PERK modulation, with GSK2606414, Sephin1 or salubrinal, failed to produce therapeutic benefits in the SOD1G93A mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) has been linked to overactivity of the protein kinase RNA-like ER kinase (PERK) branch of the unfolded protein response (UPR) pathway, both in ALS patients and mouse models. However, attempts to pharmacologically modulate PERK for therapeutic benefit have yielded inconsistent and often conflicting results. This study sought to address these discrepancies by comprehensively evaluating three commonly used, CNS-penetrant, PERK modulators (GSK2606414, salubrinal, and Sephin1) in the same experimental models, with the goal of assessing the viability of targeting the PERK pathway as a therapeutic strategy for ALS. To achieve this goal, a tunicamycin-challenge assay was developed using wild-type mice to monitor changes in liver UPR gene expression in response to PERK pathway modulation. Subsequently, multiple dosing regimens of each PERK modulator were tested in standardized, well-powered, gender-matched, and litter-matched survival efficacy studies using the SOD1G93A mouse model of ALS. The alpha-2-adrenergic receptor agonist clonidine was also tested to elucidate the results obtained from the Sephin1, and of the previously reported guanabenz studies, by comparing the effects of presence or absence of α-2 agonism. The results revealed that targeting PERK may not be an ideal approach for ALS treatment. Inhibiting PERK with GSK2606414 or activating it with salubrinal did not confer therapeutic benefits. While Sephin1 showed some promising therapeutic effects, it appears that these outcomes were mediated through PERK-independent mechanisms. Clonidine also produced some favorable therapeutic effects, which were unexpected and not linked to the UPR. In conclusion, this study highlights the challenges of pharmacologically targeting PERK for therapeutic purposes in the SOD1G93A mouse model and suggests that exploring other targets within, and outside, the UPR may be more promising avenues for ALS treatment.

Reduced C9orf72 expression exacerbates polyGR toxicity in patient iPSC-derived motor neurons and a Type I protein arginine methyltransferase inhibitor reduces that toxicity.

Introduction: Intronic repeat expansions in the C9orf72 gene are the most frequent known single genetic causes of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). These repeat expansions are believed to result in both loss-of-function and toxic gain-of-function. Gain-of-function results in the production of toxic arginine-rich dipeptide repeat proteins (DPRs), namely polyGR and polyPR. Small-molecule inhibition of Type I protein arginine methyltransferases (PRMTs) has been shown to protect against toxicity resulting from polyGR and polyPR challenge in NSC-34 cells and primary mouse-derived spinal neurons, but the effect in human motor neurons (MNs) has not yet been explored. Methods: To study this, we generated a panel of C9orf72 homozygous and hemizygous knockout iPSCs to examine the contribution of C9orf72 loss-of-function toward disease pathogenesis. We differentiated these iPSCs into spinal motor neurons (sMNs). Results: We found that reduced levels of C9orf72 exacerbate polyGR15 toxicity in a dose-dependent manner. Type I PRMT inhibition was able to partially rescue polyGR15 toxicity in both wild-type and C9orf72-expanded sMNs. Discussion: This study explores the interplay of loss-of-function and gain-of-function toxicity in C9orf72 ALS. It also implicates type I PRMT inhibitors as a possible modulator of polyGR toxicity.

A machine-learning based objective measure for ALS disease severity.

Amyotrophic Lateral Sclerosis (ALS) disease severity is usually measured using the subjective, questionnaire-based revised ALS Functional Rating Scale (ALSFRS-R). Objective measures of disease severity would be powerful tools for evaluating real-world drug effectiveness, efficacy in clinical trials, and for identifying participants for cohort studies. We developed a machine learning (ML) based objective measure for ALS disease severity based on voice samples and accelerometer measurements from a four-year longitudinal dataset. 584 people living with ALS consented and carried out prescribed speaking and limb-based tasks. 542 participants contributed 5814 voice recordings, and 350 contributed 13,009 accelerometer samples, while simultaneously measuring ALSFRS-R scores. Using these data, we trained ML models to predict bulbar-related and limb-related ALSFRS-R scores. On the test set (n = 109 participants) the voice models achieved a multiclass AUC of 0.86 (95% CI, 0.85-0.88) on speech ALSFRS-R prediction, whereas the accelerometer models achieved a median multiclass AUC of 0.73 on 6 limb-related functions. The correlations across functions observed in self-reported ALSFRS-R scores were preserved in ML-derived scores. We used these models and self-reported ALSFRS-R scores to evaluate the real-world effects of edaravone, a drug approved for use in ALS. In the cohort of 54 test participants who received edaravone as part of their usual care, the ML-derived scores were consistent with the self-reported ALSFRS-R scores. At the individual level, the continuous ML-derived score can capture gradual changes that are absent in the integer ALSFRS-R scores. This demonstrates the value of these tools for assessing disease severity and, potentially, drug effects.

Hypothesis and Theory: Roles of Arginine Methylation in C9orf72-Mediated ALS and FTD.

Hexanucleotide repeat expansion (G4C2n) mutations in the gene C9ORF72 account for approximately 30% of familial cases of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), as well as approximately 7% of sporadic cases of ALS. G4C2n mutations are known to result in the production of five species of dipeptide repeat proteins (DRPs) through non-canonical translation processes. Arginine-enriched dipeptide repeat proteins, glycine-arginine (polyGR), and proline-arginine (polyPR) have been demonstrated to be cytotoxic and deleterious in multiple experimental systems. Recently, we and others have implicated methylation of polyGR/polyPR arginine residues in disease processes related to G4C2n mutation-mediated neurodegeneration. We previously reported that inhibition of asymmetric dimethylation (ADMe) of arginine residues is protective in cell-based models of polyGR/polyPR cytotoxicity. These results are consistent with the idea that PRMT-mediated arginine methylation in the context of polyGR/polyPR exposure is harmful. However, it remains unclear why. Here we discuss the influence of arginine methylation on diverse cellular processes including liquid-liquid phase separation, chromatin remodeling, transcription, RNA processing, and RNA-binding protein localization, and we consider how methylation of polyGR/polyPR may disrupt processes essential for normal cellular function and survival.

Type I PRMT Inhibition Protects Against C9ORF72 Arginine-Rich Dipeptide Repeat Toxicity.

Repeat expansion mutations in the C9ORF72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeat-associated non-AUG translation of this expansion produces dipeptide repeat proteins (DRPs). The arginine containing DRPs, polyGR and polyPR, are consistently reported to be the most toxic. Here we demonstrated that small molecule inhibition of type I protein arginine methyltransferases (PRMT) protects against polyGR and polyPR toxicity. Furthermore, our findings suggest that asymmetric dimethylation of polyGR and polyPR by Type I PRMTs plays important roles in their cytotoxicity.

Primary Neurons and Differentiated NSC-34 Cells Are More Susceptible to Arginine-Rich ALS Dipeptide Repeat Protein-Associated Toxicity than Non-Differentiated NSC-34 and CHO Cells.

A repeat expansion mutation in the C9orf72 gene is the most common known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In this study, using multiple cell-based assay systems, we reveal both increased dipeptide repeat protein (DRP) toxicity in primary neurons and in differentiated neuronal cell lines. Using flow cytometry and confocal laser scanning microscopy of cells treated with fluorescein isothiocyanate (FITC)-labeled DRPs, we confirm that poly-glycine-arginine (GR) and poly-proline-arginine (PR) DRPs entered cells more readily than poly-glycine-proline (GP) and poly-proline-alanine (PA) DRPs. Our findings suggest that the toxicity of C9-DRPs may be influenced by properties associated with differentiated and aging motor neurons. Further, our findings provide sensitive cell-based assay systems to test phenotypic rescue ability of potential interventions.

SOD1-positive aggregate accumulation in the CNS predicts slower disease progression and increased longevity in a mutant SOD1 mouse model of ALS.

Non-natively folded variants of superoxide dismutase 1 (SOD1) are thought to contribute to the pathogenesis of familial amyotrophic lateral sclerosis (ALS), however the relative toxicities of these variants are controversial. Here, we aimed to decipher the relationships between the different SOD1 variants (aggregated, soluble misfolded, soluble total) and the clinical presentation of ALS in the SOD1G93A mouse. Using a multi-approach strategy, we found that the CNS regions least affected by disease had the most aggregated SOD1. We also found that the levels of aggregated SOD1 in the spinal cord were inversely correlated with the disease progression. Conversely, in the most affected regions, we observed that there was a high soluble misfolded/soluble total SOD1 ratio. Taken together, these findings suggest that soluble misfolded SOD1 may be the disease driver in ALS, whereas aggregated SOD1 may serve to sequester the toxic species acting in a neuroprotective fashion.

A High-throughput qPCR-based Method to Genotype the SOD1G93A Mouse Model for Relative Copy Number.

The most commonly used mouse model in ALS preclinical research expresses multiple copies of the human SOD1 (G93A) transgene. During the course of breeding, successive generations of mice can lose copies of the transgene. Because shorter lifespan of these mice is dependent on transgene copy number, it is essential to ensure that no low-copy, and therefore longer-lived, mice are included in preclinical studies. Existing techniques for SOD1G93A mouse genotyping are broadly based on creating a standard curve using a reference gene and deducing the relative amount of SOD1 by comparison with the standard curve. This type of technique is used in Alexander et al. (2004) , Vieira et al. (2017) and Maier et al. (2018) . However, it is not described in detail (see Note 1). This paper provides a detailed protocol for determining the relative copy number of the human SOD1 transgene. Briefly, the protocol involves first the extraction of high-quality genomic DNA from mouse ear tissue, creation of a genomic DNA concentration-based standard curve, and qPCR analysis of up to 88 samples at once alongside the standard curve with Gapdh as a reference gene. Analysis involves the normalization of each unknown sample using the standard curve followed by determination of the copy number of the sample relative to the cohort median. This protocol has been optimized to produce high-quality genomic DNA and consistent results, and the relative copy number cutoffs have been optimized and validated empirically by comparison of relative copy number and mouse lifespan.

A human-derived antibody targets misfolded SOD1 and ameliorates motor symptoms in mouse models of amyotrophic lateral sclerosis.

Mutations in the gene encoding superoxide dismutase 1 (SOD1) lead to misfolding and aggregation of SOD1 and cause familial amyotrophic lateral sclerosis (FALS). However, the implications of wild-type SOD1 misfolding in sporadic forms of ALS (SALS) remain unclear. By screening human memory B cells from a large cohort of healthy elderly subjects, we generated a recombinant human monoclonal antibody (α-miSOD1) that selectively bound to misfolded SOD1, but not to physiological SOD1 dimers. On postmortem spinal cord sections from 121 patients with ALS, α-miSOD1 antibody identified misfolded SOD1 in a majority of cases, regardless of their SOD1 genotype. In contrast, the α-miSOD1 antibody did not bind to its epitope in most of the 41 postmortem spinal cord sections from non-neurological control (NNC) patients. In transgenic mice overexpressing disease-causing human SOD1G37R or SOD1G93A mutations, treatment with the α-miSOD1 antibody delayed the onset of motor symptoms, extended survival by up to 2 months, and reduced aggregation of misfolded SOD1 and motor neuron degeneration. These effects were obtained whether α-miSOD1 antibody treatment was administered by direct brain infusion or peripheral administration. These results support the further development of α-miSOD1 antibody as a candidate treatment for ALS involving misfolding of SOD1.

CuATSM efficacy is independently replicated in a SOD1 mouse model of ALS while unmetallated ATSM therapy fails to reveal benefits.

A copper chelator known as diacetylbis(N(4)-methylthiosemicarbazonato) copper II (CuATSM), has been reported to be efficacious in multiple transgenic SOD1 models of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder affecting motor neurons. Here we report that we also observed CuATSM efficacy on disease onset and progression in a standardized litter-matched and gender-balanced efficacy study using B6SJL-SOD1G93A/1Gur mice. We also report improved survival trends with CuATSM treatment. In addition, we report a lack of efficacy by unmetallated ATSM in the same model using the same standardized study design. These results add to existing evidence supporting an efficacious role for copper delivery using chaperone molecules in mouse models of ALS.

A Quick Phenotypic Neurological Scoring System for Evaluating Disease Progression in the SOD1-G93A Mouse Model of ALS.

The SOD1-G93A transgenic mouse is the most widely used animal model of amyotrophic lateral sclerosis (ALS). At ALS TDI we developed a phenotypic screening protocol, demonstrated in video herein, which reliably assesses the neuromuscular function of SOD1-G93A mice in a quick manner. This protocol encompasses a simple neurological scoring system (NeuroScore) designed to assess hindlimb function. NeuroScore is focused on hindlimb function because hindlimb deficits are the earliest reported neurological sign of disease in SOD1-G93A mice. The protocol developed by ALS TDI provides an unbiased assessment of onset of paresis (slight or partial paralysis), progression and severity of paralysis and it is sensitive enough to identify drug-induced changes in disease progression. In this report, the combination of a detailed manuscript with video minimizes scoring ambiguities and inter-experimenter variability thus allowing for the protocol to be adopted by other laboratories and enabling comparisons between studies taking place at different settings. We believe that this video protocol can serve as an excellent training tool for present and future ALS researchers.

Guanabenz Treatment Accelerates Disease in a Mutant SOD1 Mouse Model of ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by loss of motor neurons. The mechanisms leading to motor neuron degeneration in ALS are unclear. However, there is evidence for involvement of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in ALS, notably in mutant SOD1 mediated models of ALS. Stress induced phosphorylation of the eIF2 alpha subunit by eukaryotic translation initiation factor 2-alpha kinase 3 Perk activates the UPR. Guanabenz is a centrally acting alpha2 adrenergic receptor agonist shown to interact with a regulatory subunit of the protein phosphatase, Pp1/Gadd34, and selectively disrupt the dephosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eif2alpha). Here we demonstrate that guanabenz is protective in fibroblasts expressing G93A mutant SOD1 when they are exposed to tunicamycin mediated ER stress. However, in contrast to other reports, guanabenz treatment accelerated ALS-like disease progression in a strain of mutant SOD1 transgenic ALS mice. This study highlights challenges of pharmacological interventions of cellular stress responses in whole animal models of ALS.

Dexpramipexole is ineffective in two models of ALS related neurodegeneration.

Treatment options for people living with amyotrophic lateral sclerosis (ALS) are limited and ineffective. Recently, dexpramipexole (RPPX) was advanced into human ALS clinical trials. In the current studies, we investigated RPPX in two parallel screening systems: 1) appropriately powered, sibling-matched, gender-balanced survival efficacy screening in high-copy B6-SJL-SOD1G93A/Gur1 mice, and 2) high-content neuronal survival screening in primary rat cortical neurons transfected with wild-type human TDP43 or mutant human TDP43. In both cases, we exposed the test systems to RPPX levels approximating those achieved in human Phase II clinical investigations. In SOD1G93A mice, no effect was observed on neuromotor disease progression or survival. In primary cortical neurons transfected with either mutant or wild-type human TDP43, a marginally significant improvement in a single indicator of neuronal survival was observed, and only at the 10 µM RPPX treatment. These systems reflect both mutant SOD1- and TDP43-mediated forms of neurodegeneration. The systems also reflect both complex non-cell autonomous and neuronal cell autonomous disease mechanisms. The results of these experiments, taken in context with results produced by other molecules tested in both screening systems, do not argue positively for further study of RPPX in ALS.

C57BL/6J congenic Prp-TDP43A315T mice develop progressive neurodegeneration in the myenteric plexus of the colon without exhibiting key features of ALS.

ALS therapy development has been hindered by the lack of rodent animal models. The discovery of TDP-43, a transcription factor that accumulates in the cytoplasm of motor neurons (MNs) in most cases of ALS, prompted attempts to develop TDP-43-based models of the disease. The current study sought to examine, in extensive detail, the emerging disease phenotype of a transgenic mouse model that overexpresses a mutant human TDP-43 (hTDP-43) gene under mouse prion promoter control. Careful attention was given to ALS-like characteristics to determine the appropriateness of this model for testing therapies for ALS. In light of previous reports that gastrointestinal (GI) dysfunction is responsible for early death in these mice, gut immunohistochemistry (IHC) and longitudinal gut motility assays were used to identify the onset and the progression of these defects. IHC studies revealed that site-specific overexpression of the hTDP-43 transgene in colonic myenteric plexes resulted in progressive neurodegeneration in this region. This change was associated with progressively reduced GI motility, culminating in frank stasis that was primarily responsible for decreasing longevity in these mice. The disease phenotype was gender- and genetic background-dependent, with congenic C57BL/6J male mice exhibiting the most aggressive form of the disease. Spinal cord IHC revealed ubiquitin-positive inclusions, but not TDP-43 aggregates, in the cytoplasm of MNs. Neither gender exhibited compelling ALS-like neuromuscular deficits, irrespective of age. While this model may be useful for studying GI tract neurodegeneration, in its present state it does not display a phenotype suitable for testing ALS therapeutics.

From transcriptome analysis to therapeutic anti-CD40L treatment in the SOD1 model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of motor neurons. Using unbiased transcript profiling in an ALS mouse model, we identified a role for the co-stimulatory pathway, a key regulator of immune responses. Furthermore, we observed that this pathway is upregulated in the blood of 56% of human patients with ALS. A therapy using a monoclonal antibody to CD40L was developed that slows weight loss, delays paralysis and extends survival in an ALS mouse model. This work demonstrates that unbiased transcript profiling can identify cellular pathways responsive to therapeutic intervention in a preclinical model of human disease.

Design, power, and interpretation of studies in the standard murine model of ALS.

Identification of SOD1 as the mutated protein in a significant subset of familial amyotrophic lateral sclerosis (FALS) cases has led to the generation of transgenic rodent models of autosomal dominant SOD1 FALS. Mice carrying 23 copies of the human SOD1(G93A) transgene are considered the standard model for FALS and ALS therapeutic studies. To date, there have been at least 50 publications describing therapeutic agents that extend the lifespan of this mouse. However, no therapeutic agent besides riluzole has shown corresponding clinical efficacy. We used computer modeling and statistical analysis of 5429 SOD1(G93A) mice from our efficacy studies to quantify the impact of several critical confounding biological variables that must be appreciated and should be controlled for when designing and interpreting efficacy studies. Having identified the most critical of these biological variables, we subsequently instituted parameters for optimal study design in the SOD1(G93A) mouse model. We retested several compounds reported in major animal studies (minocycline, creatine, celecoxib, sodium phenylbutyrate, ceftriaxone, WHI-P131, thalidomide, and riluzole) using this optimal study design and found no survival benefit in the SOD1(G93A) mouse for any compounds (including riluzole) administered by their previously reported routes and doses. The presence of these uncontrolled confounding variables in the screening system, and the failure of these several drugs to demonstrate efficacy in adequately designed and powered repeat studies, leads us to conclude that the majority of published effects are most likely measurements of noise in the distribution of survival means as opposed to actual drug effect. We recommend a minimum study design for this mouse model to best address and manage this inherent noise and to facilitate more significant and reproducible results among all laboratories employing the SOD1(G93A) mouse.

Impact of IL-1beta on flow-induced outward arterial remodeling.

BACKGROUND: Flow reduction upregulates arterial wall interleukin 1beta (IL-1beta), and IL-1beta independently modulates intimal hyperplasia under low flow conditions. We hypothesized that IL-1beta expression is also augmented under high flow, and outward remodeling occurs by way of IL-1beta-dependent mechanisms. METHODS: Carotid artery (CA) flow was surgically augmented in rabbits (n = 20). CAs were harvested at 1, 3, 7, and 14 days, and assayed via quantitative reverse transcriptase-polymerase chain reaction. IL-1 receptor I null mice (KO) and wild-type controls underwent unilateral CA ligation and harvest 4 weeks later to assess the impact of increased flow on the contralateral CA (n = 82). RESULTS: The rabbit model led to an immediate 36% increase in contralateral flow (P = .01) with an 80% increase at 14 days (P = .016) with subsequent positive remodeling. High flow induced IL-1beta messenger RNA expression (114-fold at 1 day, P < .05), with levels remaining elevated through 14 days (61-fold, P < .05). In murine experiments, CA ligation resulted in a 44% increase in contralateral flow. Wild-type and KO animals responded with equivalent 83% and 78% increases in luminal area (P = .87). CONCLUSIONS: Positive and negative perturbations of arterial blood flow induce IL-1beta in a time-dependent fashion. However, as opposed to intimal hyperplasia after flow reduction, positive arterial remodeling in response to increased flow occurs via IL-1beta independent mechanisms.