Clinically relevant mouse models of Charcot-Marie-Tooth type 2S.

Charcot-Marie-Tooth disease is an inherited peripheral neuropathy that is clinically and genetically heterogenous. Mutations in IGHMBP2, a ubiquitously expressed DNA/RNA helicase, have been shown to cause the infantile motor neuron disease spinal muscular atrophy with respiratory distress type 1 (SMARD1), and, more recently, juvenile-onset Charcot-Marie-Tooth disease type 2S (CMT2S). Using CRISPR-cas9 mutagenesis, we developed the first mouse models of CMT2S [p.Glu365del (E365del) and p.Tyr918Cys (Y918C)]. E365del is the first CMT2S mouse model to be discovered and Y918C is the first human CMT2S allele knock-in model. Phenotypic characterization of the homozygous models found progressive peripheral motor and sensory axonal degeneration. Neuromuscular and locomotor assays indicate that both E365del and Y918C mice have motor deficits, while neurobehavioral characterization of sensory function found that E365del mutants have mechanical allodynia. Analysis of femoral motor and sensory nerves identified axonal degeneration, which does not impact nerve conduction velocities in E365del mice, but it does so in the Y918C model. Based on these results, the E365del mutant mouse, and the human allele knock-in, Y918C, represent mouse models with the hallmark phenotypes of CMT2S, which will be critical for understanding the pathogenic mechanisms of IGHMBP2. These mice will complement existing Ighmbp2 alleles modeling SMARD1 to help understand the complex phenotypic and genotypic heterogeneity that is observed in patients with IGHMBP2 variants.

Identification of quantitative trait loci for survival in the mutant dynactin p150Glued mouse model of motor neuron disease.

Amyotrophic lateral sclerosis (ALS) is the most common degenerative motor neuron disorder. Although most cases of ALS are sporadic, 5-10% of cases are familial, with mutations associated with over 40 genes. There is variation of ALS symptoms within families carrying the same mutation; the disease may develop in one sibling and not in another despite the presence of the mutation in both. Although the cause of this phenotypic variation is unknown, it is likely related to genetic modifiers of disease expression. The identification of ALS causing genes has led to the development of transgenic mouse models of motor neuron disease. Similar to families with familial ALS, there are background-dependent differences in disease phenotype in transgenic mouse models of ALS suggesting that, as in human ALS, differences in phenotype may be ascribed to genetic modifiers. These genetic modifiers may not cause ALS rather their expression either exacerbates or ameliorates the effect of the mutant ALS causing genes. We have reported that in both the G93A-hSOD1 and G59S-hDCTN1 mouse models, SJL mice demonstrated a more severe phenotype than C57BL6 mice. From reciprocal intercrosses between G93A-hSOD1 transgenic mice on SJL and C57BL6 strains, we identified a major quantitative trait locus (QTL) on mouse chromosome 17 that results in a significant shift in lifespan. In this study we generated reciprocal intercrosses between transgenic G59S-hDCTN1 mice on SJL and C57BL6 strains and identified survival QTLs on mouse chromosomes 17 and 18. The chromosome 17 survival QTL on G93A-hSOD1 and G59S-hDCTN1 mice partly overlap, suggesting that the genetic modifiers located in this region may be shared by these two ALS models despite the fact that motor neuron degeneration is caused by mutations in different proteins. The overlapping region contains eighty-seven genes with non-synonymous variations predicted to be deleterious and/or damaging. Two genes in this segment, NOTCH3 and Safb/SAFB1, have been associated with motor neuron disease. The identification of genetic modifiers of motor neuron disease, especially those modifiers that are shared by SOD1 and dynactin-1 transgenic mice, may result in the identification of novel targets for therapies that can alter the course of this devastating illness.

A microfluidic approach to rescue ALS motor neuron degeneration using rapamycin.

TAR DNA-binding protein-43 (TDP-43) is known to accumulate in ubiquitinated inclusions of amyotrophic lateral sclerosis affected motor neurons, resulting in motor neuron degeneration, loss of motor functions, and eventually death. Rapamycin, an mTOR inhibitor and a commonly used immunosuppressive drug, has been shown to increase the survivability of Amyotrophic Lateral Sclerosis (ALS) affected motor neurons. Here we present a transgenic, TDP-43-A315T, mouse model expressing an ALS phenotype and demonstrate the presence of ubiquitinated cytoplasmic TDP-43 aggregates with > 80% cell death by 28 days post differentiation in vitro. Embryonic stem cells from this mouse model were used to study the onset, progression, and therapeutic remediation of TDP-43 aggregates using a novel microfluidic rapamycin concentration gradient generator. Results using a microfluidic device show that ALS affected motor neuron survival can be increased by 40.44% in a rapamycin dosage range between 0.4-1.0 µM.

Convergence of mammalian RQC and C-end rule proteolytic pathways via alanine tailing.

Incompletely synthesized nascent chains obstructing large ribosomal subunits are targeted for degradation by ribosome-associated quality control (RQC). In bacterial RQC, RqcH marks the nascent chains with C-terminal alanine (Ala) tails that are directly recognized by proteasome-like proteases, whereas in eukaryotes, RqcH orthologs (Rqc2/NEMF [nuclear export mediator factor]) assist the Ltn1/Listerin E3 ligase in nascent chain ubiquitylation. Here, we study RQC-mediated proteolytic targeting of ribosome stalling products in mammalian cells. We show that mammalian NEMF has an additional, Listerin-independent proteolytic role, which, as in bacteria, is mediated by tRNA-Ala binding and Ala tailing. However, in mammalian cells Ala tails signal proteolysis indirectly, through a pathway that recognizes C-terminal degrons; we identify the CRL2KLHDC10 E3 ligase complex and the novel C-end rule E3, Pirh2/Rchy1, as bona fide RQC pathway components that directly bind to Ala-tailed ribosome stalling products and target them for degradation. As Listerin mutation causes neurodegeneration in mice, functionally redundant E3s may likewise be implicated in molecular mechanisms of neurodegeneration.

Spinal motor neuron loss occurs through a p53-and-p21-independent mechanism in the Smn2B/- mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a pediatric neuromuscular disease caused by genetic deficiency of the survival motor neuron (SMN) protein. Pathological hallmarks of SMA are spinal motor neuron loss and skeletal muscle atrophy. The molecular mechanisms that elicit and drive preferential motor neuron degeneration and death in SMA remain unclear. Transcriptomic studies consistently report p53 pathway activation in motor neurons and spinal cord tissue of SMA mice. Recent work has identified p53 as an inducer of spinal motor neuron loss in severe Δ7 SMA mice. Additionally, the cyclin-dependent kinase inhibitor P21 (Cdkn1a), an inducer of cell cycle arrest and mediator of skeletal muscle atrophy, is consistently increased in motor neurons, spinal cords, and other tissues of various SMA models. p21 is a p53 transcriptional target but can be independently induced by cellular stressors. To ascertain whether p53 and p21 signaling pathways mediate spinal motor neuron death in milder SMA mice, and how they affect the overall SMA phenotype, we introduced Trp53 and P21 null alleles onto the Smn2B/- background. We found that p53 and p21 depletion did not modulate the timing or degree of Smn2B/- motor neuron loss as evaluated using electrophysiological and immunohistochemical methods. Moreover, we determined that Trp53 and P21 knockout differentially affected Smn2B/- mouse lifespan: p53 ablation impaired survival while p21 ablation extended survival through Smn-independent mechanisms. These results demonstrate that p53 and p21 are not primary drivers of spinal motor neuron death in Smn2B/- mice, a milder SMA mouse model, as motor neuron loss is not alleviated by their ablation.

Author Correction: NEMF mutations that impair ribosome-associated quality control are associated with neuromuscular disease.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

NEMF mutations that impair ribosome-associated quality control are associated with neuromuscular disease.

A hallmark of neurodegeneration is defective protein quality control. The E3 ligase Listerin (LTN1/Ltn1) acts in a specialized protein quality control pathway-Ribosome-associated Quality Control (RQC)-by mediating proteolytic targeting of incomplete polypeptides produced by ribosome stalling, and Ltn1 mutation leads to neurodegeneration in mice. Whether neurodegeneration results from defective RQC and whether defective RQC contributes to human disease have remained unknown. Here we show that three independently-generated mouse models with mutations in a different component of the RQC complex, NEMF/Rqc2, develop progressive motor neuron degeneration. Equivalent mutations in yeast Rqc2 selectively interfere with its ability to modify aberrant translation products with C-terminal tails which assist with RQC-mediated protein degradation, suggesting a pathomechanism. Finally, we identify NEMF mutations expected to interfere with function in patients from seven families presenting juvenile neuromuscular disease. These uncover NEMF's role in translational homeostasis in the nervous system and implicate RQC dysfunction in causing neurodegeneration.

Overlapping spectrums: The clinicogenetic commonalities between Charcot-Marie-Tooth and other neurodegenerative diseases.

Charcot-Marie-Tooth (CMT) disease is a progressive and heterogeneous inherited peripheral neuropathy. A myriad of genetic factors have been identified that contribute to the degeneration of motor and sensory axons in a length-dependent manner. Emerging biological themes underlying disease include defects in axonal trafficking, dysfunction in RNA metabolism and protein homeostasis, as well deficits in the cellular stress response. Moreover, genetic contributions to CMT can have overlap with other neuropathies, motor neuron diseases (MNDs) and neurodegenerative disorders. Recent progress in understanding the molecular biology of CMT and overlapping syndromes aids in the search for necessary therapeutic targets.

Functional rescue in a mouse model of congenital muscular dystrophy with megaconial myopathy.

Congenital muscular dystrophy with megaconial myopathy (MDCMC) is an autosomal recessive disorder characterized by progressive muscle weakness and wasting. The observation of megamitochondria in skeletal muscle biopsies is exclusive to this type of MD. The disease is caused by loss of function mutations in the choline kinase beta (CHKB) gene which results in dysfunction of the Kennedy pathway for the synthesis of phosphatidylcholine. We have previously reported a rostrocaudal MD (rmd) mouse with a deletion in the Chkb gene resulting in an MDCMC-like phenotype, and we used this mouse to test gene therapy strategies for the rescue and alleviation of the dystrophic phenotype. Introduction of a muscle-specific Chkb transgene completely rescues motor and behavioral function in the rmd mouse model, confirming the cell-autonomous nature of the disease. Intramuscular gene therapy post-disease onset using an adeno-associated viral 6 (AAV6) vector carrying a functional copy of Chkb is also capable of rescuing the dystrophy phenotype. In addition, we examined the ability of choline kinase alpha (Chka), a gene paralog of Chkb, to improve dystrophic phenotypes when upregulated in skeletal muscles of rmd mutant mice using a similar AAV6 vector. The sum of our results in a preclinical model of disease suggest that replacement of the Chkb gene or upregulation of endogenous Chka could serve as potential lines of therapy for MDCMC patients.

Analysis of BMAA enantiomers in cycads, cyanobacteria, and mammals: in vivo formation and toxicity of D-BMAA.

Chronic dietary exposure to the cyanobacterial toxin ß-N-methylamino-L-alanine (BMAA) triggers neuropathology in non-human primates, providing support for the theory that BMAA causes a fatal neurodegenerative illness among the indigenous Chamorro people of Guam. However, since there are two stereoisomers of BMAA, it is important to know if both can occur in nature, and if so, what role they might play in disease causation. As a first step, we analysed both BMAA enantiomers in cyanobacteria, cycads, and in mammals orally dosed with L-BMAA, to determine if enantiomeric changes could occur in vivo. BMAA in cyanobacteria and cycads was found only as the L-enantiomer. However, while the L-enantiomer in mammals was little changed after digestion, we detected a small pool of D-BMAA in the liver (12.5%) of mice and in the blood plasma of vervets (3.6%). Chiral analysis of cerebrospinal fluid of vervets and hindbrain of mice showed that the free BMAA in the central nervous system was the D-enantiomer. In vitro toxicity investigations with D-BMAA showed toxicity, mediated through AMPA rather than NMDA receptors. These findings raise important considerations concerning the neurotoxicity of BMAA and its relationship to neurodegenerative disease.

Severity of Demyelinating and Axonal Neuropathy Mouse Models Is Modified by Genes Affecting Structure and Function of Peripheral Nodes.

Charcot-Marie-Tooth (CMT) disease is a clinically and genetically heterogeneous group of inherited polyneuropathies. Mutations in 80 genetic loci can cause forms of CMT, resulting in demyelination and axonal dysfunction. The clinical presentation, including sensory deficits, distal muscle weakness, and atrophy, can vary greatly in severity and progression. Here, we used mouse models of CMT to demonstrate genetic interactions that result in a more severe neuropathy phenotype. The cell adhesion molecule Nrcam and the Na+ channel Scn8a (NaV1.6) are important components of nodes. Homozygous Nrcam and heterozygous Scn8a mutations synergized with both an Sh3tc2 mutation, modeling recessive demyelinating Charcot-Marie-Tooth type 4C, and mutations in Gars, modeling dominant axonal Charcot-Marie-Tooth type 2D. We conclude that genetic variants perturbing the structure and function of nodes interact with mutations affecting the cable properties of axons by thinning myelin or reducing axon diameter. Therefore, genes integral to peripheral nodes are candidate modifiers of peripheral neuropathy.

Development-on-chip: in vitro neural tube patterning with a microfluidic device.

Embryogenesis is a highly regulated process in which the precise spatial and temporal release of soluble cues directs differentiation of multipotent stem cells into discrete populations of specialized adult cell types. In the spinal cord, neural progenitor cells are directed to differentiate into adult neurons through the action of mediators released from nearby organizing centers, such as the floor plate and paraxial mesoderm. These signals combine to create spatiotemporal diffusional landscapes that precisely regulate the development of the central nervous system (CNS). Currently, in vivo and ex vivo studies of these signaling factors present some inherent ambiguity. In vitro methods are preferred for their enhanced experimental clarity but often lack the technical sophistication required for biological realism. In this article, we present a versatile microfluidic platform capable of mimicking the spatial and temporal chemical environments found in vivo during neural tube development. Simultaneous opposing and/or orthogonal gradients of developmental morphogens can be maintained, resulting in neural tube patterning analogous to that observed in vivo.

Neuromuscular Disease Models and Analysis.

Neuromuscular diseases can affect the survival of peripheral neurons, their axons extending to peripheral targets, their synaptic connections onto those targets, or the targets themselves. Examples include motor neuron diseases such as Amyotrophic Lateral Sclerosis, peripheral neuropathies such as Charcot-Marie-Tooth diseases, myasthenias, and muscular dystrophies. Characterizing these phenotypes in mouse models requires an integrated approach, examining both the nerve and muscle histologically, anatomically, and functionally by electrophysiology. Defects observed at these levels can be related back to onset, severity, and progression, as assessed by "Quality of life measures" including tests of gross motor performance such as gait or grip strength. This chapter describes methods for assessing neuromuscular disease models in mice, and how interpretation of these tests can be complicated by the inter-relatedness of the phenotypes.

Absence of UCHL 1 function leads to selective motor neuropathy.

OBJECTIVE: The aim of this study was to investigate the role of ubiquitin C-terminal hydrolase-L1 (UCHL1) for motor neuron circuitry and especially in spinal motor neuron (SMN) health, function, and connectivity. METHODS: Since mutations in UCHL1 gene leads to motor dysfunction in patients, we investigated the role of UCHL1 on SMN survival, axon health, and connectivity with the muscle, by employing molecular and cellular marker expression analysis and electrophysiological recordings, in healthy wild-type and Uchl1 (nm3419) (UCHL1-/-) mice, which lack all UCHL1 function. RESULTS: There is pure motor neuropathy with selective degeneration of the motor, but not sensory axons in the absence of UCHL1 function. Neuromuscular junctions (NMJ) are impaired in muscle groups that are innervated by slow-twitch or fast-twitch SMN. However, unlike corticospinal motor neurons, SMN cell bodies remain intact with no signs of elevated endoplasmic reticulum (ER) stress. INTERPRETATION: Presence of NMJ defects and progressive retrograde axonal degeneration in the absence of major SMN soma loss suggest that defining pathology as a function of neuron number is misleading and that upper and lower motor neurons utilize UCHL1 function in different cellular events. In line with findings in patients with mutations in UCHL1 gene, our results suggest a unique role of UCHL1, especially for motor neuron circuitry. SMN require UCHL1 to maintain NMJ and motor axon health, and that observed motor dysfunction in the absence of UCHL1 is not due to SMN loss, but mostly due to disintegrated circuitry.

Hyperactive somatostatin interneurons contribute to excitotoxicity in neurodegenerative disorders.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are overlapping neurodegenerative disorders whose pathogenesis remains largely unknown. Using TDP-43(A315T) mice, an ALS and FTD model with marked cortical pathology, we found that hyperactive somatostatin interneurons disinhibited layer 5 pyramidal neurons (L5-PNs) and contributed to their excitotoxicity. Focal ablation of somatostatin interneurons efficiently restored normal excitability of L5-PNs and alleviated neurodegeneration, suggesting a new therapeutic target for ALS and FTD.

Effect of genetic background on the dystrophic phenotype in mdx mice.

Genetic background significantly affects phenotype in multiple mouse models of human diseases, including muscular dystrophy. This phenotypic variability is partly attributed to genetic modifiers that regulate the disease process. Studies have demonstrated that introduction of the γ-sarcoglycan-null allele onto the DBA/2J background confers a more severe muscular dystrophy phenotype than the original strain, demonstrating the presence of genetic modifier loci in the DBA/2J background. To characterize the phenotype of dystrophin deficiency on the DBA/2J background, we created and phenotyped DBA/2J-congenic Dmdmdx mice (D2-mdx) and compared them with the original, C57BL/10ScSn-Dmdmdx (B10-mdx) model. These strains were compared with their respective control strains at multiple time points between 6 and 52 weeks of age. Skeletal and cardiac muscle function, inflammation, regeneration, histology and biochemistry were characterized. We found that D2-mdx mice showed significantly reduced skeletal muscle function as early as 7 weeks and reduced cardiac function by 28 weeks, suggesting that the disease phenotype is more severe than in B10-mdx mice. In addition, D2-mdx mice showed fewer central myonuclei and increased calcifications in the skeletal muscle, heart and diaphragm at 7 weeks, suggesting that their pathology is different from the B10-mdx mice. The new D2-mdx model with an earlier onset and more pronounced dystrophy phenotype may be useful for evaluating therapies that target cardiac and skeletal muscle function in dystrophin-deficient mice. Our data align the D2-mdx with Duchenne muscular dystrophy patients with the LTBP4 genetic modifier, making it one of the few instances of cross-species genetic modifiers of monogenic traits.

Exome sequencing reveals pathogenic mutations in 91 strains of mice with Mendelian disorders.

Spontaneously arising mouse mutations have served as the foundation for understanding gene function for more than 100 years. We have used exome sequencing in an effort to identify the causative mutations for 172 distinct, spontaneously arising mouse models of Mendelian disorders, including a broad range of clinically relevant phenotypes. To analyze the resulting data, we developed an analytics pipeline that is optimized for mouse exome data and a variation database that allows for reproducible, user-defined data mining as well as nomination of mutation candidates through knowledge-based integration of sample and variant data. Using these new tools, putative pathogenic mutations were identified for 91 (53%) of the strains in our study. Despite the increased power offered by potentially unlimited pedigrees and controlled breeding, about half of our exome cases remained unsolved. Using a combination of manual analyses of exome alignments and whole-genome sequencing, we provide evidence that a large fraction of unsolved exome cases have underlying structural mutations. This result directly informs efforts to investigate the similar proportion of apparently Mendelian human phenotypes that are recalcitrant to exome sequencing.

Genetic background effects on disease onset and lifespan of the mutant dynactin p150Glued mouse model of motor neuron disease.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease primarily affecting motor neurons in the central nervous system. Although most cases of ALS are sporadic, about 5-10% of cases are familial (FALS) with approximately 20% of FALS caused by mutations in the Cu/Zn superoxide dismutase (SOD1) gene. We have reported that hSOD1-G93A transgenic mice modeling this disease show a more severe phenotype when the transgene is bred on a pure SJL background and a milder phenotype when bred on a pure B6 background and that these phenotype differences link to a region on mouse Chromosome 17.To examine whether other models of motor neuron degeneration are affected by genetic background, we bred the mutant human dynactin p150Glued (G59S-hDCTN1) transgene onto inbred SJL and B6 congenic lines. This model is based on an autosomal dominant lower motor neuron disease in humans linked to a mutation in the p150Glued subunit of the dynactin complex. As seen in hSOD1-G93A mice, we observed a more severe phenotype with earlier disease onset (p<0.001) and decreased survival (p<0.00001) when the G59S-hDCTN1 transgene was bred onto the SJL background and delayed onset (p<0.0001) with increased survival (p<0.00001) when bred onto the B6 background. Furthermore, B6 mice with an SJL derived chromosome 17 interval previously shown to delay disease onset in hSOD1-G93A mice also showed delays onset in G59S-hDCTN1 mice suggesting that at least some genetic modifiers are shared. We have shown that genetic background influences phenotype in G59S-hDCTN1 mice, in part through a region of chromosome 17 similar to the G93-hSOD1 ALS mouse model. These results support the presence of genetic modifiers in both these models some of which may be shared. Identification of these modifiers will highlight intracellular pathways involved in motor neuron disease and provide new therapeutic targets that may be applicable to motor neuron degeneration.

Corticospinal Motor Neurons Are Susceptible to Increased ER Stress and Display Profound Degeneration in the Absence of UCHL1 Function.

Corticospinal motor neurons (CSMN) receive, integrate, and relay cerebral cortex's input toward spinal targets to initiate and modulate voluntary movement. CSMN degeneration is central for numerous motor neuron disorders and neurodegenerative diseases. Previously, 5 patients with mutations in the ubiquitin carboxy-terminal hydrolase-L1 (UCHL1) gene were reported to have neurodegeneration and motor neuron dysfunction with upper motor neuron involvement. To investigate the role of UCHL1 on CSMN health and stability, we used both in vivo and in vitro approaches, and took advantage of the Uchl1(nm3419) (UCHL1(-/-)) mice, which lack all UCHL1 function. We report a unique role of UCHL1 in maintaining CSMN viability and cellular integrity. CSMN show early, selective, progressive, and profound cell loss in the absence of UCHL1. CSMN degeneration, evident even at pre-symptomatic stages by disintegration of the apical dendrite and spine loss, is mediated via increased ER stress. These findings bring a novel understanding to the basis of CSMN vulnerability, and suggest UCHL1(-/-) mice as a tool to study CSMN pathology.