfunction of mitochondria and activation of mitochondrial apoptotic pathways; inhibition of microtubule‐based dynein‐mediated intracellular and axonal transport, and; inhibition of the ubiquitin‐proteasome system.