Post on 19-Oct-2020
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.