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\begin{document}

\title{Novel FKBP12 ligand promotes functional improvement in SOD1G93A ALS mice}



\author[1]{Laura Moreno-Martínez}%
\author[2]{Núria Gaja-Capdevila}%
\author[3]{Laura Mosqueira-Martin}%
\author[2]{Mireia Herrando-Grabulosa}%
\author[3]{Klaudia Gonzalez-Imaz}%
\author[1]{Ana Cristina Calvo}%
\author[4]{Maialen Sagartzazu-Aizpurua}%
\author[5]{Leticia Moreno-García}%
\author[6]{Jose Fuentes}%
\author[7]{Abraham Acevedo-Arozena}%
\author[4]{Jesus Aizpurua}%
\author[4]{Jose Miranda}%
\author[8]{Adolfo López de Munaín}%
\author[9]{Ainara Vallejo-Illarramendi}%
\author[1]{Rosario Osta}%
\author[2]{Xavier  Navarro}%
\author[3]{Francisco Gil-Bea}%
\affil[1]{University of Zaragoza}%
\affil[2]{Universitat Autònoma de Barcelona}%
\affil[3]{Biodonostia Health Research Institute}%
\affil[4]{UPV/EHU QO-I}%
\affil[5]{University of Zaragoza Department of Anatomy Embryology and Animal Genetics}%
\affil[6]{Universidad de Extremadura Facultad de Enfermeria y Terapia Ocupacional}%
\affil[7]{University Hospital of the Canary Islands}%
\affil[8]{Hospital Universitario de Donostia}%
\affil[9]{UPV/EHU Facultad de Medicina y Enfermería}%


\vspace{-1em}



  
  \date{May 25, 2024}


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\selectlanguage{english}
\begin{abstract}
Background and Purpose: Amyotrophic lateral sclerosis (ALS) is a
devastating neurodegenerative disease with limited treatment options.
ALS pathogenesis involves intricate processes within motor neurons
(MNs), characterized by dysregulated Ca2+ influx and buffering in early
ALS-affected MNs. This study proposes the modulation of ryanodine
receptors (RyRs), key mediators of intracellular Ca2+, as a therapeutic
target. Experimental Approach: A novel class of novel FKBP12 ligands
that show activity as cytosolic calcium modulators through stabilizing
RyR channel activity, were tested in the SOD1G93A mouse model of ALS.
Different outcomes were used to assess treatment efficacy including
electrophysiology, histopathology, neuromuscular function, and survival.
Key Results: Among the novel FKBP12 ligands, MP-010 was chosen for its
central nervous system availability. Chronic administration of MP-010 to
SOD1G93A mice produced a dose-dependent preservation of motor nerve
conduction, with the 61 mg/kg dose significantly delaying the onset of
motor impairment. This was accompanied by improved motor coordination,
increased innervated endplates, and significant preservation of MNs in
the spinal cord of treated mice. Notably, MP-010 treatment significantly
extended lifespan by an average of 10 days compared to vehicle.
Conclusions and Implications: FKBP12 ligands, particularly MP-010,
exhibit promising neuroprotective effects in ALS, highlighting their
potential as novel therapeutic agents. Further investigations into the
molecular mechanisms and clinical translatability of these compounds are
needed for their application in ALS treatment.%
\end{abstract}%



\sloppy


\textbf{INTRODUCTION}

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative
disease that targets both upper and lower motor neurons within the brain
and spinal cord. Tragically, ALS is characterized by a brief survival
span of 2-3 years, with only 10-20\% of patients surpassing a decade of
life post-diagnosis. Familial cases, attributable to genetic mutations,
account for 5-10\% of all ALS cases, with the rest classified as
sporadic . Despite intense ongoing research, current medications such as
Riluzole, Edaravone, Relyvrio, and Qalsody provide only a modest delay
in disease progression. The pathogenesis of ALS is a complex interplay
of multiple processes occurring within motor neurons (MNs), encompassing
amongst other proposed mechanism: excitotoxicity, endoplasmic reticulum
(ER) stress, mitochondrial dysfunction, oxidative stress, RNA processing
aberrations, protein misfolding, endosomal trafficking disruptions,
impaired axonal transport or neuroinflammation . Computational models
have underscored the significance of disturbances in calcium
(Ca\textsuperscript{2+}) homeostasis and energy imbalances as pivotal
factors predicting neuronal dysfunction and degeneration in ALS .

One distinguishing feature in early ALS-affected MNs is the excessive
influx of Ca\textsuperscript{2+} ions due to high-frequency rhythmic
activity and an increased expression of Ca\textsuperscript{2+}-permeable
AMPA receptors . These MNs exhibit low expression of
Ca\textsuperscript{2+} buffering proteins like parvalbumin and calbindin
. Overexpression of these proteins has been shown to confer resistance
to ALS-induced toxicity in MNs . Conversely, MNs that exhibit resistance
to ALS degeneration, such as those of oculomotor and Onuf's nuclei,
possess higher levels of Ca\textsuperscript{2+} buffering proteins .
Notably, ALS-associated mutations, including those in \emph{VAPB}
,\emph{SIGMAR1} and \emph{SOD1} can potentiate
Ca\textsuperscript{2+}deregulation and heighten vulnerability to the
effects of Ca\textsuperscript{2+} influx . These findings emphasize the
inherent susceptibility of MNs to intracellular Ca\textsuperscript{2+}
overload as a significant risk factor for degeneration .

Mitochondria play a pivotal role in regulating intracellular
Ca\textsuperscript{2+} levels through the mitochondrial uniporter,
particularly crucial in MNs with low intrinsic cytosolic
Ca\textsuperscript{2+} buffering capacity. In these vulnerable MNs,
mitochondria are responsible for absorbing over 50\% of intracellular
Ca\textsuperscript{2+} increases, setting them apart from other cell
types. Consequently, the pronounced demand placed on specialized
Ca\textsuperscript{2+} storage organelles makes MNs even more
susceptible to Ca\textsuperscript{2+} imbalances , especially in ALS
where mitochondria are frequently damaged . Indeed, impaired
mitochondrial Ca\textsuperscript{2+} buffering in MNs derived from
patient-induced pluripotent stem cells carrying mutations
in\emph{TARDBP} or \emph{C9orf72} has been reported .

Apart from excessive Ca\textsuperscript{2+} influx and diminished
Ca\textsuperscript{2+} buffering capacity, augmented
Ca\textsuperscript{2+} leakage from ER stores in MNs can further
contribute to cytosolic Ca\textsuperscript{2+} overload. Ryanodine
receptors (RyR) are tetrameric channels that play a crucial role in
mediating Ca\textsuperscript{2+}-induced Ca\textsuperscript{2+} release
from the ER, play a crucial role in this process . RyR channels are
activated by Ca\textsuperscript{2+} and can also be triggered by the
oxidation of redox-sensing thiols by reactive oxygen species (ROS),
creating a positive feedback loop exacerbating pathological cytoplasmic
Ca\textsuperscript{2+} overload . The endogenous ligand of RyR, FKBP12
(calstabin), normally stabilizes RyR, preventing
Ca\textsuperscript{2+}leakage from the ER. Reduced FKBP12 levels in MNs
of ALS patients underscore the importance of maintaining equilibrium
between FKBP12 and RyR in neurodegeneration .

Overall, this cumulative evidence suggests the prospect of RyRs as a
potential therapeutic targets for ameliorating intracellular
Ca\textsuperscript{2+} dysregulation and forestalling
Ca\textsuperscript{2+}-induced neurodegeneration within susceptible
neurons and neuromuscular units in ALS. In this context, we introduce a
novel class of triazole molecules, empirically validated as FKBP12
ligands to facilitate FKBP12-RyR binding. This binding capability serves
to stabilize the Ca\textsuperscript{2+} flux mediated by RyR, as
demonstrated in our previous works . In this study, we introduce MP
compounds, novel FKBP12 ligands that exhibit cytosolic calcium
modulating activity, with the potential to be used for the treatment of
ALS. This proposal is based on the encouraging results observed in
transgenic SOD1\textsuperscript{G93A} mice, a mouse model that
recapitulates the key clinical, electrophysiological, and
histopathological features of the disease. .

\textbf{METHODS}

Preparation of MP compounds

Compounds MP-001 and MP-002, strategically designed to target the
interaction site of FKBP12/RyR1, were synthesized using CuAAC ''click
chemistry'' methodologies. Specifically, aryl propargyl sulfides and
methyl azidoglycinate were utilized for MP-001, while
2-azidoethyl-N,N-dimethylammonium hydrochloride was employed for MP-002
synthesis . Compound MP-010 was prepared by adding phthaloyl peroxide to
a solution of MP-002, followed by stirring at room temperature,
basification with 7 N NH\textsubscript{3} in MeOH, and evaporation under
reduced pressure. The resulting crude product underwent purification
through column chromatography (silica gel; eluent:
CH\textsubscript{2}C\textsubscript{l2} / MeOH (7N NH\textsubscript{3})).
The yield of the purified white solid was 66\%, with\textsuperscript{1}H
and \textsuperscript{13}C NMR spectra confirming the chemical structure:
\textsuperscript{1}H NMR (400 MHz, CD\textsubscript{3}OD) \selectlanguage{greek}δ \selectlanguage{english}7.81 (s,
1H), 7.49 (d, \emph{J} = 8.8 Hz, 2H), 7.95 (d, \emph{J} = 8.8 Hz, 2H),
4.51 (t, \emph{J} = 6.3 Hz, 2H), 4.30 (dd, \emph{J} = 13.7, 1.6 Hz, 2H),
3.86 (s, 3H), 2.82 (t, \emph{J} = 6.3 Hz, 2H), 2.31 (s, 6H).
\textsuperscript{13}C NMR (101 MHz, CD\textsubscript{3}OD) \selectlanguage{greek}δ \selectlanguage{english}164.1,
137.5, 133.5, 127.7, 126.7, 115.9, 59.5, 56.1, 53.6, 45.4. IR
(cm\textsuperscript{-1}): 1592, 1495, 1459, 1303, 1249, 1172, 1086,
1022, 829, 524.

To obtain aqueous 10 mM stock solutions of these compounds for
pharmaceutical application, the free carboxylic acid of MP-001 or the
tertiary amine moiety of MP-010 was neutralized to pH = 6.8 using 2 M
NaOH and 2 M HCl, respectively. These neutralized solutions were further
diluted with drinking water to achieve pharmaceutically acceptable
concentrations of 1 mM or 2 mM prior to administration.

Recordings of ER and cytoplasmic Ca\textsuperscript{2+} in HEK293 cells

HEK293 cells, stably and inducible-expressing WT and mutant R2474S RyR2,
were generated as described and provided by Dr. Takashi Murayama
(Juntendo University). Cells co-express R-CEPIA1er, a calcium-measuring
organelle-entrapped protein indicator located at the ER, and they have
an inducible expression for wild-type RyR2 and mutant RyR2 R2473S
isoforms. 60.000 cells/well were seeded onto 96-well plates coated with
50 \selectlanguage{greek}µ\selectlanguage{english}g/ml poly-D-Lysine, as described previously. Cells were grown in
10\% FBS, DMEM media, at 37\selectlanguage{ngerman}ºC and 5\% CO2 for 24 hours. On day 2, RyR
expression was induced with 2 \selectlanguage{greek}µ\selectlanguage{english}g/ml doxycycline. Induction was achieved
after 24-28 hours, when calcium measurements were performed in
HEPES-buffered Krebs solution (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM
CaCl2, 11 mM D-(+)-Glucose, 5 mM HEPES, distilled H2O to 450 mL, pH
7.4). Fluorescent ratios (F/F0) were calculated as the ratio of the
fluorescence intensities between the initial (F0) and the last (F) 100
seconds by R-CEPIA1er in the Glomax Discover Microplate Reader
(Promega). Caffeine-induced Ca2+ transients were also measured with
Fura2 AM at room temperature using an ECLIPSE Ti/L100 epifluorescence
microscope (Nikon) equipped with a 20X S-Fluor objective, a lambda-DG4
illumination system, an Orca-Flash 2.8 camera (Hamamatsu) and
NisElements-AR software.

In vivo pharmacokinetics in mice

A bolus of 30 mg/kg of test compounds were administered via oral gavage
to isoflurane-anesthetized control mice. Tissue samples, including
muscle, brain, and serum, were collected at 2-, 4-, 6-, and 18-h
post-administration, from three mice at each time point. Collected
tissues were promptly preserved at -80\selectlanguage{ngerman}ºC until further processing.
Frozen muscle and brain samples were pulverized in liquid nitrogen using
a steel mortar immersed in dry ice. Approximately 100 mg of pulverized
tissue and 100 \selectlanguage{greek}µ\selectlanguage{english}L of serum were then incubated with a 1\% formic
acid-acetonitrile solution, with equal volumes of tissue mass for muscle
and brain, and three times for serum. Following a 90-s ultrasound bath
for muscle and a 10-s bath for serum and brain samples, the extracts
underwent centrifugation at 9600 g and 4\selectlanguage{ngerman}ºC for 5 minutes. The resulting
supernatants were collected and stored at -80ºC until further
quantification. Quantitative determination of compounds in the
biological samples was carried out using liquid chromatography-tandem
mass spectrometry (LC-MS/MS) at the Research General Services SGIker of
UPV/EHU (Vitoria-Gasteiz). The pharmacokinetic parameters following oral
administration were calculated using the PKSolver add-in program .
Noncompartmental analysis was employed to compute the pharmacokinetic
parameters of the parent compound. The terminal slope was automatically
estimated using regression with the largest adjusted
R\textsuperscript{2}. The parameters inferred included the terminal
half-life (t\textsubscript{1/2}), maximum concentration
(C\textsubscript{max}), and the time taken to reach the maximum
concentration (T\textsubscript{max}). Subsequently, the brain-to-serum
(Cb:Cs) was calculated based on the obtained pharmacokinetic parameters
.

Animals and experimental design: Treatment of SOD1\textsuperscript{G93A}
mice

We used the hybrid strain of SOD1\textsuperscript{G93A} mouse as an ALS
model (B6SJL-Tg{[}SOD1-G93A{]}1Gur). Non-transgenic wild-type (WT)
littermates were used as controls. The animals used were from colonies
that had been bred in different animal facilities for years, as
specified below. The transgenic offspring was identified by polymerase
chain reaction (PCR) amplification of DNA extracted from the tail, to
identify hemizygous transgenic mice and WT. The colony was maintained
under stander conditions (food and water ad libitum, room temperature of
22 ± 2 °C and 12:12-h light--dark cycle) at the Animal Service of the
UAB and at the animal facilities in Centro de Investigación Biomédica de
Aragón University of Zaragoza. Mice were handled in accordance with the
guidelines of the European Union Council (Directive 2010/63/EU) and
Spanish regulations (RD53/2013) on the use of laboratory animals. All
experimental procedures were approved by the Ethics Committee of the
Universitat Autònoma de Barcelona (CEEAH-UAB: 2969 and 4273) and by the
Ethic Committee for Animal Experiments from the University of Zaragoza
(PI45/18, PI15/21, PI51/21, PI02/23). Humane endpoint criterium in the
survival studies was considered when animals lost the righting reflex
for longer than 30 s.

\emph{Trial 1: Effect of MP-010 on neuromuscular outcomes}

This trial was conducted at the animal facility of the Universitat
Autònoma de Barcelona using the colony of SOD1\textsuperscript{G93A}mice
that has been bred over years by Dr. Navarro's group. At 8 weeks of age
(before drug treatment started), WT and SOD1\textsuperscript{G93A}mice
were distributed among 6 experimental groups, according to their
progenitors, sex, weight, and electrophysiology baseline values in
balanced groups, receiving either vehicle or MP-010 at two different
doses. The compound MP-010 was added to the drinking water at
concentrations of 1 mM or 2 mM, resulting in average daily dosages of 61
mg/kg or 122 mg/kg, respectively. The mice were distributed in six
groups for assessment of neuromuscular outcomes: 1) WT vehicle, this
group consists of WT mice with no treatment (n=4/4; females/males ); 2)
WT MP-010 61 mg/kg (n=5/4); 3) WT MP-010 122 mg/kg (n=2/3); 4)
SOD1\textsuperscript{G93A} vehicle (n=8/8); 5)
SOD1\textsuperscript{G93A} MP-010 61 mg/kg (n=7/8); 6)
SOD1\textsuperscript{G93A} MP-010 122 mg/kg (n=5/4). The treatment was
given from the 8th until the 16th week of age and electrophysiological,
behavioral and histological analyses were performed during this
follow-up.

\emph{Trial 2: Effect of MP-010 on survival, behavioral tests, and
genetic markers}

This trial was conducted at the animal facility of the University of
Zaragoza using the colony of SOD1\textsuperscript{G93A} mice that has
been bred for years by Dr. Osta's group. MP-010 was given to 21
SOD1\textsuperscript{G93A} mice (n=8/13 females/males) in the drinking
water at a concentration of 1 mM, treating the mice at a dose of 61
mg/kg. Simultaneously, another group of 20 SOD1\textsuperscript{G93A}
mice (n=9/11 females/males) was treated with vehicle as the control
group. The treatment started at 8 weeks of age and was weekly renewed
until each mouse reached its respective humane endpoint. Throughout the
treatment, all mice underwent weekly weight measurements and motor
behavioral tests, including rotarod and hanging-wire tests. All
behavioral experiments in the three trials were conducted under blind
conditions regarding the treatment.

\emph{Trial 3: Effect of MP-001} \emph{on survival, behavioral tests and
genetic markers}

This trial was conducted at the animal facility of the University of
Zaragoza using the colony of SOD1\textsuperscript{G93A} mice that has
been bred for years by Dr. Osta's group. This experimental study
included one group of 19 SOD1\textsuperscript{G93A} mice (n=9/10
females/males) to which compound MP-001 was administered in the drinking
water at a concentration of 0.85 mM, resulting in 61 mg/kg dose, and
another similar group of 17 SOD1\textsuperscript{G93A} mice (n=9/8
females/males) treated with vehicle as control. All mice underwent
weekly weight measurements and behavioral tests, including rotarod and
hanging-wire tests, and the survival time was monitored.

Functional evaluation

\emph{Rotarod}

The Rotarod test (ROTA-ROD/RS, LE8200, LETICA, Scientific Instruments;
Panlab, Barcelona, Spain) was conducted on a weekly basis, spanning from
8/9 to 16 weeks of age or until mice were unable to perform the test in
the case of mice from the survival experiment, to assess the motor
coordination, muscle strength, and balance of the experimental animals.
Specifically, the test involved measuring the duration for which each
mouse could maintain its position on a rotating rod set at a constant
speed of 14 revolutions per minute (rpm). Each animal underwent a series
of five trials, and the longest latency achieved without falling was
recorded. An arbitrary cut-off time of 180 seconds was applied in the
assessment. The symptomatic disease onset for each mouse was determined
as the first week when the mouse was unable to keep walking 180 seconds
on the rod.

\emph{Hanging-wire test}

Hanging-wire test was carried out in mice involved in the survival study
to assess the muscular strength. Briefly, each mouse was placed on an
inverted wire lid, and the time it took for the mouse to fall was
recorded. Similar to the rotarod test, the mice had three attempts to
remain on the wire for a maximum of 180 seconds per trial, and the
longest latency was noted.

Nerve conduction tests

Motor nerve conduction tests were performed by percutaneously sciatic
nerve stimulation (with single square pulses of 20 \selectlanguage{greek}μ\selectlanguage{english}s duration)
delivered through a pair of needle electrodes placed at the sciatic
notch. Compound muscle action potentials (CMAP) were recorded from the
tibialis anterior (TA) and plantar interossei (PL) muscles utilizing
microneedle electrodes. Recorded potentials were amplified and displayed
with settings adjusted to measure amplitude from baseline to the maximal
negative peak, latency from the stimulus to the onset of the first
negative deflection, and the duration of the wave . Throughout the
tests, pentobarbital (50 mg/kg i.p.) was used to anesthetize and the
mice body temperature was maintained at a constant level using a
thermostated heating pad. Electrophysiological tests were conducted at 8
(prior to the initiation of drug administration), 12, 14 and 16 weeks of
age.,

Histology

At the 16-week of age mice sacrificed with an overdose of pentobarbital
and underwent transcardial perfusion with a 4\% paraformaldehyde
solution. Following perfusion, the lumbar segment of the spinal cord was
carefully extracted, post-fixed for 2 hours, and subsequently
cryopreserved in a solution of 30\% sucrose in phosphate buffer (PB) at
4\selectlanguage{ngerman}ºC. Then, transverse 20-\selectlanguage{greek}µ\selectlanguage{english}m thick sections of the spinal cord were
serially cut with a cryostat. For assessing the number of MNs, slices of
L4-L5 spinal cord segments separated 100 \selectlanguage{greek}μ\selectlanguage{english}m were stained with a solution
of 3.1mM cresyl violet for 3 h. MNs were identified by their anatomical
localization in the ventral horn of the spinal cord and counted
following strict criteria of their size (diameters lager than 20\selectlanguage{greek}µ\selectlanguage{english}m) and
morphological characteristics (polygonal shape and prominent nucleoli).

Neuromuscular junction assessment

After perfusion, the TA muscle was collected and underwent
cryoprotection in PB 30\% sucrose solution. To label neuromuscular
junctions (NMJ), the muscle was sectioned in longitudinal slices of 50
\selectlanguage{greek}µ\selectlanguage{english}m thickness, collected in sequential series. Following a blocking step
using 5\% normal donkey serum, the sections were incubated for 48 hours
at 4\selectlanguage{ngerman}°C with primary antibodies, chicken anti-neurofilament 200 (NF200,
1:1000; AB5539, Millipore) and rabbit anti-synaptophysin (1:500;
AB32127, Abcam). After washing steps, the sections were incubated
overnight with secondary antibodies Alexa Fluor 594-conjugated secondary
antibody (1:200; A11042-A21207, Invitrogen) and Alexa Fluor
488-conjugated \selectlanguage{greek}α-\selectlanguage{english}bungarotoxin (1:500; B13422, Life Technologies). The
prepared slides were then mounted using Fluoromount-G. Images were
captured under confocal microscopy (LSM 700 Axio Observer, Carl Zeiss,
20x with a numerical aperture of 0.5), and maximum projection images
were generated from z projections with a thickness of 1.3 \selectlanguage{greek}µ\selectlanguage{english}m. To
evaluate the proportion of innervated NMJs, each individual endplate was
classified as either occupied (when presynaptic terminals covered the
endplate) or vacant (when there was no presynaptic label in contact with
the endplate). A total of at least 60 endplates from four different
fields were analyzed for each mouse.

Statistical methods

Data are shown as mean \selectlanguage{ngerman}± SEM. Electrophysiological and functional
measurements were analyzed with Two-way ANOVA, applying Bonferroni
post-hoc test for multiple comparisons. Histological data were analyzed
using One-way ANOVA with Bonferroni multiple comparisons test. The
survival time of mice was evaluated using Kaplan-Meier analysis and
Log-Rank test. Comparisons of means of gene and protein expression were
analyzed by unrelated t-test. The computer-assisted analysis of the
bands obtained in western blot was performed with AlphaEase FC software
(Bonsai Technologies Group, S.A.). Statistical analysis was performed
using SPSS (version 20, IBM, Armonk, NY, USA), and graphs were made
using GraphPad Prism Software (version 5, La Jolla, CA, USA) and
Microsoft Excel. Statistical significance was set at p\textless{}0.05.

\textbf{RESULTS}

Pharmacokinetics and target engagement of novel FKBP12 ligands

The newly designed 4-arylthiomethyl-1-carboxyalkyl-1,2,3-triazoles were
synthesized with the aim of serving as potential ligands that can
concurrently bind to both the ionic and hydrophobic pockets of the
FKBP12/RyR1 complex . These compounds demonstrated in vitro target
engagement with FKBP12 through the rapamycin-inducible FKBP/FRP binding
domain protein fragment complementation assay, thereby stabilizing
cytosolic Ca\textsuperscript{2+} levels in human myotubes under
nitro-oxidative stress . Within this class of compounds, MP-001 (Fig.
1A) demonstrated superior binding affinity to FKBP12 and elicited
co-localization of FKBP12/RyR1 in human myotubes under nitro-oxidative
stress conditions . MP-001 not only attenuated Ca\textsuperscript{2+} ER
leakage through the RyR induced by nitro-oxidative stress but also
demonstrated the capacity to rectify the heightened susceptibility to
spontaneous Ca\textsuperscript{2+} release observed in conditions of ER
Ca\textsuperscript{2+} overload resulting from the RyR2 R2474S mutation,
which disrupts the interaction with the FKBP12.6 subunit . This
manifestation is evidenced by the increased
Ca\textsuperscript{2+}content released upon stimulation with caffeine
(Suppl. Fig. 1).

Polar group exchange from carboxylic acid to N,N-dimethylamino moiety
yielded compound MP-002 (Fig. 1A) with improved blood-brain-brain
barrier (BBB) permeability. However, MP-002 exhibits very rapid
metabolism, which results in low circulating levels, making it
unsuitable for chronic treatment of the central nervous system (CNS).
Concretely, MP-002 displayed a C\textsubscript{max} of 5.1 ± 2.4 ng/mL
with an inappreciable half-life in serum after an oral dosing of 30
mg/kg (data not shown).

Subsequently, for chronic treatment in the
SOD1\textsuperscript{G93A}mouse model, we selected compound MP-010 (Fig.
1A) as an improved version of MP-002, incorporating a sulfoxide
functional group to enhance its pharmacokinetic properties. MP-010
demonstrated favorable oral absorption, with a mean C\textsubscript{max}
of 1144.4 ± 397.2 ng/mL and a half-life of 3.0 h in serum (Fig. 1B), a
C\textsubscript{max} of 1173.1 ± 279.6 ng/g and a half-life of 2.5 h in
muscle (Fig. 1C), and a C\textsubscript{max} of 340.8 ± 166.9 ng/g and a
half-life of 3.7 h in the brain after a dosing of 30 mg/kg (Fig. 1D).
The brain-to-serum concentration ratio (Cb:Cs) of MP-010 was
significantly greater than 0.04, even after 18 hours following dosing
(Fig. 1D). This value is the threshold at which a molecule is commonly
considered to be ''brain penetrant,'' as blood brain volume approximates
4\% of total brain volume .

With respect to the stabilizing properties of RyR, both compounds MP-002
and MP-010 showed a complete rectification of the
Ca\textsuperscript{2+}ER leak caused by the RyR2 R2474S mutation, as
evidenced by the increased Ca\textsuperscript{2+} content released upon
stimulation with caffeine (Suppl. Fig. 1).

Furthermore, we sought to evaluate another compound with in vivo
stabilities comparable to those of MP-010 but with reduced or
insignificant tissue distribution. Our aim was to determine that any
positive effects of MP-010 are attributable to its mechanisms of action
within the CNS. Based on this assumption, we chose MP-001 as an
alternative compound, with a C\textsubscript{max} and half-life of
2179.8 ± 305.9 ng/mL and 2.5 hours in serum (Fig. 1B), 163.0 ± 22.1 ng/g
and 2.5 hours in muscle (Fig. 1C), and 30.3 ± 5.6 ng/g and 1.2 hours in
the brain (Fig. 1D), respectively. Its Cb:Cs ratio was found to be less
than 0.04 (Fig. 1D).

Therefore, we used MP-010 and MP-001 to investigate the hypothesis that
modulating intracellular Ca\textsuperscript{2+} levels by facilitating
the interaction between FKBP12 and RyR may confer neuroprotective
effects in ALS.

Evaluating the efficacy of compound MP-010 in alleviating neuromuscular
deficits in SOD1\textsuperscript{G93A} mice

In the first phase of our study, we examined the potential of MP-010 to
ameliorate motor impairments and neuromuscular dysfunction in
SOD1\textsuperscript{G93A} transgenic mice. To this end, the animals
were given two different doses of MP-010 (61 and 122 mg/kg) (Fig. 2A).
Remarkably, the chronic administration of MP-010 at both doses examined
did not result in any discernible differences in body weight between
treated and vehicle groups of WT or SOD1\textsuperscript{G93A} mice
(Fig. 2B). A notable increase in body weight was noted in the WT groups
compared to the SOD1\textsuperscript{G93A} vehicle-treated group,
particularly at the 12 to 16-week time points (Fig. 2B). This emphasizes
the expected divergence in body weight trajectories between the WT and
SOD1\textsuperscript{G93A} cohorts throughout the study.

Electrophysiological assessments during the follow-up revealed that
MP-010 treatment at a dose of 61 mg/kg prevented the decline of CMAP
amplitude compared to untreated SOD1\textsuperscript{G93A} mice (Fig.
2C). Notably, at early (12 weeks), intermediate (14 weeks), and advanced
(16 weeks) disease stages of the disease, MP-010 at 61 mg/kg exerted
significant effects on CMAP amplitude preservation in the TA muscle of
SOD1\textsuperscript{G93A} mice (Fig. 2C). In contrast, mice treated
with the 122 mg/kg dose did not manifest a substantial improvement
compared to the vehicle-treated group (Fig. 2C). In the PL muscle there
were no significant improvements with any of the doses of MP-010 in the
treated SOD1\textsuperscript{G93A} mice (Fig. 2D).

The rotarod test was used to assess locomotion and coordination, and to
pinpoint the initiation of the symptomatic phase of the disease. The
administration of MP-010 at the dose of 61 mg/kg yielded a significant
improvement in rotarod performance at an advanced disease stage (14-16
weeks of age in SOD1\textsuperscript{G93A} mice) (Fig. 2E), delaying the
onset of motor impairment by 2 weeks compared to untreated animals (13
weeks). In contrast, in accordance with the CMAP data, there was no
change in rotarod performance between SOD1\textsuperscript{G93A} mice
treated with 122 mg/kg and vehicle control. The beneficial effects of
the 61 mg/kg regime were indicative of a deceleration in the progression
of the disease, highlighting the potential therapeutic impact of MP-010
on mitigating motor deficits in SOD1\textsuperscript{G93A} mice.

At the end of the functional follow-up (16 weeks of age), a significant
increase in the number of innervated NMJ within the TA muscle in
SOD1\textsuperscript{G93A} mice receiving MP-010 treatment at both doses
was noticed, compared to the vehicle-treated groups (Fig. 3A,B). This
finding supports the observed preservation of CMAP amplitude in nerve
conduction tests.

Evaluating the efficacy of compound MP-010 in preserving MN loss in
SOD1\textsuperscript{G93A} mice

At the end of functional follow-up, spinal cord was harvested for
histological studies. The quantification of alpha MNs in the ventral
horn of spinal cord sections stained with cresyl violet revealed that
untreated control SOD1\textsuperscript{G93A} mice experienced a loss of
over 60\% of MNs compared to WT mice at 16 weeks of age (Fig. 3C,D). The
administration of MP-010 at 61mg/kg significantly increased the number
of surviving MNs in SOD1\textsuperscript{G93A} mice, whereas the dose of
122 mg/kg MP-010 did not mitigate MN loss (Fig. 3C,D).

Evaluating the efficacy of compound MP-010 in lifespan in
SOD1\textsuperscript{G93A} mice

After observing that MP-010 treatment confers motor and
electrophysiological benefits, leading to reduced MN loss, a second
trial in SOD1\textsuperscript{G93A} mice was conducted to investigate
its potential to extend survival, particularly at the dosage of 61 mg/kg
(Fig. 4A). Our observations revealed that MP-010-treated animals
exhibited reduced weight loss compared to vehicle-treated controls, with
significant differences becoming apparent at 18-19 weeks of age (Fig.
4B). The results obtained in the survival study indicated that MP-010
delayed mortality by approximately 7 days, extending the average
lifespan from 132 ± 8.35 days in vehicle-treated
SOD1\textsuperscript{G93A} mice to 139 ± 10.92 days in MP-010-treated
mice (Fig. 4C).

Analyzing the outcomes of the behavioral tests, we again observed that
MP-010-treated SOD1\textsuperscript{G93A} mice at the dose of 61 mg/kg
demonstrated enhanced rotarod performance at advanced stages compared to
vehicle-treated counterparts (Fig. 4D). Motor strength, assessed in the
hanging-wire test, revealed no significant differences between the two
groups of animals (Fig. 4E).

Evaluating the efficacy of compound MP-001 in survival, motor function
and muscular markers in SOD1\textsuperscript{G93A} mice

We also investigated on SOD1\textsuperscript{G93A} mice the therapeutic
potential of the compound MP-001 (Fig. 5A), characterized by limited or
negligible CNS availability (Fig. 1), thereby confining its mechanism of
action to peripheral tissues. The body weight of all animals was
monitored weekly from 8 weeks of age until 22 weeks. We observed reduced
weight loss in the treated animals, with significant differences at the
13th and 15th weeks of age (Fig. 5B). However, as depicted in the
survival analysis, MP-001 treatment did not significantly extend the
survival of SOD1\textsuperscript{G93A} mice (Fig. 5C). Regarding the
results obtained in the motor tests, no discernible improvement was
observed in the SOD1\textsuperscript{G93A} mice treated with MP-001
(Figure 5D,E).

\textbf{DISCUSSION AND CONCLUSIONS}

Our investigation centered on modulating RyR activity, specifically
through a novel class of triazole molecules known as MP compounds that
act as FKBP12 ligands . The primary focus of our study was on MP-010, a
RyR stabilizer with favorable pharmacokinetics and CNS permeability. Our
results in SOD1\textsuperscript{G93A} mice, a widely accepted ALS model,
demonstrated significant benefits following MP-010 administration.
Notably, increased survival, protection against MN loss, muscle
denervation, and enhanced neuromuscular function were observed,
highlighting the potential of MP-010 as a therapeutic intervention for
ALS. The robustness of our findings is emphasized by their replication
in two distinct colonies of SOD1\textsuperscript{G93A} mice with a mixed
hybrid B6SJL genetic background, across two independent laboratories.
This replication is particularly noteworthy considering the potential
genetic drift that hybrid ALS SOD1 colonies may undergo among different
research labs, leading to variability in disease onset or symptom
severity . This potential confounding factor makes the replication of
our findings in two independent laboratories even more significant.
Remarkably, despite differing ages of motor symptom onset between the
two colonies (14 weeks in one laboratory and 17 weeks in the other),
MP-010 demonstrated beneficial effects in both settings at the dose of
61 mg/kg. The compound exhibited remarkable tolerability and appeared
devoid of apparent toxicity even at high doses. Doubling the dose did
not induce a change in body weight, a recognized clinical symptom of
pharmacological toxicity. Nevertheless, the compound's therapeutic
efficacy was compromised at this higher dose. This unexpected loss of
efficacy could be attributed to off-target effects. These unintended
interactions with non-target proteins could be interfering with the
compound's ability to reach its intended target and exert its
therapeutic effects. Further investigations are warranted to identify
and characterize specific off-target mechanisms, enabling the
development of strategies to mitigate them and optimize the compound's
efficacy.

As previously introduced, cytosolic Ca\textsuperscript{2+} overload
assumes particular importance in the vulnerability of spinal MNs
subjected to ALS toxicity , and deficient Ca\textsuperscript{2+}handling
from ER and mitochondrial stores has emerged as a key player in the
pathogenesis of ALS. This sets the stage for a positive feedback loop
that amplifies pathological Ca\textsuperscript{2+} overload,
intensifying the neurodegenerative processes associated with the disease
. Mutations in genes such as \emph{SIGMA1R} and \emph{VAPB} , identified
in ALS patients, underscore the significance of ER and mitochondrial
proteins in Ca\textsuperscript{2+} homeostasis . In the context of the
present study, the rationale for targeting RyR stemmed from its central
role in Ca\textsuperscript{2+}-induced Ca\textsuperscript{2+} release
from the ER, aiming to prevent excessive cytosolic
Ca\textsuperscript{2+} accumulation, especially in the most vulnerable
MNs associated with ALS. The outcomes of the current study,
demonstrating the therapeutic benefits of a drug promoting the
interaction between RyR and its endogenous ligand FKBP12 in the
SOD1\textsuperscript{G93A} mouse model of ALS, are in accordance with
earlier research that has emphasized reductions in FKBP12 in the context
of ALS . Also in the SOD1\textsuperscript{G93A} mouse, various
alternative strategies to reduce the Ca\textsuperscript{2+} burden on
MNs have been successfully implemented. These strategies include the
application of the AMPA receptor antagonist talampanel and SIGMA1R
agonists , both exerting their effects through the modulation of
extracellular Ca\textsuperscript{2+} influx to cytosol.

The dysregulation of RyR signaling emerges as a fundamental contributor
to the pathogenesis of various neurodegenerative disorders, notably
Alzheimer's disease (AD). Specifically, RyRs have been demonstrated to
interact with amyloid-beta (A\selectlanguage{greek}β\selectlanguage{english}) peptide, a protein known to play a
pivotal role in AD development. The binding of A\selectlanguage{greek}β \selectlanguage{english}to RyRs can modulate
their function, resulting in heightened Ca\textsuperscript{2+} release
from the ER, subsequently leading to excitotoxicity, neuronal death, and
cognitive deficits . Although our investigation cannot definitively
ascertain the existence of Ca\textsuperscript{2+} leakage through RyR in
ALS MNs or whether this pathological phenomenon is replicated in the
SOD1\textsuperscript{G93A} mouse model, the administration of the
compound MP-010 emphasizes the potential therapeutic significance of
targeting FKBP12 to safeguard the RyR-dependent proper release of
Ca\textsuperscript{2+} and alleviate the downstream effects of disrupted
Ca2+ homeostasis. This approach offers a targeted intervention for ALS.
However, while it is important to note that an aberrant increase in
RyR-mediated Ca\textsuperscript{2+} release can contribute to disrupting
neuronal homeostasis, triggering excitotoxicity, conversely, diminished
RyR function may compromise the crucial Ca\textsuperscript{2+} signaling
required for proper neuronal operation. This impairment might lead to a
breakdown in synaptic transmission and plasticity, potentially
contributing to the manifestation of motor deficits in individuals with
ALS. Consequently, inhibiting RyR under this premise may not be a
plausible strategy for treating neurons susceptible to ALS. Indeed,
prior research utilizing dantrolene, an RyR1 inhibitor clinically used
to treat malignant hyperthermia, failed to demonstrate significant
improvements when administered to the SOD1\textsuperscript{G93A} mouse
model , confirming the inadequacy of RyR inhibition as a therapeutic
avenue. In contrast, compounds exhibiting RyR-stabilizing activity,
previously recognized under the nomenclature ''rycals'' , adopt a
distinct approach by modulating RyR channel opening. These compounds
achieve this modulation by facilitating the interaction between the
topoisomerase FKBP12 and RyR, particularly in pathological scenarios
characterized by persistent Ca\textsuperscript{2+} leakage . Thus, the
strategic targeting of RyR modulation through rycal compounds offers a
more nuanced and potentially effective therapeutic strategy compared to
direct inhibition.

Comparative analysis with MP-001, another compound characterized by
limited CNS availability, provides additional insights. The lack of
MP-001 effect underscore the significance of targeting
Ca\textsuperscript{2+} homeostasis at the CNS level to achieve
therapeutic efficacy. Thus, it dismisses the possibility that the
therapeutic effects could be mediated by the modulation of
Ca\textsuperscript{2+} dynamics in cells of the innate immune system. It
is crucial to consider this aspect, especially given that immune cells
heavily rely on Ca\textsuperscript{2+}-dependent processes and tightly
regulated Ca\textsuperscript{2+} homeostasis , and that the innate
immune response appears to play an active role in the pathophysiology of
ALS and the SOD1G93A mouse model .

While our study presents promising results, several avenues for future
research emerge. Elucidating the molecular mechanisms underlying the
observed effects of MP-010, particularly its impact on
Ca\textsuperscript{2+} dynamics and neuronal survival, is critical for a
comprehensive understanding of ALS pathology. Additionally,
considerations of toxicity, dosage optimization, and potential side
effects are paramount before translating these findings into clinical
trials. In conclusion, our study contributes to the evolving landscape
of ALS research by highlighting the therapeutic potential of targeting
intracellular Ca\textsuperscript{2+} dynamics through the modulation of
RyR activity. MP-010, with its favorable preclinical outcomes, offers a
promising avenue for further exploration and potential clinical
development.

\textbf{AUTHOR CONTRIBUTION}

X.N, R.O., A.L.M., A.V-I., F.J.G-B., J.F. and A.A-A. contributed to
design the study. J.I.M., J.M.A., and M.S-A. designed and synthesized
the compounds. L.M-M., N.G-C., L.M. and M.H-G. performed experiments and
analyzed data. X.N, R.O., A.L.M., A.V-I., F.J.G-B, L.M-M., N.G-C., L.M.
contributed to the preparation of the manuscript. X.N, R.O., A.L.M., and
F.J.G-B. supervised and coordinated all the work.

\textbf{FUNDING}

This work was funded by project CIBER-CALS PI2020/08-1, and Grants
CB06/05/1105 and CB06/05/0041 from the Instituto de Salud Carlos III of
Spain, co-funded by European Union (NextGenerationEU, Recovery,
Transformation and Resilience Plan. F.J.G.-B. was funded by the Roche
Stop Fuga de Cerebros program (BIO19/ROCHE/017/BD) and received support
from the IKERBASQUE Research Foundation (IKERBASQUE/PP/2022/003).
A.V.-I. received funding from MCIN/AEI/10.13039/501100011033 (PID
2020-119780RB-I00) and from the Basque Government (IT1732-22). L.M.-M.
holds a PhD fellowship from the UPV/EHU (PIF19/184).

\textbf{ACKNOWLEDGEMENTS}

Dr. Takashi Sakurai is gratefully acknowledged for his generous gift of
HEK293 cells stably and inducibly expressing mutant and WT forms of RyR2
and co-expressing R-CEPIA1er, which were instrumental in the successful
completion of this work. The authors thank for technical and human
support provided by M\selectlanguage{ngerman}ª Carmen Sampedro from SGIker of UPV/EHU and
European funding (ERDF and ESF), and by Neus Hernández, Mónica Espejo
and Jessica Jaramillo from the UAB for their excellent technical
support.

\textbf{CONFLICT OF INTEREST}

F.J.G.-B., A.V.I, J.M.A., A.L.M have equity ownership in Miramoon Pharma
S.L., which is developing novel triazole molecules related to the
research being reported. The terms of this arrangement have been
reviewed and approved by the University of the Basque Country and BIOEF.

\textbf{REFERENCES}

\textbf{FIGURES}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1. Chemical Structure of RyR Stabilizers and
Concentration-Time Profiles in Mice Following a Single Oral Bolus of
MP-010 or MP-001 at a Dose of 30 mg/kg.}

\textbf{A} ) Chemical structures of FKBP12 ligands MP-001, MP-002 and
MP-010. Compound MP-001 restored cytosolic calcium homeostasis in
myotubes but show limited BBB permeability (See Fig. 1A-C). Modification
to MP-002 improved BBB permeability, although the compound displayed too
short serum half-time for systemic administration. Finally, the chemical
incorporation of the sulfoxide function into MP-010 provided favorable
pharmacokinetic parameters for development.

\textbf{B,C,D} ) Control mice were administered a single dose of MP-010
or MP-001 at 30 mg/kg by oral gavage, and their concentrations were
assessed at 2, 4, 6, and 18 h post-dosing in the plasma (\textbf{B} ),
skeletal muscle (\textbf{C} ), and brain (\textbf{D} ).

\textbf{D} ) The calculated mean brain-to-serum (Cb:Cs) ratios were
significantly higher for MP-010 than MP-001 and exhibited values above
the threshold determining penetrability to the brain (Cb:Cs
\textgreater{} 0.04, dashed line).

The presented concentrations and values are the mean \selectlanguage{ngerman}± S.E.M. (n=3).
*p\textless{} 0.05 vs. MP-001-treated mice.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2. Improvement of Motor Function in
SOD1\textsuperscript{G93A} Mice upon MP-010 Treatment.}

\textbf{A)} Schematic diagram of the experimental design for the trial
whose data are shown in this figure and figure 3.

\textbf{B)} Plot of the percentage of body weight for the different mice
groups during the follow-up period. Body weight results are expressed as
a percentage relative to baseline values for each mouse.

\textbf{C-D)} Electrophysiological tests illustrating the preservation
of compound muscle action potentials (CMAP) amplitude in the tibialis
anterior (TA) muscle but not in the plantar interosseous (PL) muscle at
16 weeks of age. Amplitude values of CMAP are presented as a percentage
relative to the baseline value for each mouse.

\textbf{E)} Graph depicting the impact of different MP-010 treatments on
functional outcome in the rotarod test.

Mean \selectlanguage{ngerman}± SEM are represented in the plots. Two-way ANOVA with Bonferroni's
multiple comparisons test was employed. *p \textless{} 0.05 compared to
the SOD1\textsuperscript{G93A} vehicle group. Each colored * represents
the comparison between each group (defined by colors on panel A) and the
SOD\textsuperscript{G93A} vehicle group (grey).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3. Preservation of NMJ Innervation and Spinal MNs in
SOD1\textsuperscript{G93A} Mice upon MP-010 Treatment.}

\textbf{A)} Plot illustrating the percentage of neuromuscular junctions
(NMJ) innervated (overlap of signals) in the different mouse groups.

\textbf{B)} Representative confocal images of NMJ in tibialis anterior
(TA) muscles of mice at 16 weeks of age, presented as maximum
projections generated from 1.3 \selectlanguage{greek}μ\selectlanguage{english}m z projections. Immunolabeling against
\selectlanguage{greek}α-\selectlanguage{english}bungarotoxin (nicotine receptor, in green), neurofilament 200 +
synaptophysin (synapse, in red) was performed to assess NMJ innervation.
Scale bar: 100 \selectlanguage{greek}µ\selectlanguage{english}m.

\textbf{C)} Representative images of lumbar spinal sections depicting
MNs stained with cresyl violet from mice at 16 weeks of age. Scale bar:
100 \selectlanguage{greek}μ\selectlanguage{english}m.

\textbf{D)} Plot showing the number of surviving spinal MNs (mean number
of MNs per section \selectlanguage{ngerman}± SEM) in L4-L5 segments at 16 weeks of age in the
different mouse groups.

Mean ± SEM are represented in the plots. Statistical analysis employed
one-way ANOVA with Bonferroni multiple comparisons test. *p \textless{}
0.05 compared to the SOD1\textsuperscript{G93A} vehicle group.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4. Enhanced Survival and Amelioration of Motor Symptoms
in SOD1\textsuperscript{G93A} Mice Treated with MP-010.}

\textbf{A)} Schematic diagram of the experimental design of this trial.

\textbf{B)} Plot illustrating the percentage of body weight for
different mouse groups during the follow-up period. Body weight results
are expressed as a percentage relative to baseline values for each
mouse.

\textbf{C)} Survival curves indicate that treated mice exhibited a
longer lifespan compared to untreated counterparts, as determined by the
Log-rank (Mantel-Cox) test. *p = 0.017 compared to the
SOD1\textsuperscript{G93A} vehicle group.

\textbf{D)} MP-010 treatment resulted in an improvement in motor
coordination, as assessed by the rotarod test.

\textbf{E)} MP-010 treatment did not lead to improved motor strength, as
evaluated in the hanging-wire test.

Mean \selectlanguage{ngerman}± SEM are represented in the plots. Two-way ANOVA with Bonferroni's
multiple comparisons test was employed. *p \textless{} 0.05 compared to
the SOD1\textsuperscript{G93A} vehicle group.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 5. Marginal extension of survival and attenuation of
weight loss in SOD1\textsuperscript{G93A} mice upon MP-001
administration.}

\textbf{A)} Schematic diagram of the experimental design for the trial
to test MP-001.

\textbf{B)} Mice treated with MP-001 exhibited reduced weight loss at
13-15 weeks of age.

\textbf{C)} Treated mice displayed a slightly longer lifespan compared
to untreated counterparts, but the differences were not statistically
significant, as determined by the Log-rank (Mantel-Cox) Test.

\textbf{D)} The treated and control animals demonstrated comparable
results concerning motor coordination in the rotarod test.

\textbf{E)} SOD1\textsuperscript{G93A} mice under treatment did not
exhibit an improvement in motor strength, as assessed in the
hanging-wire test.

Mean \selectlanguage{ngerman}± SEM are represented in the plots. Two-way ANOVA with Bonferroni's
multiple comparisons test was employed. *p \textless{} 0.05 compared to
the SOD1\textsuperscript{G93A} vehicle group.

\textbf{BULLET POINT SUMMARY}

\textbf{What is already known}

Increased intracellular calcium levels contribute to the pathogenesis of
ALS.

Targeted therapy towards modulating intracellular calcium homeostasis
may have potential in treating ALS.

\textbf{What this study adds}

Introduces a novel class of FKBP12 ligands showing neuroprotection in
the SOD1\textsuperscript{G93A} mouse model of ALS.

Demonstrates the potential therapeutic impact of stabilizing RyR channel
activity for ALS treatment.

\textbf{Clinical significance}

Stabilizing RyR channel activity presents a promising avenue for ALS
treatment with implications for disease progression.

\selectlanguage{english}
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