Mice with endogenous TDP‐43 mutations exhibit gain of splicing function and characteristics of amyotrophic lateral sclerosis

TDP‐43 (encoded by the gene TARDBP) is an RNA binding protein central to the pathogenesis of amyotrophic lateral sclerosis (ALS). However, how TARDBP mutations trigger pathogenesis remains unknown. Here, we use novel mouse mutants carrying point mutations in endogenous Tardbp to dissect TDP‐43 function at physiological levels both in vitro and in vivo. Interestingly, we find that mutations within the C‐terminal domain of TDP‐43 lead to a gain of splicing function. Using two different strains, we are able to separate TDP‐43 loss‐ and gain‐of‐function effects. TDP‐43 gain‐of‐function effects in these mice reveal a novel category of splicing events controlled by TDP‐43, referred to as “skiptic” exons, in which skipping of constitutive exons causes changes in gene expression. In vivo, this gain‐of‐function mutation in endogenous Tardbp causes an adult‐onset neuromuscular phenotype accompanied by motor neuron loss and neurodegenerative changes. Furthermore, we have validated the splicing gain‐of‐function and skiptic exons in ALS patient‐derived cells. Our findings provide a novel pathogenic mechanism and highlight how TDP‐43 gain of function and loss of function affect RNA processing differently, suggesting they may act at different disease stages.     

Gene Targeting of Mouse Tardbp Negatively Affects Masp2 Expression

Amyotrophic Lateral Sclerosis (ALS) is a devastating adult onset neurodegenerative disease affecting both upper and lower motor neurons. TDP-43, encoded by the TARDBP gene, was identified as a component of motor neuron cytoplasmic inclusions in both familial and sporadic ALS and has become a pathological signature of the disease. TDP-43 is a nuclear protein involved in RNA metabolism, however in ALS, TDP-43 is mislocalized to the cytoplasm of affected motor neurons, suggesting that disease might be caused by TDP-43 loss of function. To investigate this hypothesis, we attempted to generate a mouse conditional knockout of the Tardbp gene using the classical Cre-loxP technology. Even though heterozygote mice for the targeted allele were successfully generated, we were unable to obtain homozygotes. Here we show that although the targeting vector was specifically designed to not overlap with Tardbp adjacent genes, the homologous recombination event affected the expression of a downstream gene, Masp2. This may explain the inability to obtain homozygote mice with targeted Tardbp.     

Mitochondrial Dysfunction and Decrease in Body Weight of a Transgenic Knock-in Mouse Model for TDP-43

The majority of amyotrophic lateral sclerosis (ALS) cases as well as many patients suffering from frontotemporal lobar dementia (FTLD) with ubiquitinated inclusion bodies show TDP-43 pathology, the protein encoded by the TAR DNA-binding protein (Tardbp) gene. We used recombinase-mediated cassette exchange to introduce an ALS patient cDNA into the mouse Tdp-43 locus. Expression levels of human A315T TDP-43 protein were 300% elevated in heterozygotes, whereas the endogenous mouse Tdp-43 was decreased to 20% of wild type levels as a result of disturbed feedback regulation. Heterozygous TDP-43^A315TKi mutants lost 10% of their body weight and developed insoluble TDP-43 protein starting as early as 3 months after birth, a pathology that was exacerbated with age. We analyzed the splicing patterns of known Tdp-43 target genes as well as genome-wide gene expression levels in different tissues that indicated mitochondrial dysfunction. In heterozygous mutant animals, we observed a relative decrease in expression of Parkin (Park2) and the fatty acid transporter CD36 along with an increase in fatty acids, HDL cholesterol, and glucose in the blood. As seen in transmission electron microscopy, neuronal cells in motor cortices of TDP-43^A315TKi animals had abnormal neuronal mitochondrial cristae formation. Motor neurons were reduced to 90%, but only slight motoric impairment was detected. The observed phenotype was interpreted as a predisease model, which might be valuable for the identification of further environmental or genetic triggers of neurodegeneration.     

Unmasking the skiptic task of TDP‐43

The mechanism by which mutations in TAR DNA‐binding protein 43 (TDP‐43) cause neurodegeneration remains incompletely understood. In this issue of The EMBO Journal, Fratta et al ( 2018 ) describe how a point mutation in the C‐terminal low complexity domain of TDP‐43 leads to the skipping of otherwise constitutively conserved exons. In vivo, this mutation triggers late‐onset progressive neuromuscular disturbances, as seen in amyotrophic lateral sclerosis (ALS), suggesting that TDP‐43 splicing gain‐of‐function contributes to ALS pathogenesis.     

The Pathological Phenotypes of Human TDP-43 Transgenic Mouse Models Are Independent of Downregulation of Mouse Tdp-43

Tar DNA binding protein 43 (TDP-43) is the major component of pathological deposits in frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) and in amyotrophic lateral sclerosis (ALS). It has been reported that TDP-43 transgenic mouse models expressing human TDP-43 wild-type or ALS-associated mutations recapitulate certain ALS and FTLD pathological phenotypes. Of note, expression of human TDP-43 (hTDP-43) reduces the levels of mouse Tdp-43 (mTdp-43). However, it remained unclear whether the mechanisms through which TDP-43 induces ALS or FTLD-like pathologies resulted from a reduction in mTdp-43, an increase in hTDP-43, or a combination of both. In elucidating the role of mTdp-43 and hTDP-43 in hTDP-43 transgenic mice, we observed that reduction of mTdp-43 in non-transgenic mice by intraventricular brain injection of AAV1-shTardbp leads to a dramatic increase in the levels of splicing variants of mouse sortilin 1 and translin. However, the levels of these two abnormal splicing variants are not increased in hTDP-43 transgenic mice despite significant downregulation of mTdp-43 in these mice. Moreover, further downregulation of mTdp-43 in hTDP-43 hemizygous mice, which are asymptomatic, to the levels equivalent to that of mTdp-43 in hTDP-43 homozygous mice does not induce the pathological phenotypes observed in the homozygous mice. Lastly, the number of dendritic spines and the RNA levels of TDP-43 RNA targets critical for synapse formation and function are significantly decreased in symptomatic homozygous mice. Together, our findings indicate that mTdp-43 downregulation does not lead to a loss of function mechanism or account for the pathological phenotypes observed in hTDP-43 homozygous mice because hTDP-43 compensates for the reduction, and associated functions of mTdp-43. Rather, expression of hTDP-43 beyond a certain threshold leads to abnormal metabolism of TDP-43 RNA targets critical for neuronal structure and function, which might be responsible for the ALS or FTLD-like pathologies observed in homozygous hTDP-43 transgenic mice.     

Reduced Life- and Healthspan in Mice Carrying a Mono-Allelic BubR1 MVA Mutation

Mosaic Variegated Aneuploidy (MVA) syndrome is a rare autosomal recessive disorder characterized by inaccurate chromosome segregation and high rates of near-diploid aneuploidy. Children with MVA syndrome die at an early age, are cancer prone, and have progeroid features like facial dysmorphisms, short stature, and cataracts. The majority of MVA cases are linked to mutations in BUBR1, a mitotic checkpoint gene required for proper chromosome segregation. Affected patients either have bi-allelic BUBR1 mutations, with one allele harboring a missense mutation and the other a nonsense mutation, or mono-allelic BUBR1 mutations combined with allelic variants that yield low amounts of wild-type BubR1 protein. Parents of MVA patients that carry single allele mutations have mild mitotic defects, but whether they are at risk for any of the pathologies associated with MVA syndrome is unknown. To address this, we engineered a mouse model for the nonsense mutation 2211insGTTA (referred to as GTTA) found in MVA patients with bi-allelic BUBR1 mutations. Here we report that both the median and maximum lifespans of the resulting BubR1 ^+/GTTA mice are significantly reduced. Furthermore, BubR1 ^+/GTTA mice develop several aging-related phenotypes at an accelerated rate, including cataract formation, lordokyphosis, skeletal muscle wasting, impaired exercise ability, and fat loss. BubR1 ^+/GTTA mice develop mild aneuploidies and show enhanced growth of carcinogen-induced tumors. Collectively, these data demonstrate that the BUBR1 GTTA mutation compromises longevity and healthspan, raising the interesting possibility that mono-allelic changes in BUBR1 might contribute to differences in aging rates in the general population.     

Spreading of pathological TDP-43 along corticospinal tract axons induces ALS-like phenotypes in Atg5+/- mice

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease, characterized by phosphorylated TDP-43 (pTDP-43)-positive inclusions in neurons and glial cells. However, the pathogenic mechanism that underlies ALS remains largely unknown. To investigate the effects of autophagy deficiency in the formation and spreading of pathological TDP-43 along corticospinal tract axons, TDP-43 preformed fibrils (PFFs) were prepared and unilaterally injected into the fifth layer of the left primary motor cortex (M1) or the left anterior horn of the seventh cervical spinal cord segment (C7) of Atg5^+/- mice. After the injection of TDP-43 PFFs, the elevated levels of pTDP-43 were present in several pyramidal tract-associated regions of Atg5^+/- mice. Additionally, the occurrence of spontaneous potentials detected by electromyography demonstrates evidence of lower motor neuron dysfunction in M1-TDP-43 PFFs-injected Atg5^+/- mice, and prolonged central motor conduction time detected by motor evoked potentials provides evidence of upper motor neuron dysfunction in C7-TDP-43 PFFs-injected Atg5^+/- mice. These results show that injection of TDP-43 PFFs into the M1 or C7 of Atg5^+/- mice induces the spreading of pathological TDP-43 along corticospinal tract axons in both an anterograde and retrograde manner. Importantly, TDP-43 PFFs-injected Atg5^+/- mice also display ALS-like motor dysfunction. Taken together, our findings provide direct evidence that TDP-43 PFFs-injected Atg5^+/- mice exhibited ALS-like neuropathology and motor phenotypes, suggesting that autophagy deficiency promotes the formation and spreading of pathological TDP-43 in vivo.     

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-SOD1^G93A/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 SOD1^G93A 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.     

A mouse model for Li-Fraumeni-Like Syndrome with cardiac angiosarcomas associated to POT1 mutations

The shelterin protein POT1 has been found mutated in many different familial and sporadic cancers, however, no mouse models to understand the pathobiology of these mutations have been developed so far. To address the molecular mechanisms underlying the tumorigenic effects of POT1 mutant proteins in humans, we have generated a mouse model for the human POT1^R117C mutation found in Li-Fraumeni-Like families with cases of cardiac angiosarcoma by introducing this mutation in the Pot1a endogenous locus, knock-in for Pot1a^R117C. We find here that both mouse embryonic fibroblasts (MEFs) and tissues from Pot1a^+/ki mice show longer telomeres than wild-type controls. Longer telomeres in Pot1a^+/ki MEFs are dependent on telomerase activity as they are not found in double mutant Pot1a^+/ki Tert^-/- telomerase-deficient MEFs. By using complementation assays we further show that POT1a pR117C exerts dominant-negative effects at telomeres. As in human Li-Fraumeni patients, heterozygous Pot1a^+/ki mice spontaneously develop a high incidence of angiosarcomas, including cardiac angiosarcomas, and this is associated to the presence of abnormally long telomeres in endothelial cells as well as in the tumors. The Pot1a^+/R117C mouse model constitutes a useful tool to understand human cancers initiated by POT1 mutations.     

Aberrant localization of FUS and TDP43 is associated with misfolding of SOD1 in amyotrophic lateral sclerosis

Background

Amyotrophic lateral sclerosis (ALS) is incurable and characterized by progressive paralysis of the muscles of the limbs, speech and swallowing, and respiration due to the progressive degeneration of voluntary motor neurons. Clinically indistinguishable ALS can be caused by genetic mutations of Cu/Zn superoxide dismutase (SOD1), TAR-DNA binding protein 43 (TDP43), or fused in sarcoma/translocated in liposarcoma (FUS/TLS), or can occur in the absence of known mutation as sporadic disease. In this study, we tested the hypothesis that FUS/TLS and TDP43 gain new pathogenic functions upon aberrant accumulation in the cytosol that directly or indirectly include misfolding of SOD1.

Methodology/Principal Findings

Patient spinal cord necropsy immunohistochemistry with SOD1 misfolding-specific antibodies revealed misfolded SOD1 in perikarya and motor axons of SOD1-familial ALS (SOD1-FALS), and in motor axons of R521C-FUS FALS and sporadic ALS (SALS) with cytoplasmic TDP43 inclusions. SOD1 misfolding and oxidation was also detected using immunocytochemistry and quantitative immunoprecipitation of human neuroblastoma SH-SY5Y cells as well as cultured murine spinal neural cells transgenic for human wtSOD1, which were transiently transfected with human cytosolic mutant FUS or TDP43, or wtTDP43.

Conclusion/Significance

We conclude that cytosolic mislocalization of FUS or TDP43 in vitro and ALS in vivo may kindle wtSOD1 misfolding in non-SOD1 FALS and SALS. The lack of immunohistochemical compartmental co-localization of misfolded SOD1 with cytosolic TDP43 or FUS suggests an indirect induction of SOD1 misfolding followed by propagation through template directed misfolding beyond its site of inception. The identification of a final common pathway in the molecular pathogenesis of ALS provides a treatment target for this devastating disease.     

Activation of p38 MAPK pathway in the skull abnormalities of Apert syndrome Fgfr2 +P253R mice

Background

Apert syndrome is characterized by craniosynostosis and limb abnormalities and is primarily caused by FGFR2 +/P253R and +/S252W mutations. The former mutation is present in approximately one third whereas the latter mutation is present in two-thirds of the patients with this condition. We previously reported an inbred transgenic mouse model with the Fgfr2 +/S252W mutation on the C57BL/6J background for Apert syndrome. Here we present a mouse model for the Fgfr2+/P253R mutation.

Results

We generated inbred Fgfr2 ^+/P253R mice on the same C56BL/6J genetic background and analyzed their skeletal abnormalities. 3D micro-CT scans of the skulls of the Fgfr2 ^+/P253R mice revealed that the skull length was shortened with the length of the anterior cranial base significantly shorter than that of the Fgfr2 ^+/S252W mice at P0. The Fgfr2 ^+/P253R mice presented with synostosis of the coronal suture and proximate fronts with disorganized cellularity in sagittal and lambdoid sutures. Abnormal osteogenesis and proliferation were observed at the developing coronal suture and long bones of the Fgfr2 ^+/P253R mice as in the Fgfr2 ^+/S252W mice. Activation of mitogen-activated protein kinases (MAPK) was observed in the Fgfr2 ^+/P253R neurocranium with an increase in phosphorylated p38 as well as ERK1/2, whereas phosphorylated AKT and PKCα were not obviously changed as compared to those of wild-type controls. There were localized phenotypic and molecular variations among individual embryos with different mutations and among those with the same mutation.

Conclusions

Our in vivo studies demonstrated that the Fgfr2 +/P253R mutation resulted in mice with cranial features that resemble those of the Fgfr2 ^+/S252W mice and human Apert syndrome. Activated p38 in addition to the ERK1/2 signaling pathways may mediate the mutant neurocranial phenotype. Though Apert syndrome is traditionally thought to be a consistent phenotype, our results suggest localized and regional variations in the phenotypes that characterize Apert syndrome.     

A single N‐terminal phosphomimic disrupts TDP‐43 polymerization,phase separation,and RNA splicing

TDP‐43 is an RNA‐binding protein active in splicing that concentrates into membraneless ribonucleoprotein granules and forms aggregates in amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. Although best known for its predominantly disordered C‐terminal domain which mediates ALS inclusions, TDP‐43 has a globular N‐terminal domain (NTD). Here, we show that TDP‐43 NTD assembles into head‐to‐tail linear chains and that phosphomimetic substitution at S48 disrupts TDP‐43 polymeric assembly, discourages liquid–liquid phase separation (LLPS) in vitro, fluidizes liquid–liquid phase separated nuclear TDP‐43 reporter constructs in cells, and disrupts RNA splicing activity. Finally, we present the solution NMR structure of a head‐to‐tail NTD dimer comprised of two engineered variants that allow saturation of the native polymerization interface while disrupting higher‐order polymerization. These data provide structural detail for the established mechanistic role of the well‐folded TDP‐43 NTD in splicing and link this function to LLPS. In addition, the fusion‐tag solubilized, recombinant form of TDP‐43 full‐length protein developed here will enable future phase separation and in vitro biochemical assays on TDP‐43 function and interactions that have been hampered in the past by TDP‐43 aggregation.     

Generation and Characterization of a Mouse Model Harboring the Exon-3 Deletion in the Cardiac Ryanodine Receptor

A large genomic deletion in human cardiac ryanodine receptor (RYR2) gene has been detected in a number of unrelated families with various clinical phenotypes, including catecholaminergic polymorphic ventricular tachycardia (CPVT). This genomic deletion results in an in-frame deletion of exon-3 (Ex3-del). To understand the underlying disease mechanism of the RyR2 Ex3-del mutation, we generated a mouse model in which the RyR2 exon-3 sequence plus 15-bp intron sequences flanking exon-3 were deleted. Heterozygous Ex3-del mice (Ex3-del^+/−) survived, but no homozygous Ex3-del mice were born. Unexpectedly, the Ex3-del^+/− mice are not susceptible to CPVT. Ex3-del^+/− cardiomyocytes exhibited similar amplitude but altered dynamics of depolarization-induced Ca²⁺ transients compared to wild type (WT) cells. Immunoblotting analysis revealed markedly reduced expression of RyR2 protein in the Ex3-del^+/− mutant heart, indicating that Ex3-del has a major impact on RyR2 protein expression in mice. Cardiac specific, conditional knockout of the WT RyR2 allele in Ex3-del^+/− mice led to bradycardia and death. Thus, the absence of CPVT and other phenotypes in Ex3-del^+/− mice may be attributable to the predominant expression of the WT RyR2 allele as a result of the markedly reduced expression of the Ex3-del mutant allele. The effect of Ex3-del on RyR2 protein expression is discussed in relation to the phenotypic variability in individuals with the RyR2 exon-3 deletion.     

Dietary Regimens Modify Early Onset of Obesity in Mice Haploinsufficient for Rai1

Smith-Magenis syndrome is a complex genomic disorder in which a majority of individuals are obese by adolescence. While an interstitial deletion of chromosome 17p11.2 is the leading cause, mutation or deletion of the RAI1 gene alone results in most features of the disorder. Previous studies have shown that heterozygous knockout of Rai1 results in an obese phenotype in mice and that Smith-Magenis syndrome mouse models have a significantly reduced fecundity and an altered transmission pattern of the mutant Rai1 allele, complicating large, extended studies in these models. In this study, we show that breeding C57Bl/6J Rai1^+/− mice with FVB/NJ to create F1 Rai1^+/− offspring in a mixed genetic background ameliorates both fecundity and Rai1 allele transmission phenotypes. These findings suggest that the mixed background provides a more robust platform for breeding and larger phenotypic studies. We also characterized the effect of dietary intake on Rai1^+/− mouse growth during adolescent and early adulthood developmental stages. Animals fed a high carbohydrate or a high fat diet gained weight at a significantly faster rate than their wild type littermates. Both high fat and high carbohydrate fed Rai1^+/− mice also had an increase in body fat and altered fat distribution patterns. Interestingly, Rai1^+/− mice fed different diets did not display altered fasting blood glucose levels. These results suggest that dietary regimens are extremely important for individuals with Smith- Magenis syndrome and that food high in fat and carbohydrates may exacerbate obesity outcomes.     

Characterization of the MeCP2R168X Knockin Mouse Model for Rett Syndrome

Rett syndrome, one of the most common causes of mental retardation in females, is caused by mutations in the X chromosomal gene MECP2. Mice deficient for MeCP2 recapitulate some of the symptoms seen in patients with Rett syndrome. It has been shown that reactivation of silent MECP2 alleles can reverse some of the symptoms in these mice. We have generated a knockin mouse model for translational research that carries the most common nonsense mutation in Rett syndrome, R168X. In this article we describe the phenotype of this mouse model. In male MeCP2^R168X mice life span was reduced to 12–14 weeks and bodyweight was significantly lower than in wild type littermates. First symptoms including tremor, hind limb clasping and inactivity occurred at age 27 days. At age 6 weeks nest building, rotarod, open-field and elevated plus maze experiments showed impaired motor performance, reduced activity and decreased anxiety-like behavior. Plethysmography at the same time showed apneas and irregular breathing with reduced frequency. Female MeCP2^R168X mice showed no significant abnormalities except decreased performance on the rotarod at age 9 months. In conclusion we show that the male MeCP2^R168X mice have a phenotype similar to that seen in MECP2 knockout mouse models and are therefore well suited for translational research. The female mice, however, have a much milder and less constant phenotype making such research with this mouse model more challenging.     

Metabolic Enzyme Alterations and Astrocyte Dysfunction in a Murine Model of Alexander Disease With Severe Reactive Gliosis

Alexander disease (AxD) is a rare and fatal neurodegenerative disorder caused by mutations in the gene encoding glial fibrillary acidic protein (GFAP). In this report, a mouse model of AxD (GFAP^Tg;Gfap^+/R236H) was analyzed that contains a heterozygous R236H point mutation in murine Gfap as well as a transgene with a GFAP promoter to overexpress human GFAP. Using label-free quantitative proteomic comparisons of brain tissue from GFAP^Tg;Gfap^+/R236H versus wild-type mice confirmed upregulation of the glutathione metabolism pathway and indicated proteins were elevated in the peroxisome proliferator-activated receptor (PPAR) signaling pathway, which had not been reported previously in AxD. Relative protein-level differences were confirmed by a targeted proteomics assay, including proteins related to astrocytes and oligodendrocytes. Of particular interest was the decreased level of the oligodendrocyte protein, 2-hydroxyacylsphingosine 1-beta-galactosyltransferase (Ugt8), since Ugt8-deficient mice exhibit a phenotype similar to GFAP^Tg;Gfap^+/R236H mice (e.g., tremors, ataxia, hind-limb paralysis). In addition, decreased levels of myelin-associated proteins were found in the GFAP^Tg;Gfap^+/R236H mice, consistent with the role of Ugt8 in myelin synthesis. Fabp7 upregulation in GFAP^Tg;Gfap^+/R236H mice was also selected for further investigation due to its uncharacterized association to AxD, critical function in astrocyte proliferation, and functional ability to inhibit the anti-inflammatory PPAR signaling pathway in models of amyotrophic lateral sclerosis (ALS). Within Gfap⁺ astrocytes, Fabp7 was markedly increased in the hippocampus, a brain region subjected to extensive pathology and chronic reactive gliosis in GFAP^Tg;Gfap^+/R236H mice. Last, to determine whether the findings in GFAP^Tg;Gfap^+/R236H mice are present in the human condition, AxD patient and control samples were analyzed by Western blot, which indicated that Type I AxD patients have a significant fourfold upregulation of FABP7. However, immunohistochemistry analysis showed that UGT8 accumulates in AxD patient subpial brain regions where abundant amounts of Rosenthal fibers are located, which was not observed in the GFAP^Tg;Gfap^+/R236H mice.     

The voltage‐gated calcium channel blocker lomerizine is neuroprotective in motor neurons expressing mutant SOD1, but not TDP‐43

Excitotoxicity and disruption of Ca²⁺ homeostasis have been implicated in amyotrophic lateral sclerosis (ALS) and limiting Ca²⁺ entry is protective in models of ALS caused by mutation of SOD1. Lomerizine, an antagonist of L‐ and T‐type voltage‐gated calcium channels and transient receptor potential channel 5 transient receptor potential channels, is well tolerated clinically, making it a potential therapeutic candidate. Lomerizine reduced glutamate excitotoxicity in cultured motor neurons by reducing the accumulation of cytoplasmic Ca²⁺ and protected motor neurons against multiple measures of mutant SOD1 toxicity: Ca²⁺ overload, impaired mitochondrial trafficking, mitochondrial fragmentation, formation of mutant SOD1 inclusions, and loss of viability. To assess the utility of lomerizine in other forms of ALS, calcium homeostasis was evaluated in culture models of disease because of mutations in the RNA‐binding proteins transactive response DNA‐binding protein 43 (TDP‐43) and Fused in Sarcoma (FUS). Calcium did not play the same role in the toxicity of these mutant proteins as with mutant SOD1 and lomerizine failed to prevent cytoplasmic accumulation of mutant TDP‐43, a hallmark of its pathology. These experiments point to differences in the pathogenic pathways between types of ALS and show the utility of primary culture models in comparing those mechanisms and effectiveness of therapeutic strategies.