1-s2.0-S2589004226009405-main-html.html

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Neuromuscular Junction Innervation and Motor Function Are Preserved by Restoring

Muscarinic Signaling in Perisynaptic Glia in ALS

Elsa Tremblay, Danielle Arbour, Joanne Vallée, Roberta Piovesana, Geneviève

Vallières, Emine Mechichi, Richard Robitaille
PII:

S2589-0042(26)00940-5

DOI:

https://doi.org/10.1016/j.isci.2026.115565

Reference:

ISCI 115565

To appear in:

iScience

Received Date: 10 February 2025
Revised Date: 19 December 2025
Accepted Date: 30 March 2026

Please cite this article as: Tremblay, E., Arbour, D., Vallée, J., Piovesana, R., Vallières, G., Mechichi,

E., Robitaille, R., Neuromuscular Junction Innervation and Motor Function Are Preserved by Restoring

Muscarinic Signaling in Perisynaptic Glia in ALS,

iScience

(2026), doi:

https://doi.org/10.1016/

j.isci.2026.115565

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Neuromuscular Junction Innervation and Motor Function Are Preserved by Restoring

Muscarinic Signaling in Perisynaptic Glia in ALS

Elsa Tremblay

1,2,3

, Danielle Arbour

1,2,3,4

, Joanne Vallée

, Roberta Piovesana

1,2,3,4

, Geneviève

Vallières

1,2,3,4

, Emine Mechichi

, Richard Robitaille*

1,2,3,4

1. Université de Montréal, Département de Neurosciences, Montréal, Canada

2. Centre Interdisciplinaire de Recherche sur le Cerveau et l’Apprentissage (CIRCA)

3. Groupe de recherche sur la signalisation neurale et la circuiterie (GRSNC)

4. Institut Courtois d’innovation biomédicale, Faculté de médecine, Université de Montréal

5. Université de Montréal, Faculté de l’apprentissage continu, Montréal, Canada

Lead contact: Richard Robitaille (richard.robitaille@umontreal.ca).

* Address for correspondence:

Richard Robitaille

Département de neurosciences

Université de Montréal

PO box 6128, station « centre-ville »

Montreal, Canada, H3C 3J7

Email: richard.robitaille@umontreal.ca

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SUMMARY

Neuromuscular junction (NMJ) denervation is an early pathological event in amyotrophic lateral

sclerosis (ALS) causing motor dysfunction and paralysis. Glial cells at the NMJ, perisynaptic Schwann

cells (PSCs), ensure a balance between maintenance and repair via muscarinic receptor signaling. However,

in ALS mouse models, PSCs show an aberrant muscarinic hyperactivation. We posited that this excessive

activation impairs the PSC capacity to support NMJ repair in ALS. Beginning at symptoms onset,

SOD1

G37R

mice received daily oral administration of darifenacin, a clinically approved type 3 muscarinic receptor

antagonist, to reduce PSC hyperactivation. The treatment improved locomotion and preserved NMJ

innervation in male mice, with comparable effects observed in females, and extended survival in males.

Functional benefits were supported by signs of glial repair and enhanced survival of lumbar motor neurons.

These preclinical data indicate that pathological PSC hyperactivity contributes to NMJ denervation in ALS

and support therapeutic strategies targeting NMJs in ALS.

KEYWORDS

Neuromuscular junction, glial cells, perisynaptic Schwann cells, M3 muscarinic receptors, amyotrophic

lateral sclerosis, preclinical study, therapeutic strategy

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INTRODUCTION

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder which causes the

progressive loss of upper and lower motor neurons (MNs), leading to gradual paralysis and death in 2 to 5

years. Neuromuscular junction (NMJ) denervation is a hallmark of ALS that starts before clinical

symptoms, precedes the death of MNs

1-4

and is observed in patients

1,5

and in numerous ALS mouse models

2,4,6-12

. NMJs undergo multiple cycles of denervation and reinnervation in males and females before the final

loss of MNs

, with a larger axonal sprouting in females

. This highlights NMJ remodeling before and

during the symptomatic stage of the disease. Importantly, it was shown that rescue of neuronal loss does

not improve NMJ innervation and motor function, and consequently the prevention of MN loss alone failed

to significantly delay disease onset and progression

14-17

. These observations point to the importance of

early and long-lasting local NMJ events in ALS and emphasize the relevance for therapeutic interventions

at the NMJ.

ALS is a non-cell autonomous disease, with significant involvement of glial cells in disease

pathogenesis and progression

18-21

. Glial cells at the NMJ, perisynaptic Schwann cells (PSCs), are essential

for its maintenance and repair. Juxtaposed on top of the nerve terminal, surrounding the synaptic cleft, PSCs

are important synaptic partners of the NMJ

as they detect synaptic activity through activation of their

muscarinic and purinergic receptors. Activation of PSC muscarinic receptors leads to a Ca

rise

12,22-25

triggering their modulation of synaptic transmission

23,26

. PSCs muscarinic receptors also regulate NMJ

stability and repair

27,28

whereby muscarinic signaling is decreased following NMJ injury and denervation,

leading to gene regulation necessary for NMJ repair

. PSCs then extend fine processes that support nerve

terminal sprouting towards denervated NMJs

28,30-32

. Hence, muscarinic receptors act as a master switch for

PSCs such that a balanced level of activation leads PSCs into a maintenance mode to support NMJ

functions, while a low muscarinic activity leads PSCs to switch to a repair mode. Our previous work

demonstrated that PSCs have a high muscarinic excitability observed throughout disease progression

observed in

SOD1

G37R

mice

12,33

even at denervated NMJs

, and at disease onset of

Profilin-1

mice

(

PrP-

hPFN1

G118V

)

(Figure S1). Importantly, many reported PSCs malfunctions in ALS, including phagocytic

defects and a compromised repair capacity, that are all under the tight regulation by the muscarinic receptor

activity

Hence, owing to their key regulation of PSCs functions, we postulate that the muscarinic hyper-

excitability seen throughout disease progression could prevent PSCs from performing their normal

functions, leading to NMJ instability and denervation in ALS. This could be an important cause of NMJ

deficits and disease pathogenesis and progression. We therefore posit that dampening the muscarinic

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activity of PSCs will help maintain NMJ innervation, and ultimately maintain motor functions and survival,

offering a promising therapeutic strategy to target PSCs and the NMJ in ALS.

To this end, we performed a preclinical trial study using darifenacin, a highly selective type 3

muscarinic ACh receptor (M3) antagonist currently used for the treatment of overactive bladder

34-37

. Using

SOD1

G37R

mice, we found that chronic treatment with darifenacin started at symptoms onset improved

behavioral and locomotor function, and survival in male mice. This was associated with improved

neuromuscular function, NMJ innervation and lumbar motor neuron survival. Comparable impacts on NMJ

innervation and locomotion were observed in female mice. Our strategy represents an innovative and

accessible therapy for ALS, pointing towards NMJ preservation as a relevant therapeutic target that could

help improve motor function and the quality of life of ALS patients.

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RESULTS

To determine the contribution of the glial muscarinic signaling to ALS pathogenesis, we performed a

preclinical testing of darifenacin, a highly selective muscarinic M3 receptor antagonist

34,37

. In addition to

its high selectivity, darifenacin was chosen for its safety and tolerability profile in humans

34-37

and very

low blood-brain-barrier permeability which limit potential detrimental effects, including central nervous

system (CNS) side effects

38,39

Darifenacin reduces PSCs muscarinic activation

To evaluate the efficacy and selectivity of darifenacin, we measured PSCs activity using Ca

imaging as a reporter of the mAChRs activation, first in

ex vivo

Soleus

(SOL) preparations, then

in vivo

both WT and

SOD1

G37

. Bath -applications of doses ranging from 50 nM to 1000 nM completely blocked

responses following mAChRs activation by local application of the agonist muscarine (10 µM) (Figure

S2), demonstrating that darifenacin is a very potent blocker of PSCs activation. Efficacy of

in vivo

treatment

with darifenacin for 1 week in

SOD1

G37R

mice showed that an oral dose of 10 mg/kg daily reduced

ex vivo

responses amplitude, resulting in the required partial blockade (Figure S3).

In vivo

target engagement was then confirmed using this dosage (10 mg/kg daily for 1 week with

either vehicle or darifenacin). We observed a 50% reduction of

ex vivo

response amplitude elicited by

application of muscarine (10 μM) in darifenacin-treated mice compared to vehicle-treated mice (Figure 1a,

b’, b’’, c). Interestingly, large and small PSCs Ca

responses were observed on the same NMJs following

darifenacin treatment, consistent with PSCs intrinsic heterogeneity

, and indicative that all NMJs had

PSCs that were altered by the darifenacin treatment. Furthermore, darifenacin treatment did not alter the

other main receptor system driving PSCs activity since PSCs purinergic responses elicited by local

application of ATP were unchanged (10 µM; Figure 1d’, d’’, e) Together, these experiments show that M3

receptors are significant activators of PSCs and validate darifenacin as an efficient strategy to dampen PSCs

muscarinic activity

in vivo

Darifenacin improves locomotor function and gait

We first investigated the functional impact of darifenacin treatment on locomotor behavior. We

treated a cohort of

SOD1

G37R

mice from disease onset (P440-460) and performed behavioral tests and

measurements (grip strength, rotarod, gait and body weight).

Using the grip strength test we observed that darifenacin treatment improved the overall strength

of fore- and hindlimbs of the animals (Figure 2a). Indeed, while mice of the darifenacin and vehicle-treated

groups began the trial with a similar grip strength force, the darifenacin-treated mice performed better than

the vehicle-treated group from P490 until the end of the preclinical trial (Figure 2a).

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Darifenacin treatment also improved the motor performance, balance and coordination. Using a

standard acceleration protocol on the Rotarod, we observed that the performance of darifenacin-treated and

vehicle-treated mice progressively declined, demonstrating the expected progression of ALS motor

phenotype (Figure 2b). However, the motor performance of darifenacin-treated mice was maintained at

higher levels compared to vehicle-treated mice during the late phase of the disease, at age P520 and P525

(Figure 2b). In fact, at this age, most of the vehicle-treated mice were no longer able to perform adequately

the test with a mean latency to fall of 8.53 ± 2.49 seconds while more than half of the darifenacin-treated

group were still able to stand for several seconds on the rotating rod, with a mean of 18.74 ± 3.49 seconds

(Figure 2b).

The improved motor performance was further confirmed by a gait analysis. We quantified, at

the end of the treatment, stride length and step width that are gradually reduced during disease progression

as gait becomes impaired

(Figure 2c, d). Stride length and step width were significantly increased for

mice treated with darifenacin compared to the vehicle-treated ones at P520 (Figure 2c, d).

Darifenacin slows disease progression and extends mice survival

Finally, we monitored the changes in the mice body weight, a metric that is directly related to the

progression of the disease and survival (Figure S4). As shown in Figure 3a, body weight loss at P520 was

significantly reduced in the darifenacin-treated group compared to the vehicle-treated mice.

The impact of darifenacin on disease progression was determined by comparing the neurological

score (NS; Figure S4) of the mice at the time of sacrifice (Figure 3b). The NS of darifenacin-treated mice

were substantially less advanced than the control group, with a median NS of 3 for treated animals and a

median NS of 4 for vehicle-treated animals (Figure 3b), despite having the same mean age of onset. Ten

(10) animals out of 13 (77%) presented a NS of 2 or 3 at P520, compared to only 7 out of 17 (41%) in

vehicle-treated mice. Hence, darifenacin slowed down disease progression in

SOD1

G37R

mice. Consistent

with this observation, mice treated with darifenacin were still active, exploring their environment while the

vehicle-treated mice mostly showed a severe hindlimb paralysis at the endpoint (Video S1, video S2).

Importantly, we treated a small cohort of female animals and confirmed that disease onset occurred

at a similar age in females and males, and that chronic treatment was well tolerated in females. Disease

progression under treatment was comparable between sexes, with no significant differences in grip strength

decline, weight loss, NS and gait impairment at sacrifice, or treatment duration (Figure S5a-g; Mann-

Whitney test, p > 0.05).

Next, we tested if the treatment with darifenacin increased survival. We initiated a survival cohort

that received the same darifenacin treatment regimen that was continued until the end stage, corresponding

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to the level 5 of the disease (Figure S4). Treatment with darifenacin significantly extended survival from

disease onset by 23%, with a mean of 22.44 ± 10.49 days (Figure 3c). An additional analysis indicated a

large effect size (Cohen’s

= 0.95; 95% CI, 0.02–1.82), consistent with a marked difference in survival

between darifenacin-treated and vehicle-treated mice.

Darifenacin improves EDL muscle force, NMJ efficacy and fatigue properties

Owing to the improved locomotor behavior and enhanced survival by the darifenacin chronic

treatment, we then tested if these outcomes were associated with preserved neuromuscular functions at age

of sacrifice (P520). Following chronic treatment with darifenacin (10 mg/kg), using a force transducer

(Figure 4a, b), we observed at P520 that stimulation of the motor nerve and NMJ activation generated

significantly higher mean twitch forces from the darifenacin-treated group (139.3 ± 9.2 mN) than from the

vehicle-treated group (80.1 ± 4.6 mN) in EDL. These values represent respectively 70% and 41% of the

WT mean twitch force value (196.3 ± 12.9 mN). There was a significant interaction effect between

frequency of stimulation and treatment, and darifenacin-treated mice were significantly distinct from

vehicle-treated from 60 Hz to 300 Hz (Figure 4b). A similar pattern was observed with direct muscle

stimulation, with a significant interaction between treatment group and stimulation frequency (Figure 4c).

Vehicle-treated

SOD1

G37R

mice generated lower force than WT mice across all frequencies, whereas

darifenacin-treated mice were again not significantly different from WT mice at higher frequencies (Figure

4c). For direct muscle stimulation, significant differences between darifenacin-treated and vehicle-treated

SOD1

G37R

mice were detected at higher stimulation frequencies (140–300 Hz), indicating preservation of

fast-twitch properties of the EDL muscle (Figure 4c).

We next determined the NMJ contractile capacity

which is the proportion of the whole muscle

force capacity that is used specifically by the neuromuscular system upon nerve stimulation (normally

100% in a WT).

We posit that darifenacin should improve the contractile capacity by improving contractile

force generated following nerve and muscle stimulation. Indeed, the NMJ contractile capacity was

significantly higher in darifenacin-treated mice, with 86.1 ± 2.8% compared to only 55.7 ± 7.8% for vehicle-

treated mice (Figure 4d). Interestingly, we also observed a better preservation of the EDL muscle weight in

treated animals (Figure 4e).

In addition to the large contractile twitch force generated, fatigable fast-twitch muscles like the

EDL are also characterized by a high level of fatigability

. In ALS, alterations of NMJ innervation with

compensatory motor units reinnervating fibers alter the muscle fatigue properties, paradoxically rendering

them more resistant to fatigue

42-44

. Since the treatment by darifenacin preserved muscle strength, we posit

that darifenacin would also preserve the normal fatigue properties of the EDL.

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Following the high frequency fatigue protocol at 120 Hz (Figure 4f), while force generated at the

beginning of the protocol was higher in darifenacin-treated than vehicle-treated mice following nerve

stimulation (Figure 4h), there was no difference in the rate of nerve-evoked muscle fatigue between vehicle-

treated and darifenacin-treated mice. However, only vehicle-treated mice were significantly less fatigable

than WT muscles at the end of the fatigue protocol (Figure 4h, i). In addition, direct muscle stimulation was

superimposed (every 10

stimulations) to the nerve stimulation to evaluate muscle fatigue during the

protocol. As can be seen in figure 4j, k, direct muscle stimulation in WT mice initially elicited an elevated

contractile force but, by the end of the protocol, EDL WT muscles showed a typical reduction in force

amplitude, probably due to their full recruitment by repeated nerve stimulations and their high fatigability

(44.7% decrease of force, 55.3 ± 2.7% of the baseline force). In contrast, only a minimal reduction in force

was observed in the EDL of vehicle-treated

SOD1

G37R

mice (23% decrease in force; 76.6 ± 4.7% of

baseline), and these values were significantly different from those of WT mice (Figure 4j, k).

This reduced

fatigue likely reflects impaired neuromuscular transmission and the lower force generated by vehicle-

treated muscles during repeated nerve stimulation throughout the protocol. Importantly, however,

darifenacin-treated muscles developed a larger decrease in force (38.6% decrease of force, 61.4 ± 2.8% of

the baseline force) than the vehicle-treated ones following nerve stimulation, but were not different from

WT mice (Figure 4i, j).

To further assess neurotransmission (NT) failure during each train—representing the component

of fatigue associated with denervation and NMJ fatigue—we subtracted the fatigue observed during direct

muscle stimulation from that measured during nerve stimulation, normalized to the initial maximal force

obtained with combined nerve and muscle stimulation. This difference was then expressed for each train as

a percentage of the initial maximal muscle force at the start of the train

43,45

. Analysis revealed a significant

treatment × time interaction: NT failure was significantly increased in the vehicle-treated group compared

with WT vehicle–treated and darifenacin-treated mice up to 44 s of the protocol (Figure 4g). This early

increase in NT failure likely reflects a higher rate of denervation or NMJ deficits in vehicle-treated mice

compared with darifenacin-treated mice. However, there was no significant difference between WT

vehicle-treated and darifenacin-treated mice regarding NT failure (Figure 4g).

Darifenacin preserves SOL contractile force and nerve fatigue properties

Darifenacin had no significant impact on the more resistant slow-twitch SOL muscle when

parameters were analyzed at P520 (Figure S6). However, owing to the SOL slower denervation rate in ALS

, we posit that a more significant readout would be obtained at the end stage. At this age, darifenacin-

treated SOL force generated tended to be higher than vehicle-treated mice (Figure 5a, b), with more strength

generated by direct muscle stimulation in darifenacin-treated than vehicle-treated mice, at 50 Hz and 80 Hz

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(Figure 5e-f). Moreover, we observed that the nerve-evoked maximal contractile force (80 Hz, 500 ms) was

not significantly reduced between P520 and end stage in darifenacin-treated SOL (loss of 24.9%, Figure

5c), while it was significantly reduced in vehicle-treated mice (reduction of 40,6%). The same phenomenon

was observed for muscle stimulation (Figure 5d). These results indicate that darifenacin slowed down the

deterioration of the SOL muscle and showed efficiency at a later disease stage on a more resistant muscle.

In addition, SOL from darifenacin-treated mice presented the same level of fatigue normally

observed in SOL of WT mice (72% fatigue at the end of the protocol, 27.7 ± 3.2% from baseline) (Figure

5g, h). Baseline contractile force at the end of the protocol was 33.8 ± 2.1% for vehicle-treated mice (66.2%

fatigue), which was significantly different from the darifenacin-treated mice (75.2% fatigue, 24.8 ± 2.0%

from baseline) (Figure 5g, h). Muscle force after direct muscle stimulation (every 10 stimulations), which

is independent from the recruitment of the NMJ, was not different between all groups (Figure 5h).

Darifenacin treatment improves innervation of NMJs of vulnerable motor units

We next tested the possibility that the improvement of motor behavior and neuromuscular function

by darifenacin underlies enhanced NMJ innervation, structure and integrity. Indeed, dampening PSCs

excitability with darifenacin should restore PSCs balanced regulation of NMJs, thus improving and/or

maintaining NMJ innervation. Consistent with our hypothesis, darifenacin had a global beneficial impact

on NMJ innervation of EDL muscles, and the proportion of fully innervated NMJs was higher in

darifenacin-treated in comparison to vehicle-treated mice (Figure 6a, b). In addition, the number of

denervated NMJs was lower in darifenacin-treated compared to vehicle-treated animals (Figure 6b, c).

There was no change in the number of partially innervated NMJs in the darifenacin-treated vs vehicle-

treated animals (Figure 6b). Similar results were obtained in darifenacin-treated females where a high level

of fully and partially innervated NMJs (80.03 ± 5.95%) and a low percentage of denervated NMJs (19.97

± 5.95%) (Figure S5h-i; n = 209, N = 3) were observed.

Darifenacin increases signs of NMJ repair

The improved innervation observed in the EDL is consistent with our initial hypothesis that

dampening PSCs excitability with darifenacin should restore PSCs ability to engage in NMJ repair as shown

by the presence of glial processes

9,12

and improve or maintain innervation. To test for the presence of glial

signs of repair, we focused on the presence of PSCs processes that are extended upon NMJ impairment (29,

30), driving nerve terminal sprouting triggered by these glial extensions.

As shown in Figure 6, there was a significant increase in NMJs presenting nerve sprouting profiles

(see Figure 6d) extended on PSCs processes in EDL muscles from darifenacin-treated mice, which is

indicative of NMJ remodeling (Figure 6e, f). There was no significant difference in the number of glial

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processes alone in the darifenacin-treated in comparison to vehicle, suggesting that most processes were

occupied by nerve terminal sprouts, indicative of effective glial guidance mechanisms (Figure 6e, f).

Finally, polyinnervation, which represents another sign of NMJ repair, was unchanged between the

darifenacin-treated and vehicle-treated animals (Figure 6e). Abundant signs of repair were also observed at

EDL NMJs of female darifenacin-treated mice (Figure S5h, j; n = 209, N = 3). Nerve sprouting was frequent

and even more prominent in females than in males (34.39 ± 1.29%), supporting the conclusion that

darifenacin treatment is associated with elevated repair signs in the EDL muscle, comparable to the effect

observed in males.

Consistent with our observations on the functional impact of darifenacin on the SOL, NMJ

innervation of SOL was moderately affected and presented a better preserved NMJ innervation at end stage

(Figure 7a). While fully innervated NMJs tended to be abundant in darifenacin-treated SOL, there was no

statistical difference between vehicle-treated (59.4 ± 5.5%) and darifenacin-treated (73.62 ± 5.8%) SOL

muscles (Figure 7a; vehicle, N = 9; darifenacin, N = 8; p = 0.0958, unpaired

-test). The proportion of

denervated NMJs was less than 15% and similar in both groups (Figure 7a).

Importantly, however, we observed abundant repair signs as there was significantly more nerve

sprouting events in darifenacin-treated SOL compared to the vehicle-treated (Figure 7b, c). Presence of

polyinnervation and glial process extensions without nerve terminal were unchanged between the two

groups (Figure 7b). These data suggest that darifenacin facilitated NMJ repair at the SOL, further supporting

the positive impact of the darifenacin treatment on NMJs of the SOL.

Darifenacin increases motor neuron survival and reduces microglial reactivity

Even though darifenacin poorly crosses the blood-brain barrier, we posit that it could have a

positive impact on MN survival by stabilizing and preserving NMJ innervation and limiting the detrimental

stress that the dynamic cycle of denervation – reinnervation imposes on MN

. As shown in Figure 8 (a, b,

b’), darifenacin-treated animals presented twice as many MNs in the lumbar segments 4 – 6 (L4–L6) and

in the ventral horn as a whole than the vehicle group, indicating that darifenacin treatment improved MN

survival in the lumbar spinal cord. In addition, enhanced MN survival was accompanied by a reduced

microglial reactivity as indicated by a significant reduction of Iba1 intensity labelling in L4-L6 (Figure 8c,

d’) and the lumbar spinal cord (Fig 8c, d) whereas GFAP intensity was unchanged (Figure 8e, e’).

Consistent with the reduced microglial activity, the levels of the a

ctivating transcription factor 4 (

ATF4),

as a reporter of cellular stress response

47,48

, were no longer different from WT after darifenacin treatment

whereas ATF4 levels in vehicle-treated mice differed from those in WT mice (Figure 8f).

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DISCUSSION

In this study, we report a beneficial impact of the chronic treatment with the M3 antagonist

darifenacin on several key parameters related to ALS disease progression, highlighting the clinical

relevance of this strategy. Our work was based on the innovative approach that restoring muscarinic-driven

PSCs functions would re-establish their balanced regulation of NMJ maintenance and repair

12,28

, thus

improving NMJ innervation, leading to improvement of nerve and muscle functions, MN survival and

consequently general motor function and survival in ALS.

Glial-driven improvement of NMJ innervation: maintenance or repair?

Consistent with our main hypothesis, our results show that restoring PSCs muscarinic balanced

regulation facilitated NMJ innervation by recovering PSCs contribution to NMJ maintenance and repair.

Our results indicate that NMJ innervation can be targeted to positively impact motor functions in ALS.

Considering that the early NMJ loss in ALS

2,6,7,10,11,49-52

is a dynamic process with cycles of asynchronous

and contemporaneous denervation and reinnervation

2,52

, there are two major scenarios through which

darifenacin regulation of PSCs can lead to a stronger and extended NMJ innervation. First, it could improve

PSCs plasticity, facilitating reinnervation of NMJs during the asynchronous dismantlement of NMJs

occurring during disease progression. Second, darifenacin could stabilize NMJs, reducing their denervation

rate and preserving them for a longer period. Several lines of evidence of our results suggest that both

mechanisms were at play to favor NMJ innervation.

First, as a sign of target engagement and consistent with the restoration of PSCs functions, we

observed that darifenacin treatment reduced aberrant PSC hyper-muscarinic response

2,12

leading to

increased nerve sprouting events that are associated with NMJ repair (Figure 6). This result argues in favor

of the PSC repair mode and their NMJ remodeling phenotype

27,53

. Furthermore, nerve sprouting only

occurs when PSCs first extends processes following denervation

54,55

, towards an innervated NMJ

allowing nerve sprouts to contact the denervated NMJ. In ALS, although PSCs process extension occurs, it

is mostly off-target

and poorly supports nerve sprouting to achieve reinnervation

In addition, EDL is

composed mainly of fast fatigable motor units known for their reduced ability to form nerve sprouting

following injury or ALS

7,33

. In this study, darifenacin-treated male mice presented twice as many NMJs

with nerve sprouts than in vehicle-treated ones. Nerve sprouting was also always observed on top of a PSC

process, supporting the view that PSCs of denervated NMJs first elongate processes and guide motor nerve

to reinnervate NMJs, as previously described

. This suggests that dampening muscarinic activity of PSCs

facilitated the reinnervation of vacated endplates through NMJ repair. Interestingly, the signs of repair

were also present in females following treatment where they appeared to be more abundant than in males.

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Second, analysis of the motor functions showed that the benefits of the treatment on mice motor

behavior occurred later after the start of the treatment, around P480. This observation is difficult to reconcile

with a rapid consolidation of NMJ innervation that would have been observed if PSCs would rather

maintain NMJ innervation instead of promoting NMJ repair. However, the impact of darifenacin treatment

on MN survival (Figure 8) argues in favor of better NMJ stabilization and maintenance. Hence, we propose

that PSCs might first compensate the instability of the NMJs by facilitating their repair. Eventually, PSCs

would temporarily overcome the denervation process to stabilize NMJs towards the end of the treatment.

This suggested model on the balanced contribution of NMJ maintenance

repair of darifenacin actions

could be examined using repeated

in vivo

imaging of NMJs

Muscarinic signaling and glial cells function in ALS

The

ex vivo

data obtained in this study (Figure S2) showed that M3 receptors of PSCs are major

regulators of their excitability as suggested by the very high efficacy of darifenacin (in the nM range) to

completely block PSCs Ca

responses. In addition to its

ex vivo

efficacy, our results also show that

darifenacin targeted PSCs during the

in vivo

treatment as revealed by the dampening of muscarine-induced

responses (Figure 1). Furthermore, muscarinic-driven properties of PSCs such as process extensions

and guidance of nerve terminal sprouting were also improved by the treatment (Figure 6). These results

suggest that M3 receptor malfunction contribute to the maladapted properties of PSCs leading to the demise

of NMJs in ALS. Importantly, these results further confirm target engagement of the drug, a critical

prerequisite for this pre-clinical study.

In addition to its high selective affinity for M3 receptors over M2 and M4 (59 times), it is quite

unlikely that darifenacin acts presynaptically since only M2 and M4

receptors

have been reported on the

nerve terminal

. In addition, general blockers of all muscarinic receptors, including M2 and M4,

have an

overall detrimental effect on the NMJ and results in the retraction of the nerve terminal

. However, albeit

the 9 times higher affinity of darifenacin for M3 over M1 receptors, a possible contribution of M1 receptors

cannot be excluded as they are present on PSCs and muscle fibers

. Also, a possible impact of darifenacin

on immune cells cannot be ruled out owing to the regulatory roles of M3 receptors in these cells

57-61

Improved neuromuscular function and locomotion: restoring strength and movements

Our results show that chronic treatments with a selective M3 receptors antagonist preserved the

EDL nerve and muscle-evoked contractile force

46,62,63

, thus preserving the functionality of NMJs, and

slowed down the progression of neuromuscular weakening of the SOL. Beneficial impact on innervation

and force for the EDL muscle was striking, such that this ameliorated neuromuscular function likely

contributed to the behavioral improvements. Interestingly, the increase in strength following darifenacin

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treatment occurred later in the resistant SOL, possibly due to the greater intrinsic plasticity of the NMJs of

this muscle compared to the ones of the EDL

Another key impact of the darifenacin treatment was to preserve the typical fatigue properties of

the EDL and SOL muscles (Figure 5). Indeed, while the pathological changes were apparent in the vehicle-

treated mice, including decreased contractile force and increased neuromuscular and muscle fatigue

resistance, darifenacin limited the magnitude of the changes in the force and muscle fatigue generated, both

in EDL and SOL muscles. This may suggest that darifenacin reduced the alterations in the contractile and

metabolic fibers properties during disease progression that are known to take place in ALS

44,62,64

Neurotransmission failure (NF), which allows discrimination between nerve-related fatigue or denervation

versus intrinsic muscle fatigue, was increased only in vehicle-treated EDL muscles, compared with

darifenacin-treated and WT EDL muscles. Innervation analyses suggest that this elevated NF was primarily

linked to denervation, as the difference with the other groups was highest at the beginning of the protocol

and declined over time. Conversely, muscle fatigue following nerve stimulation was reduced in vehicle-

treated mice, partially compensating for increased NF at later stages, consistent with the increased fatigue

resistance observed in damaged muscles in ALS

. It would be interesting to directly determine the muscle

cross-sectional area to further confirm the muscle contribution to these alterations. These changes may

reflect the known NMJ reinnervation by slower MU following the loss of vulnerable fast motor units

9,49,50

as the proportion of slow muscle fibers progressively increase due to the demise of the fast-fatigable motor

units. Consistent with our results, another mechanism to consider is the failure at axon branching points

that could be enhanced in ALS, due to the motor unit expansion observed during the pathology

. By

stabilizing NMJs, darifenacin may limit axonal sprouting, thus limiting failures. Our data suggest that more

fatigable MU could be preserved by the treatment, as EDL force production and fatigue properties more

closely resembled WT values, consistent with the higher MN count reported in the lumbar spinal cord.

Darifenacin treatment provides benefits to the whole motor unit

We observed a beneficial impact of the treatment on the survival of MNs in the lumbar segments

innervating the EDL and SOL and of the neighboring ones, even though darifenacin does not cross the

blood-brain barrier

38,39

. This finding underscores the pathological importance of axonal integrity and NMJ

innervation and is consistent with evidence highlighting the critical role of peripheral signaling in MN

survival in ALS

1,3,6,65,66

. Noteworthily, it would be advantageous for future studies to directly correlate the

MN survival with the innervation status of their related NMJs. Our data suggest that protection of NMJs

was beneficial for the spinal MNs and delayed their death. Consistent with this possibility is the reduction

of microglia activation observed, indicative of a reduced neurodegeneration and a diminution of

neuroinflammation, this is further supported by the decreased ATF4 signal, a marker associated with

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neuronal stress and degeneration

48,67

. Together, these findings indicate a critical therapeutic window during

which NMJ rescue is associated with enhanced MN survival and attenuated neuroinflammation, potentially

contributing to improved muscle and motor functions in ALS.

Clinical and therapeutic potentials of darifenacin in ALS

The most striking results observed in this study are the functional impacts of the peripheral therapy

darifenacin on locomotor function and survival. Indeed, all behavioral parameters analyzed (grip strength,

gait, body weight and NS) were improved following chronic darifenacin treatment, without significant

adverse side effects, alleviating motor deficits observed in ALS mouse models

40,49,68

. Furthermore,

although deeper analyses on females are warranted, particularly on longitudinal experiments such as

survival and functional assessments, our data indicate that the effects of treatment on behavioral outcomes

and NMJ innervation were comparable in male and female mice, and that the treatment was well tolerated

in both sexes. Since NMJ denervation is ubiquitous in ALS, targeting NMJs implies that all patients could

benefit from this strategy. Additionally, darifenacin is well tolerated in mice and humans

34-37,69

, owing to

its reduced permeability of the blood brain-barrier and high specificity, thus limiting neurological side

effects. In particular, darifenacin has very limited cardiovascular effects and only causes mild peripheral

nervous system side effects typical of muscarinic antagonists such as mouth dryness, urinary retention

(Figure S7) and constipation

34-39,69

. The improvement of motor functions in treated mice would argue that

darifenacin treatment might improve the quality of life of the patients and provide a window of therapeutic

intervention. This strategy is now tested in an ongoing clinical trial (trial # NCT06249867

In addition, our

approach is also amenable to a strategy of combinational therapy since targeting NMJs is complementary

to most ongoing efforts that mainly target CNS mechanisms. Hence, the therapeutic potential of darifenacin

in ALS could be potentiated by concomitant administration of one or more other therapeutic strategies

involving different molecular mechanisms. An interesting avenue could be to combine with other

successfully approved treatments for ALS, for example, with Tofersen, an FDA-approved oligonucleotide

antisense treatment which targets specifically misfolded

SOD1

in mutant

SOD1

patients

. In addition,

other preclinical studies have presented promising candidates that could potentially be combined with

darifenacin

71-73

This work highlights an innovative therapeutic strategy for ALS, targeting M3 muscarinic

regulation of glial cells at the NMJ, resulting in an amelioration of the neuromuscular system, motor

functions and locomotion and survival. This innovative strategy could provide a better innervation and

locomotion, thus improving the quality of life in ALS patients. Exploiting the NMJ as a therapeutic target

in ALS would be relevant to all forms of ALS and open possibilities to combinational strategies with other

drugs, targeting different pathological pathways to develop alternative treatment strategies for ALS.

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Limitations of the study

Sex-specific differences previously reported in the

SOD1

G37R

mice revealed a faster disease

progression

and exacerbated NMJ denervation

in female compared to male. Hence, notwithstanding

the absence of a vehicle-treated female group, the comparable behavioral performance of darifenacin-

treated females and treated males suggests that darifenacin exerts beneficial effects in females. However,

owing to the limited number of female

SOD1

G37R

mice available for this study, further experiments will be

required to fully assess the impact of darifenacin on disease progression in female mice, including NMJ

denervation and MN survival, and longitudinal experiments such as survival and functional assessments.

MN counting has limitations as it was done using density measurements on lumbar segments that only

partially innervate EDL and SOL muscles. The use of a more direct correlation between the survival of the

exact motor neurons innervating these muscles would provide a direct link between NMJ innervation and

MN survival.

RESOURCE AVAILABILITY

Lead contact

Requests for further information and resources should be directed to and will be fulfilled by the lead contact,

Richard Robitaille (richard.robitaille@umontreal.ca).

Material availability

This study did not generate new unique reagents.

Data and code availability

•

All data reported in this study is deposited in the online repository at:

http://dx.doi.org/doi: 10.17632/tj888d7mt7.1

http://dx.doi.org/doi: 10.17632/4csgm6z25y.1

http://dx.doi.org/doi: 10.17632/kzmcprftmr.1

http://dx.doi.org/doi: 10.17632/2h29gjfmyp.1

http://dx.doi.org/doi: 10.17632/nkrwysnzj6.1

•

This paper does not report original code.

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•

Any additional information required to reanalyze the data reported in this paper is available from

the lead contact upon request.

Supplemental information

Document S1. Figure S1-S7

Video S1

Vehicle-treated mouse showing a lack of hindlimb stretch reflex and paralysis on its left hindleg.

Video S2

Darifenacin-treated mouse showing a reduced hindlimb reflex but walking easily in the cage.

ACKNOWLEDGMENTS

The authors thank Dr Sandrine Da Cruz for critical reading and constructive comments on the manuscript

and Dr Philippe-Antoine Beauséjour for help with the figures and videos. This work was supported by

grants from the Canadian Institutes for Health Research (FRN # 111070 and PJT # 173415) and initially by

grants from ALS Canada and Brain Canada, and the Packard Center for ALS research. The graphical

abstract and the schematic of experimental design in figure 2 were created using BioRender.com.

AUTHOR CONTRIBUTIONS

Conceptualization, RR, ET and DA; Methodology, RR, ET, DA and JV; Investigation, ET, DA, JV, RP,

GV and EM; Formal Analysis, ET, DA, JV, RP, EM and GV; Writing – Original Draft, ET and RR; Writing

– Review & Editing, ET, RR, DA, RP and GV; Supervision, RR. All authors read and approved the final

manuscript.

DECLARATION OF INTERESTS

RR, DA, ET and JV declare a patent application related to this work (US Patent Application No. 18/779,877,

Anticholinergic compounds for use in treating neuromuscular disorders

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FIGURES TITLES AND LEGENDS

Figure 1

Oral darifenacin treatment dampens muscarinic hyperexcitability in SOD1

G37R

mice

(a) Typical endplate of a vehicle-treated (top) and darifenacin-treated NMJ (bottom), labeled with α-BTX,

and PSCs loaded with Fluo-4 AM (blue).

(b' & d') False color images of PSCs represented in A (*) loaded with fluorescent Ca

indicator Fluo-4 AM

before (1), during (2), and after (3) muscarine (b') or ATP (d') local application.

(b'' & d'' traces of representative changes in fluorescence elicited by application of muscarine (b”) and ATP

(d”) from vehicle-treated (black traces) and darifenacin-treated (red traces) mice.

(c-e) Mean amplitude of Ca

-responses elicited in PSCs by muscarine (c) or ATP application (e). **, p <

0.01, unpaired

-test and Welch’s

-test. Data are represented as mean +/- SEM. Scale bar, 10 µm.

Figure 2 Treatment with darifenacin improved grip strength and locomotor function

(a) Schematic diagram of the preclinical trial design, including behavioral and physiological experiments.

(b) Changes of grip strength during the treatment, from P430 to P525 for darifenacin- (red) and vehicle-

treated animals (grey). Grey bar represents the mean WT values at the same age for grip strength.

assess motor function, coordination, and balance in

SOD1

G37R

mice, for darifenacin (red) versus vehicle-

treated mice (grey) between 450 until 525 days of age.

(d) Raw data of the gait analysis performed by applying non-toxic ink (forelimb, blue; hindlimb, red) on

mice limbs.

(e) Stride length and step width for both groups of animals. Grey bar represents the normal WT values for

these two parameters. Data are represented as mean +/- SEM. *, p < 0.05, **, p < 0.01, unpaired

-test.

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Figure 3 Darifenacin treatment reduces body weight loss, slows disease progression, and extends

survival

(a) Percentage of body weight loss (mean ± SEM) at P520 for both groups of animals.

(b) Distribution of the neurological score at P520 for darifenacin-treated (red) vs vehicle-treated mice

(grey).

Data are represented as mean +/- SEM. * p < 0.05, Mann-Whitney test, unpaired

-test, Log-rank (Mantel-

Cox) test.

Figure 4 Darifenacin increases EDL muscle force and contractile capacity and preserve muscle

fatigue properties

(a) Photograph illustrating the muscle force transducer setup, allowing to evoke muscle contractions by

stimulating either the nerve (NMJ-induced) or the muscle directly (all muscles fibers recruited). Examples

of muscle contractions elicited by nerve or muscle stimulation, used to calculate the nerve-muscle

contractile capacity.

(b-c) Maximal twitch force generated by

the EDL upon nerve (b) or muscle stimulations (c) at different

frequencies (5 Hz-300 Hz) for darifenacin-treated

SOD1

G37R

(red) and vehicle-treated

SOD1

G37R

(grey) and

WT mice (black). Representative force traces at 80 Hz are shown for each group.

(d) Mean ± SEM of the contractile capacity expressed in percentage, representing the mean proportion of

the peak force generated by nerve stimulation over muscle stimulation (which represents 100%) for

stimulation frequencies between 5 Hz-100 Hz.

(e) Mean ± SEM of EDL muscle weight for both animal groups.

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(f) Diagram illustrating the fatigue protocol. Nine (9) out of 10 stimulations were nerve-evoked only

whereas muscle stimulation was superimposed to the nerve stimulation every 10 stimulations. The fatigue

protocol was followed by a 30-minute recovery period.

(g, i) Representative force traces showing the amplitude of contractions at the first stimulation and at the

end of the fatigue protocol for each group during nerve (g) or direct muscle stimulation (i).

(h,j) Peak contractile force during the fatigue protocol and the recovery period expressed as percentage of

the baseline force generated at the beginning, for muscle stimulation (h) and nerve stimulation (j).

Data are represented as mean +/- SEM. *, p < 0.05, **, p < 0.01, ***, p < 0.001, repeated measures two-

way ANOVA and unpaired

-test.

Figure 5

Darifenacin treatment preserves SOL muscle contractile force and fatigue properties at

P550

(a-b) Maximal twitch force of the SOL following nerve (a) or muscle stimulation (b) at various stimulation

frequencies (5 Hz-300 Hz) for darifenacin-treated (red) and vehicle-treated animals (grey) in the survival

cohort.

stimulation (c) or direct muscle stimulation (d), at P520 and end stage, for darifenacin-treated (red) and

vehicle-treated animals (grey). The mean difference between contractile force at P520 and end stage value

for each group is illustrated by a blue rectangle.

(e-f) Dot plots showing the mean ± SEM of the peak contractile force following a 2-second stimulation of

the muscle at 50 Hz (e) or 80 Hz (f). Note that direct muscle stimulation at both frequencies led to an

increased force in treated animals.

(g) Fatigue protocol of stimulation of the SOL muscle, including 300 motor nerve stimulations of 50 Hz

for 500 ms each. Muscle stimulation was superimposed to the nerve stimulation every 10 stimulations. (h)

Graph showing the peak contractile force during a fatigue protocol including a train of 300 nerve

stimulations at 50 Hz (duration 500 ms per 1.1 second) and a 30 min subsequent recovery period. Muscle

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stimulation was also superimposed every 10 stimulations. Data is expressed as the percentage of the

baseline force generated at the beginning of the fatigue protocol, for direct muscle stimulation (squares)

and nerve stimulation (circles) of Darifenacin-treated (red) and control animals (grey).

Data are represented as mean +/- SEM. *, p < 0.05, repeated measures two-way ANOVA and unpaired

test.

Figure 6 Darifenacin treatment improves NMJ innervation and signs of NMJ repair in EDL

(a) Graphical illustrations of the three types of innervation status included in the morphological analysis:

innervated (full coverage of postsynaptic receptors, in red, by the nerve terminal, in green), partially

innervated (incomplete coverage of the NMJ by the nerve terminal) and denervated (absence of presynaptic

coverage).

(b) Dot plots showing the mean ± SEM of the percentage of fully innervated, partially innervated and

denervated NMJs.

green) and postsynaptic AChRs (muscle endplate, α-BTX, in red) in the darifenacin-treated EDL (top) and

the vehicle-treated EDL (bottom).

(d) Diagram and confocal images illustrating two signs of NMJ repair: nerve sprouting (white arrow, green

and blue) and glial process extension (blue arrow, blue).

(e) Dot plots of the occurrence of nerve sprouting, polyinnervation and glial processes at NMJs of vehicle

and darifenacin treated mice. (f) Confocal images of immunofluorescent labeling of the NMJ: postsynaptic

nAChRs (muscle endplate, α-BTX, in red) and the merge including also the presynaptic terminal (SV2 +

NF-M, in green) and PSCs (S100, in cyan) for vehicle-treated (top panel) and darifenacin-treated EDL

(bottom panel). Note the presence of nerve sprouting (white arrows) in treated animals, and the absence of

NMJ repair signs in control NMJs, despite ongoing denervation.

Data are represented as mean +/- SEM. *, p < 0.05, unpaired

-Test. **, p < 0.01, unpaired

-test, Scale bar,

10 µm.

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Figure 7

Darifenacin treatment increases nerve sprouting in SOL muscle

(a) Dot plots of the percentage of NMJs innervated, partially innervated and denervated.

(b) Dot plots of the percentage of NMJs presenting nerve sprouting, polyinnervation and glial processes in

darifenacin- vs Vehicle-treated NMJs.

NF-M, in green), PSCs (S100, in cyan) and postsynaptic nAChRs (muscle endplate, α-BTX, in red) for

vehicle-treated (top panel) and darifenacin-treated EDL (bottom panel). Note the presence of nerve

sprouting, composed of the nerve terminal extension within glial processes (white arrow) in darifenacin-

treated animals, as opposed to the limited NMJ repair signs in control NMJs, despite ongoing denervation

(white asterisk).

Data are represented as mean +/- SEM. *, p < 0.05, unpaired

-test. Scale bar=10 µm.

Figure 8 Darifenacin treatment increases survival of motor neurons in the lumbar spinal cord and

decreases microglia activation

(a) Confocal fluorescence images of immunofluorescence of alpha motor neurons (αMN; arrow heads)

labeled with neuronal-specific nuclear protein (NeuN, green) and choline acetyltransferase (ChAT, red) in

lumbar spinal cord cross sections of vehicle-treated (top panel) and darifenacin-treated mice (bottom panel)

at P520.

(b) Dot plots of the number of α-MNs (mean ± SEM) in vehicle- (black) versus darifenacin-treated animals

(red) in L1-L6 and (b’) in only L4-L6 lumbar spinal cord cross sections.

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adaptor molecule 1; Iba1, in red) and astrocytes (Glial fibrillary acidic protein; GFAP, in green) activation

in the ventral horn of a lumbar spinal cord cross section of vehicle-(top panel) and darifenacin-treated mice

(bottom panel) at P520.

(d-e) Dot plots of the fluorescence intensity, in spinal cord cross sections, for Iba1 (d) and GFAP (e) for

vehicle-treated (grey) and darifenacin-treated animals (red) in L1-L6 and in only L4-L6 lumbar spinal cord

cross sections (d’-e’).

(f) Quantification of ATF4 protein levels in lumbar spinal cords by Western blot, in vehicle-treated WT

(black), vehicle-treated

SOD1

G37R

mice (grey), and darifenacin-treated

SOD1

G37R

mice (red).

Data are represented as mean +/- SEM. *, p < 0.05, **, p < 0.01, ****, p < 0.0001, Mann-Whitney test and

Kruskal-Wallis one-way ANOVA. Scale bar = 100 µm.

STAR

★

METHODS

Key resources table

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Antibodies

Chicken anti-NFM

Rockland

Immunochemicals

Inc.

#212-901-D84

mouse IgG1 anti-SV2

Developmental

Studies

Hybridoma Bank

RRID:

AB_2315387

Alexa 594 conjugated α-bungarotoxin

Invitrogen

#B13423

Rabbit anti-S100

Agilent

IR504

Donkey anti-chicken Alexa 488

Jackson

Immunoresearch

RRID: AB_234

0375

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Goat anti-mouse IgG1 Alexa 488

Jackson

Immunoresearch

RRID: AB_233

8854

Donkey anti-rabbit Alexa 647

Jackson

Immunoresearch

RRID: AB_249

2288

Chicken anti-GFAP

Abcam

AB134436

Goat anti-ChAT

Millipore

AB144P

Mouse IgG1 anti-NeuN

Millipore

MAB377

Donkey anti-goat Alexa 594

Jackson

Immunoresearch

RRID:

AB_2340433

Rabbit anti-Iba1

FUJIFILM Wako

Pure Chemical

Corporation

#019-19741

Rabbit anti-ATF4

Cell Signaling

#11815

GAPDH (14C10) Rabbit Monoclonal Antibody

Cell Signaling

#2118

Peroxidase AffiniPure® Donkey Anti-Rabbit IgG (H+L)

Jackson

Immunoresearch

RRID: AB_100

15282

Chemicals, peptides, and recombinant proteins

Darifenacin hydrobromide

Sigma-Aldrich

SML1102

Prolong Gold antifade mounting medium

Thermo Fisher

Scientific

#P36930

Triton

Sigma-Aldrich

#X100-500ML

Muscarine

Sigma-Aldrich

#M6532

ATP

Sigma-Aldrich

#A9187

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Normal Donkey Serum

Jackson

ImmunoResearch

#017-000-121

Normal Goat Serum

Jackson

ImmunoResearch

#005-000-121

Fluo-4, AM

Invitrogen

F14202

Pluronic

F-127

Invitrogen

P6867

Tubocurarine hydrochloride pentahydrate

Sigma-Aldrich

T2379

Dimethyl sulfoxide (DMSO)

Sigma-Aldrich

D1435

Sodium chloride (NaCl)

Sigma-Aldrich

S7653

Potassium chloride (KCl)

Sigma-Aldrich

P9541

Magnesium chloride hexahydrate (MgCl

.6H

Sigma-Aldrich

M2670

Sodium bicarbonate (NaHCO

)

Sigma-Aldrich

S5761

Calcium chloride dihydrate (CaCl

.2H

Sigma-Aldrich

C5080

D-(+)-Glucose

Sigma-Aldrich

G5767

L-Glutamic acid

Sigma-Aldrich

G8415

L-Glutamine

Sigma-Aldrich

G3126

BES

Sigma-Aldrich

B9879

Thiamine pyrophosphate

Sigma-Aldrich

C8754

Choline chloride

Sigma-Aldrich

C7527

Formaldehyde

Mecalab Ltee

#1045

Sigma Fast inhibitor tablet

Sigma-Aldrich

S8820

Tris Base

Thermo Fisher

Scientific

BP152

Glycine

Thermo Fisher

Scientific

J64365A1

Ponceau S

Thermo Fisher

Scientific

J60744.18

Tween 20

Thermo Fisher

Scientific

BP337

Glycerol

Sigma-Aldrich

G5516

Bromophenol Blue

Sigma-Aldrich

114391

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2-Mercaptoethanol

Thermo Fisher

Scientific

O3446l

Methanol

Thermo Fisher

Scientific

BP1105

Bovine Serum Albumin

Sigma-Aldrich

A7906

NP-40 lysis buffer

Thermo Fisher

Scientific

J60766.AK

Sodium Dodecyl Sulfate (SDS)

Bio-Rad

1610301

Hydrochloric Acid Concentrate

Thermo Fisher

Scientific

SA49

Ethylenediaminetetraacetic acid (EDTA)

Sigma-Aldrich

Critical commercial assays

Pierce

BCA Protein Assay Kit

ThermoFisher

Scientific

23225

Deposited data

Raw data

Figures 1, S2, S3,

S6 and S7

Raw data

Figures 2-3-4-7

and statistical

information

Raw data

Figure 6

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Raw data

Figure 8 (a, b, f)

and figure S5

Raw data

Figure 8 (c, d, e)

and figure S1

Experimental models: Organisms/strains

Mouse line :

B6.Cg-Tg(SOD1*G37R)29Dpr/J

The Jackson

laboratory

RRID:IMSR_JA

X:008229

Mouse line :

C57BL/6N-

Mdga2

Tg(Prnp-PFN1*G118V)838Kiaei

The Jackson

laboratory

RRID:IMSR_JA

X:030568

Software and algorithms

Prism (version 8-10.0)

GraphPad

Software

www.graphpad.

com

Fluo-View FV10-ASW Viewer

Evident Scientific

https://evidentsc

ientific.com/en/

downloads

ImageJ (version 1.52a)

National Institutes

of Health

https://imagej.ne

t/ij/

Zen Desk

Zeiss Software

WinWCP (V.5.7.2)

Strathclyde

Electrophysiology

Software

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Photoshop (version CC 2019-2025)

Adobe

https://www.ado

be.com/ca/produ

cts/photoshop.ht

Illustrator (version 7-10 and CC 2019-2025)

Adobe

Other

Superfrost plus Gold Slides

Fisher Scientific

22-035813

PVDF blotting membranes

Thermo Fisher

Scientific

Cat#88518

4-15% Mini protean tgx-stain-Free gels

Bio-Rad

Laboratories

Canada Ltd

#4568083

EXPERIMENTAL MODELS AND STUDY DETAILS

SOD1

G37R

mouse model

Mice overexpressing the human mutated

SOD1

G37R

transgene, line 29, were obtained from The Jackson

Laboratory and bred at our animal facilities on a C57BL/6 background (

RRID:IMSR_JAX:008229

). This

mouse model is a late onset (P440-P460), slowly progressing model of ALS that recapitulates the human

phenotype of the disease. The characterization of this strain phenotype has been previously published

9,12,33,75

. A total of 84

SOD1

G37R

mice (81 male and 3 female) as well as 9 male WT C57BL/6 littermates,

aged P380-P600, were included in this study to assess the impact of darifenacin treatment in male and

female mice. Experiments were performed in accordance with the guidelines of the Canadian Council of

Animal Care and the Comité de déontologie de l’expérimentation sur les animaux (CDEA) of Université

de Montréal (protocol #23-027).

Profilin-1 mouse model

Profilin-1

mice

(

PrP-hPFN1

G118V

)

were

obtained

from

the

Jackson

Laboratory

(RRID:IMSR_JAX:030568

). A cohort of 8 male

Profilin-1

mice were put into calorie restriction (4-

5g/day) until motor symptoms developed. Experiments were performed between P119-P127.

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METHOD DETAILS

Drug

The chosen compound for the preclinical study was darifenacin, a type 3 mAChR antagonist

already approved for the treatment of overactive bladder

34-37

. Darifenacin has a high selectivity for the M3

muscarinic receptor subtype, with a 59-times higher selectivity over M2 and M4 receptors and a 1-fold

higher selectivity over M1 and M5. This significantly contributes to the minimal side effects (cognitive and

cardiac) and limited CNS/heart-related issues. Darifenacin (#SML1102, Sigma-Aldrich, diluted in DMSO

at 100 mg/mL) was administered orally 5 days a week at a concentration of 10mg/kg based on the extensive

pre-clinical data available for the development of the use of darifenacin for the treatment of over-reactive

bladder. This dosage was chosen because of its efficacy to partly block PSC calcium response to muscarine

(Figure S1) and absence of toxicity and limited adverse side effects in toxicological studies

Paraphimosis, which reflects urinary retention, was monitored throughout disease progression. Urinary

retention at end stage did not differ significantly compared to vehicle-treated mice (Figure S7).

Preclinical trial design

The preclinical trial was designed according to guidelines for preclinical animal research in

ALS/MND

. A total of 73 male

SOD1

G37R

mice were included across the various experiments of the

preclinical trial and 9 WT C57BL/6 controls. Mice were randomly assigned to the experimental

(darifenacin) or the control (vehicle) group. In addition, a cohort of three female

SOD1

G37R

mice received

darifenacin to assess treatment safety and to confirm that females exhibited a comparable response to

treatment. Treatment, behavior monitoring, experiments and results analysis were done blindly. Standard

ALS behavioral measurements (described below) were performed weekly to measure disease progression.

A set of neurological scores (NS, levels 1 to 5, figure S4) was used to determine the onset of

symptoms and the progression and severity of symptoms during the disease. Treatment was started at

disease onset representing a neurological score of 1.

Inclusion criteria were

SOD1

G37R

mice at disease onset (NS of 1: Exclusion criteria included any

major health complications during the preclinical trial, such as severe wounds, cancer, or infection. The late

symptomatic stage, at the age of P520 (P515-535) was used to measure neuromuscular functions, NMJ

innervation and motor neuronal survival. A cohort of mice was allowed to progress to the end stage of the

disease, (NS of 5, Table S1) to test the impact of the treatment on survival and to monitor NMJ innervation

on more resistant muscles.

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Grip strength measurements

Grip strength was measured using a strength meter (Bioseb, Harvard Apparatus) calibrated for

mice. Mice were lifted by the tail and placed on a grid grabbing it with the fore and hind paws and gently

pulled to measure the force generated. Three sets of measures were obtained weekly (5 attempts for each

set) for each mouse, with a 5-minute interval. The 3 best measurements were used for statistical analysis.

Rotarod acceleration protocol

Motor coordination, strength and balance were assessed weekly using a rotarod (TSE Rotarod, TSE

Systems Gmbh, Germany). Following a warm-up period (constant speed of 4 rpm for a maximum of 300

seconds), attempts were performed using an acceleration protocol

starting at a speed of 4 rpm up to 40

rpm, in 300 seconds. Two attempts per session were performed and the longest latency to fall was used.

Gait analysis

Gait was assessed using the footprint test. Paws were inked with a nontoxic paint (forepaws in blue

and hind paws in red) and mice were placed on a restricted pathway on a white paper sheet (30 cm long,

10 cm wide) and free to walk along the walkway. Ten footsteps from two different runs were measured to

evaluate stride length (distance between front and hindlimbs) and step width (distance between left and

right paws).

Nerve–muscle preparations

Preparations of

Extensor Digitorum Longus

(EDL) and

Soleus

(SOL) muscles and their innervating

nerve were dissected in oxygenated Ree's solution (in m

): 110 NaCl, 5 KCl, 1 MgCl

, 25 NaHCO

, 2

CaCl

, 11 glucose, 0.3 glutamic acid, 0.4 glutamine, 5 BES (N,N-Bis(2-hydroxyethyl)-2-

aminoethanesulfonic acid sodium salt), 0.036 choline chloride, and 4.34 × 10

−7

cocarboxylase. After

dissection, nerve muscle preparations were constantly perfused with oxygenated Ree's solution (95% O

5% CO

Calcium imaging of PSCs

Muscles and their innervation were dissected and pinned in a physiological recording chamber.

Imaging of PSCs intracellular Ca

was performed

12,33

. Nerve-muscle preparations were incubated for 90

min (2 x 45 min) in a preoxygenated physiological saline solution containing 10 µM fluo-4AM (Invitrogen),

0.02% pluronic acid (Invitrogen) and 0.15% dimethyl sulfoxide (Sigma-Aldrich) at 26°C.

Nerve-muscle

preparations were incubated with Alexa Fluor 594-conjugated α-bungarotoxin (BTX) (10



g for 10 min;

Journal Pre-proof

Invitrogen) at the end of the loading period. Ca

elevations in PSCs were elicited via two stimulating

approaches: a micropipette positioned at proximity of the cells was used to locally apply agonists muscarine

(10 µM in pipette, #M6532, Sigma-Aldrich) and ATP (10 µM in pipette, #A9187, Sigma-Aldrich) at 20

min interval. Only cells responding to both agonist applications were considered in the analysis. As reported

before

12,33

as many as 10 NMJs per muscle were tested with all loaded PSCs on the NMJs (up to 3/NMJ).

Measurements of neuromuscular properties

EDL and SOL nerve-muscle preparations were attached vertically to a fixed force transducer

(model 402 A - 500 mN, Aurora Scientific Inc.) using surgical threads. The preparations were attached at

the tendons level to the transducer at one extremity and to a homemade adaptable hook at the opposite

extremity (Figure 4a). This system was designed to elicit muscle contractions elicited from both muscle

and/or nerve stimulations. A platinum reference electrode was positioned near a tendon. To stimulate the

muscle, a second platinum electrode was juxtaposed away from the nerve entry and the band of NMJs. To

elicit muscle contractions from motor nerve stimulation, the deep peroneal nerve was suctioned into an

electrode made of PE tubing and filled with Rees’ solution. Stimulation of the motor nerve elicits muscle

contractions through NMJ efficacy, reflecting the contractile strength of innervated muscle fibers only.

Direct muscle stimulation, however, depolarizes all muscle fibers and reflects the maximal contractile force

of the whole muscle

43,44

. Motor nerve was stimulated by a single supra-maximal square-wave of 500 mV,

0.1 ms pulse. Muscle fibers were stimulated by a square pulse stimulation of 15 V, 1 ms. Optimal muscle

length was determined by gradually stretching the muscle until maximal contractile force output was

attained. Recordings were obtained with WinWCP software (Strathclyde Electrophysiology Software).

Force-frequency curve: Nerve and muscle stimulations were performed to generate a standard

force-frequency curve. Alternate nerve and muscle stimulations were executed at increased frequencies (5

Hz, 10 Hz, 20 Hz, 30 Hz, 40 Hz, 50 Hz, 60 Hz, 70 Hz, 80 Hz, 90 Hz, 100 Hz, 120 Hz, 140 Hz, 160 Hz, 180

Hz, 200 Hz, 250 Hz and 300 Hz) and the force generated monitored. There was a 1-minute rest period

between each stimulation. These frequencies cover the full range of the motor neuron activities

77,78

including higher frequencies that are design to further challenge the limits of the neuromuscular system.

Muscle fatigue: The fatigue protocol (Figure 4f) for the EDL involved a bout of 180 trains of nerve

stimulations with a duration of 300 ms, elicited at a frequency of 120Hz. The resting period between each

stimulation was 700 ms, for a total protocol duration of 3 min. Muscular stimulations were super-imposed

to nerve stimulations every 10

stimulations (18 simultaneous nerve-muscle stimulations), to evaluate

muscular reserve. The fatigue protocol was designed to challenge the NMJ, and a stimulation frequency of

120 Hz was selected because fast fatigable and fast fatigue-resistant motor units in fast-twitch muscles can

fire doublets and triplets at the onset of activation at frequencies exceeding 100 Hz.

77-79

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The fatigue protocol for the slow-twitch SOL (Figure 5g) involves a train of 300 nerve stimulations,

elicited at 50 Hz for 500 ms. Nerve fatigue stimulation was elicited at a slower stimulation frequency in the

SOL, adapted to the slow-twitch nature of the SOL characterized by a tonic slow firing rate

77,78

. The resting

period was 1.1 seconds between every stimulation. The total duration of the fatigue protocol was 5 min 30

sec. A simultaneous nerve-muscle stimulations were performed every 10 stimulations) to evaluate nerve

and muscle fatigue, NMJ contractile capacity and neurotransmission failure.

Each fatigue protocol was followed by a 30-minute recovery period during which muscular and

neuromuscular contractile force were measured at the following latencies (after the end of the fatigue

protocol): 5 s, 10 s, 15 s, 30 s, 45 sec, 1 min, 1.5 min, 2 min, 2.5 min, 5 min, 10 min, 20 min and 30 min.

NMJ contractile capacity was calculated as the ratio of nerve- to muscle-evoked contractile force

and averaged across stimulation frequencies ranging from 5 to 100 Hz:

𝑁𝑒𝑟𝑣𝑒 − 𝑒𝑣𝑜𝑘𝑒𝑑 𝑐𝑜𝑛𝑡𝑟𝑎𝑐𝑡𝑖𝑙𝑒 𝑓𝑜𝑟𝑐𝑒 (𝑚𝑁)

𝑀𝑢𝑠𝑐𝑙𝑒 − 𝑒𝑣𝑜𝑘𝑒𝑑 𝑐𝑜𝑛𝑡𝑟𝑎𝑐𝑡𝑖𝑙𝑒 𝑓𝑜𝑟𝑐𝑒 (𝑚𝑁)

𝑋 100%

The mean contractile capacity is normally 100 % in WT mice, indicating that the stimulation of the motor

nerve and the neuronal control of the muscle recruits 100 % of its maximal contractile capacity and that

NMJs are innervated and fully functional in a healthy mouse.

Neurotransmission failure (NF) was calculated during the fatigue protocol for both muscle types

using the following formula:

NF =

𝐹−𝑀𝐹

(1−𝑀𝐹)

X 100%

where fatigue (F) represents the mean force decrease measured after nerve stimulation for each stimulation

train, and muscle fatigue (MF) represents the force reduction measured following muscle stimulation at the

beginning of each train.

NMJ immunofluorescence labeling

Whole-mount muscle preparations were dissected, fixed and processed for immunostaining

2,9

Dissected EDL and SOL muscles were fixed for 10 min in 4% formaldehyde, washed three times in 1X

PBS, permeabilized in 100% cold methanol for 6 min at −20 °C, and then incubated for 1 h in blocking

solution (10% Normal Donkey Serum (NDS), 1% Triton-X100, in PBS 1X). After, muscles were incubated

with a rabbit anti-S100 antibody (1:4, #IR504, Agilent) overnight for approximately 20 hours at 4 °C, then

Journal Pre-proof

with chicken anti-neurofilament M (NF-M, 1:2000, #212-901-D84, Rockland Immunochemicals Inc.) and

mouse IgG1 anti-synaptic vesicular protein 2 (SV2, 1:2000, Developmental Studies Hybridoma Bank) for

20 hours at 4 °C. The incubation solution used for all antibodies was composed of 2% NDS, 1% Triton-

X100 in PBS 1X solution. Muscles were then incubated for 2 hours with the secondary antibodies donkey

anti-rabbit Alexa 647 (1:500, #711-605-152, Jackson Immunoresearch), simultaneously with donkey anti-

chicken Alexa 488 (1:500, #703-545-155, Jackson Immunoresearch) and goat anti-mouse IgG1 Alexa 488

(1:500, #115-545-205, Jackson Immunoresearch), as well as Alexa594-conjugated-α-BTX (1.33 µg/ml,

#B13423, Invitrogen). Muscles were washed six times for 5 min each in PBS 1X - Triton 0.01%. Finally,

EDL and SOL whole muscle preparations were mounted in Prolong Gold antifade mounting medium

(#P36930, Thermo Fisher Scientific).

Observations and image acquisition were obtained using a Zeiss LSM 880 confocal microscope,

with a 63X oil immersion objective (N.A. 1.4) or an Olympus FV 1000, with either a 60X oil immersion

objective or a 20X water immersion objective (respectively N.A. 1.42 and 0.90). NMJs were classified as

fully denervated (no colocalization between presynaptic and postsynaptic markers), partially innervated (up

to 95% colocalization), or fully innervated (95–100% colocalization between presynaptic and postsynaptic

staining). As previously reported

12,29,33

, an NMJ was classified as denervated when no presynaptic staining

was observed while a sprouting profile was determined by presence of a nerve terminal that extends beyond

the end-plate territory (more than 10 µm). No image manipulations were performed after acquisition, except

for brightness and contrast adjustments with Adobe Photoshop (version CC 2019-2025) for figure

presentation. The software Adobe Illustrator (version 7-10 and CC 2019-2025) was used to prepare figures.

Spinal cord cross-sections and motor neuron counts

Mice were perfused transcardially with PBS 1X for 5 min and 4% formaldehyde for 15 min. Mice

were post-fixed in formaldehyde for 24 hours and lumbar spinal cords were subsequently dissected. Spinal

cords were then cryoprotected in a 30% sucrose - PBS 1X solution for 72 hours and frozen using dry ice

Floating spinal cord cross sections (30 μm) were obtained using a sliding microtome (SM2000R, Leica) [4,

6].

For motor neuron counts, spinal cords cross sections were washed six times in PBS 1X and

incubated 1 hour in a blocking solution (10% NDS, 0.3% Triton-X100 in PBS 1X). Then, they were double

labeled for 17-18 hours against Choline Acetyltransferase (ChAT; 1:100; Goat;

AB144P, Millipore) and

the neuronal marker Neuronal Nuclei (NeuN; 1:300; Mouse IgG1;

MAB377, Millipore) in the 10% NDS

blocking solution at 4°C. Note that after each antibody incubation for spinal cords, sections were washed

three times for 5 min with PBS 1X. Spinal cord sections were then incubated for 1 hour with the secondary

antibody donkey anti-goat Alexa 594 (1:500; #705-585-147; Jackson Immunoresearch) in the NDS

Journal Pre-proof

blocking solution, then incubated for 1 hour in a goat blocking solution (10% Normal Goat Serum, 0.3%

Triton X-100 in PBS 1X) and finally1 hour with the goat anti-mouse IgG1 Alexa 488 secondary antibody

(1:500; #115-545-205; Jackson Immunoresearch) in the 10% goat blocking solution previously described

and mounted with Prolong Gold antifade reagent (#P36930, Thermo Fisher Scientific). Double-positive

cells expressing ChAT and NeuN from each ventral horn were identified as α-MNs [4], to differentiate

them from the γ-MNs, which do not express ChAT but express NeuN. One in 4 lumbar spinal cords sections

were imaged and quantified, and a minimum of twenty lumbar spinal cord sections per animal were

analysed for the L1-L6 lumbar sections and between twelve and eighteen for the L4-L6 lumbar sections.

For GFAP and Iba1 staining, spinal cord cross sections were again washed 6 times in PBS 1X and incubated

1 hour in a blocking solution (10% NDS, 0.3% Triton-X100 in PBS 1X). Then, they were double labeled

at 4°C overnight for 17 hours against the astroglial marker GFAP (1:500; chicken; #AB134436, Abcam)

and the microglial marker Iba1 (1:500; rabbit, #01919741, Wako) in the 10% NDS blocking solution, and

with the secondary antibody donkey anti-chicken Alexa 488 for 1 hour (1:500; #703-545-155; Jackson

Immunoresearch) in the NDS blocking solution. Finally, they were incubated for 1 hour with the donkey

anti-rabbit Alexa 647 secondary antibody (1:500; #711-605-152; Jackson Immunoresearch) in the 10% goat

blocking solution previously described and mounted with Prolong Gold antifade reagent (#P36930, Thermo

Fisher Scientific) on Superfrost plus Gold Slides (#22-035813, Fisher Scientific). After each antibody

incubation, sections were washed three times for 5 min with PBS 1X. A minimum of twenty lumbar spinal

cord sections per animal were analysed for the L1-L6 lumbar sections and between twelve and eighteen for

the L4-6 lumbar sections.

Observations and image acquisition were obtained using a Zeiss LSM 880 confocal microscope,

with a 63X oil immersion objective (N.A. 1.4) or an Olympus FV 1000, with either a 60X oil immersion

objective or a 20X water immersion objective (respectively N.A. 1.42 and 0.90). No image manipulations

were performed after acquisition, except with Adobe Photoshop (version CC 2019-2025) for brightness and

contrast adjustments for figure presentation. The software Adobe Illustrator (version 7-10 and CC 2019-

2025) was used to prepare figures.

Western blots

Spinal cords were rapidly collected and frozen in liquid N

and stored at -80

C until protein

extraction. Samples were then weighed to calculate the volume of lysis buffer to be used (5 x weight; 50

mM Tris-HCl pH 7.5, 1 mM EDTA pH 8, 150mM NaCl, 1% triton-X and 1:10 protease inhibitor (Sigma

Fast inhibitor tablet; S8820; Sigma-Aldrich). Samples were maintained on ice, and partial mechanical

digestion was done. Samples were then incubated for 30 min on ice before a second mechanical digestion

was performed. SDS/NP40 (0.5X of the weight of the original tissue) were added, and samples were

Journal Pre-proof

incubated for 10 min at room temperature. Afterwards, samples were centrifuged for 30 min at 14,800 rpm

at 4°C. Supernatant (protein extract) was then collected, and protein concentration was determined using

Pierce

BCA Protein Assay Kit (Thermo Fisher Scientific), according to the manufacturer’s protocol.

Sample buffer (4×, 240 mM Tris-HCl pH 6.8, 8% (

w/v

) SDS, 40% (

v/v

) Glycerol, 0.1% (

w/v

)

Bromophenol Blue, 10% (

v/v

) 2-mercapoethanol, pH 8.3-8.8) was added to 20 µg of protein lysates.

Samples were loaded onto 4-15% Mini protean tgx-stain-Free gels (#4568083, Bio-Rad Laboratories

Canada Ltd) and ran at 100V for 2 hours using Tris-glycine running buffer [25mM Tris, 190mM glycine,

1% (w/v) SDS]. Resolved proteins were transferred onto PVDF blotting membranes (#88518, Thermo

Fisher Scientific) at 100 V for 60 min on ice, in transfer buffer (250 mM Tris-base; 192nM glycine, 10%

(

v/v

) methanol; pH 8.3). In order to confirm successful protein transfer, membranes were stained with

Ponceau S red (Thermo Fisher Scientific), before being blocked for 1 hour in a Tris-buffer saline (TBS)-

Tween Solution (10 mM Tris pH 7.5, 100 mM NaCl, 0.1% (

v/v

) Tween 20) containing 5% (

w/v

) of non-fat

dry milk (blocking buffer). After two washes of 5 min with TBS-0.1% Tween 20 buffer, membranes were

incubated with the primary antibody (ATF4, 1:1000, #11815, Cell Signaling, overnight; diluted in TBS-

Tween Solution with 5% Bovine Serum Albumin (BSA)). Membranes were then washed 3 times for 10

min with TBS-0.1% Tween 20 buffer and incubated for 1 hour at room temperature with anti-rabbit (HRP)

secondary antibody (1:5000, #711-035-152; Jackson ImmunoResearch Lab) for chemiluminescence

detection. For detection of the housekeeping protein GAPDH (14C10) (#2118S, Cell Signalling; 1:5000, 1

hour at room temperature), membranes were stripped with a glycine solution (100 mM, pH 2.9; Sigma-

Aldrich, UK) for 15 min at room temperature and, after three washes, blocked again for 1 hour in 5% (

w/v

)

of non-fat dry milk prior to further blotting. Membranes were exposed to SuperSignal West Pico

Chemiluminescent Substrate (#34580, Thermo Fisher Scientific) for signal detection. Image acquisitions

were carried out using ChemiDoc Imaging Systems (Bio-Rad) and densitometric analysis were performed

using ImageJ software (Version 1.52a, National Institutes of Health NIH, USA); optical density values were

normalized to the corresponding reference protein band.

Quantification and statistical analysis

Results are represented as the mean ± SEM where the number of animals is identified as

(number

of replicates). The number of NMJs or number of cells for calcium imaging experiments or number of

spinal cord cross sections is represented by

(number of observations)

Unpaired

-tests were used when

comparing two independent groups. Welch’s

-test was used to compare two independent groups when

variances were unequal. One-way ANOVA was applied for comparisons among three groups. When data

did not meet the assumptions of parametric tests, the Mann–Whitney or Kruskal–Wallis tests were used.

Repeated-measures two-way ANOVA followed by Tukey’s or Sidak’s multiple comparisons

post hoc

test

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Journal Pre-proof

✱

Darifenacin

Vehicle

Body weight loss at P520

Body weight loss (%)

Survival curve

✱

Age (days)

ilit

l (

)

500

525

550

575

600

625

650

100

Vehicle
Darifenacin

Neurological score at P520

Neurological score (1 to 5)

r o

f m

ice

Vehicle

Darifenacin

Journal Pre-proof

50 100 150 200 250 300

100

150

Stimulation frequency (Hz)

Muscle stimulation

Nerve stimulation

80 Hz - 500 ms

Muscle stimulation

50 Hz - 2 sec

Vehicle

Darifenacin

100

150

200

250

✱

Muscle stimulation

80 Hz - 2 sec

Vehicle

Darifenacin

100

150

200

250

✱

Muscle stimulation

80 Hz - 500 ms

100 150 200 250 300

100

125

150

175

Vehicle

Darifenacin

Stimulation frequency (Hz)

100

150

200

250

✱✱

✱

Contractile force (mN)

100

150

200

250

✱✱

✱

P520

Vehicle

Darifenacin

endstage

P520

Vehicle

Darifenacin

endstage

Fatigue and recovery

9 Nerve stims

11 s

30 trains, 300 stim, 333 sec

270 nerve stim.

30 nerve+muscle stim.

1 Nerve +

muscle stim.

x 9/10

x 1/10

TRAIN 1

TRAIN 2

10 stimulations

Nerve stimulation

Nerve + muscle stimulation

1 stimulation =

50 Hz-500 ms

Contractile force (% Ctrl)

Contractile force (mN)

100

200

300

100

120

330 400 470 1000 2000

Muscle Darifenacin

Muscle Vehicle

Time (s)

Nerve Darifenacin

Nerve Vehicle

Nerve WT

Muscle WT

Muscle stim

Nerve stim

5 min

30 min

Fatigue

Recovery

Contractile force (mN)

Journal Pre-proof

Key resources table

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Antibodies

Chicken anti-NFM

Rockland

Immunochemicals

Inc.

#212-901-D84

mouse IgG1 anti-SV2

Developmental

Studies

Hybridoma Bank

RRID:

AB_2315387

Alexa 594 conjugated α-bungarotoxin

Invitrogen

#B13423

Rabbit anti-S100

Agilent

IR504

Donkey anti-chicken Alexa 488

Jackson

Immunoresearch

RRID: AB_234

0375

Goat anti-mouse IgG1 Alexa 488

Jackson

Immunoresearch

RRID: AB_233

8854

Donkey anti-rabbit Alexa 647

Jackson

Immunoresearch

RRID: AB_249

2288

Chicken anti-GFAP

Abcam

AB134436

Goat anti-ChAT

Millipore

AB144P

Mouse IgG1 anti-NeuN

Millipore

MAB377

Donkey anti-goat Alexa 594

Jackson

Immunoresearch

RRID:

AB_2340433

Rabbit anti-Iba1

FUJIFILM Wako

Pure Chemical

Corporation

#019-19741

Rabbit anti-ATF4

Cell Signaling

#11815

Journal Pre-proof

GAPDH (14C10) Rabbit Monoclonal Antibody

Cell Signaling

#2118

Peroxidase AffiniPure® Donkey Anti-Rabbit IgG (H+L)

Jackson

Immunoresearch

RRID: AB_100

15282

Chemicals, peptides, and recombinant proteins

Darifenacin hydrobromide

Sigma-Aldrich

SML1102

Prolong Gold antifade mounting medium

Thermo Fisher

Scientific

#P36930

Triton

Sigma-Aldrich

#X100-500ML

Muscarine

Sigma-Aldrich

#M6532

ATP

Sigma-Aldrich

#A9187

Normal Donkey Serum

Jackson

ImmunoResearch

#017-000-121

Normal Goat Serum

Jackson

ImmunoResearch

#005-000-121

Fluo-4, AM

Invitrogen

F14202

Pluronic

F-127

Invitrogen

P6867

Tubocurarine hydrochloride pentahydrate

Sigma-Aldrich

T2379

Dimethyl sulfoxide (DMSO)

Sigma-Aldrich

D1435

Sodium chloride (NaCl)

Sigma-Aldrich

S7653

Potassium chloride (KCl)

Sigma-Aldrich

P9541

Magnesium chloride hexahydrate (MgCl

.6H

Sigma-Aldrich

M2670

Sodium bicarbonate (NaHCO

)

Sigma-Aldrich

S5761

Calcium chloride dihydrate (CaCl

.2H

Sigma-Aldrich

C5080

D-(+)-Glucose

Sigma-Aldrich

G5767

L-Glutamic acid

Sigma-Aldrich

G8415

L-Glutamine

Sigma-Aldrich

G3126

BES

Sigma-Aldrich

B9879

Thiamine pyrophosphate

Sigma-Aldrich

C8754

Journal Pre-proof

Choline chloride

Sigma-Aldrich

C7527

Formaldehyde

Mecalab Ltee

#1045

Sigma Fast inhibitor tablet

Sigma-Aldrich

S8820

Tris Base

Thermo Fisher

Scientific

BP152

Glycine

Thermo Fisher

Scientific

J64365A1

Ponceau S

Thermo Fisher

Scientific

J60744.18

Tween 20

Thermo Fisher

Scientific

BP337

Glycerol

Sigma-Aldrich

G5516

Bromophenol Blue

Sigma-Aldrich

114391

2-Mercaptoethanol

Thermo Fisher

Scientific

O3446l

Methanol

Thermo Fisher

Scientific

BP1105

Bovine Serum Albumin

Sigma-Aldrich

A7906

NP-40 lysis buffer

Thermo Fisher

Scientific

J60766.AK

Sodium Dodecyl Sulfate (SDS)

Bio-Rad

1610301

Hydrochloric Acid Concentrate

Thermo Fisher

Scientific

SA49

Ethylenediaminetetraacetic acid (EDTA)

Sigma-Aldrich

Critical commercial assays

Pierce

BCA Protein Assay Kit

ThermoFisher

Scientific

23225

Deposited data

Journal Pre-proof

Raw data

Figures 1, S2, S3,

S6 and S7

Raw data

Figures 2-3-4-7

and statistical

information

Raw data

Figure 6

Raw data

Figure 8 (a,b ,f)

and figure S5

Raw data

Figure 8 (c, d, e)

and figure S1

Experimental models: Organisms/strains

Mouse line :

B6.Cg-Tg(SOD1*G37R)29Dpr/J

The Jackson

laboratory

RRID:IMSR_JA

X:008229

Mouse line :

C57BL/6N-

Mdga2

Tg(Prnp-PFN1*G118V)838Kiaei

The Jackson

laboratory

RRID:IMSR_JA

X:030568

Software and algorithms

Prism (version 8-10.0)

GraphPad

Software

www.graphpad.

com

Fluo-View FV10-ASW Viewer

Evident Scientific

https://evidentsc

ientific.com/en/

downloads

ImageJ (version 1.52a)

National Institutes

of Health

https://imagej.ne

t/ij/

Journal Pre-proof

Zen Desk

Zeiss Software

WinWCP (V.5.7.2)

Strathclyde

Electrophysiology

Software

Photoshop (version CC 2019-2025)

Adobe

https://www.ado

be.com/ca/produ

cts/photoshop.ht

Illustrator (version 7-10 and CC 2019-2025)

Adobe

Other

Superfrost plus Gold Slides

Fisher Scientific

22-035813

PVDF blotting membranes

Thermo Fisher

Scientific

Cat#88518

4-15% Mini protean tgx-stain-Free gels

Bio-Rad

Laboratories

Canada Ltd

#4568083

Journal Pre-proof