Stathmin-2 loss leads to neurofilament-dependent axonal collapse driving motor and sensory denervation.

The mRNA transcript of the human STMN2 gene, encoding for stathmin-2 protein (also called SCG10), is profoundly impacted by TAR DNA-binding protein 43 (TDP-43) loss of function. The latter is a hallmark of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Using a combination of approaches, including transient antisense oligonucleotide-mediated suppression, sustained shRNA-induced depletion in aging mice, and germline deletion, we show that stathmin-2 has an important role in the establishment and maintenance of neurofilament-dependent axoplasmic organization that is critical for preserving the caliber and conduction velocity of myelinated large-diameter axons. Persistent stathmin-2 loss in adult mice results in pathologies found in ALS, including reduced interneurofilament spacing, axonal caliber collapse that drives tearing within outer myelin layers, diminished conduction velocity, progressive motor and sensory deficits, and muscle denervation. These findings reinforce restoration of stathmin-2 as an attractive therapeutic approach for ALS and other TDP-43-dependent neurodegenerative diseases.

Focused ultrasound-mediated brain genome editing.

Gene editing in the brain has been challenging because of the restricted transport imposed by the blood-brain barrier (BBB). Current approaches mainly rely on local injection to bypass the BBB. However, such administration is highly invasive and not amenable to treating certain delicate regions of the brain. We demonstrate a safe and effective gene editing technique by using focused ultrasound (FUS) to transiently open the BBB for the transport of intravenously delivered CRISPR/Cas9 machinery to the brain.

MATR3 P154S knock-in mice do not exhibit motor, muscle or neuropathologic features of ALS.

Matrin 3 is a nuclear matrix protein that has many roles in RNA processing including splicing and transport of mRNA. Many missense mutations in the Matrin 3 gene (MATR3) have been linked to familial forms of amyotrophic lateral sclerosis (ALS) and distal myopathy. However, the exact role of MATR3 mutations in ALS and myopathy pathogenesis is not understood. To demonstrate a role of MATR3 mutations in vivo, we generated a novel CRISPR/Cas9 mediated knock-in mouse model harboring the MATR3 P154S mutation expressed under the control of the endogenous promoter. The P154S variant of the MATR3 gene has been linked to familial forms of ALS. Heterozygous and homozygous MATR3 P154S knock-in mice did not develop progressive motor deficits compared to wild-type mice. In addition, ALS-like pathology did not develop in nervous or muscle tissue in either heterozygous or homozygous mice. Our results suggest that the MATR3 P154S variant is not sufficient to produce ALS-like pathology in vivo.

Neurobehavioral deficits of mice expressing a low level of G127V mutant frataxin.

Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by reduced expression of the mitochondrial protein frataxin (FXN). Most FRDA patients are homozygous for large expansions of GAA repeats in intron 1 of FXN, while some are compound heterozygotes with an expanded GAA tract in one allele and a missense or nonsense mutation in the other. A missense mutation, changing a glycine to valine at position 130 (G130V), is prevalent among the clinical variants. We and others have demonstrated that levels of mature FXN protein in FRDA G130V samples are reduced below those detected in samples harboring homozygous repeat expansions. Little is known regarding expression and function of endogenous FXN-G130V protein due to lack of reagents and models that can distinguish the mutant FXN protein from the wild-type FXN produced from the GAA-expanded allele. We aimed to determine the effect of the G130V (murine G127V) mutation on Fxn expression and to define its multi-system impact in vivo. We used CRISPR/Cas9 to introduce the G127V missense mutation in the Fxn coding sequence and generated homozygous mice (FxnG127V/G127V). We also introduced the G127V mutation into a GAA repeat expansion FRDA mouse model (FxnGAA230/KO; KIKO) to generate a compound heterozygous strain (FxnG127V/GAA230). We performed neurobehavioral tests on cohorts of WT and Fxn mutant animals at three-month intervals for one year, and collected tissue samples to analyze molecular changes during that time. The endogenous Fxn G127V protein is detected at much lower levels in all tissues analyzed from FxnG127V/G127V mice compared to age and sex-matched WT mice without differences in Fxn transcript levels. FxnG127V/G127V mice are significantly smaller than WT counterparts, but perform similarly in most neurobehavioral tasks. RNA sequencing analysis revealed reduced expression of genes in oxidative phosphorylation and protein synthesis, underscoring the metabolic consequences in our mouse model expressing extremely low levels of Fxn. Results of these studies provide insight into the unique pathogenic mechanism of the FXN G130V mechanism and the tolerable limit of Fxn/FXN expression in vivo.

Efficient in vivo neuronal genome editing in the mouse brain using nanocapsules containing CRISPR-Cas9 ribonucleoproteins.

Genome editing of somatic cells via clustered regularly interspaced short palindromic repeats (CRISPR) offers promise for new therapeutics to treat a variety of genetic disorders, including neurological diseases. However, the dense and complex parenchyma of the brain and the post-mitotic state of neurons make efficient genome editing challenging. In vivo delivery systems for CRISPR-Cas proteins and single guide RNA (sgRNA) include both viral vectors and non-viral strategies, each presenting different advantages and disadvantages for clinical application. We developed non-viral and biodegradable PEGylated nanocapsules (NCs) that deliver preassembled Cas9-sgRNA ribonucleoproteins (RNPs). Here, we show that the RNP NCs led to robust genome editing in neurons following intracerebral injection into the healthy mouse striatum. Genome editing was predominantly observed in medium spiny neurons (>80%), with occasional editing in cholinergic, calretinin, and parvalbumin interneurons. Glial activation was minimal and was localized along the needle tract. Our results demonstrate that the RNP NCs are capable of safe and efficient neuronal genome editing in vivo.

Loss of Endothelial TDP-43 Leads to Blood Brain Barrier Defects in Mouse Models of Amyotrophic Lateral Sclerosis and Frontotemporal Dementia.

Loss of nuclear TDP-43 occurs in a wide range of neurodegenerative diseases, and specific mutations in the TARDBP gene that encodes the protein are linked to familial Frontal Temporal Lobar Dementia (FTD), and Amyotrophic Lateral Sclerosis (ALS). Although the focus has been on neuronal cell dysfunction caused by TDP-43 variants, TARDBP mRNA transcripts are expressed at similar levels in brain endothelial cells (ECs). Since increased permeability across the blood brain barrier (BBB) precedes cognitive decline, we postulated that altered functions of TDP-43 in ECs contributes to BBB dysfunction in neurodegenerative disease. To test this hypothesis, we examined EC function and BBB properties in mice with either knock-in mutations found in ALS/FTLD patients (TARDBPG348C and GRNR493X) or EC-specific deletion of TDP-43 throughout the endothelium (Cdh5(PAC)CreERT2; Tardbpff) or restricted to brain endothelium (Slco1c1(BAC)CreERT2; Tardbpff). We found that TARDBPG348C mice exhibited increased permeability to 3kDa Texas Red dextran and NHS-biotin, relative to their littermate controls, which could be recapitulated in cultured brain ECs from these mice. Nuclear levels of TDP-43 were reduced in vitro and in vivo in ECs from TARDBPG348C mice. This coincided with a reduction in junctional proteins VE-cadherin, claudin-5 and ZO-1 in isolated ECs, supporting a cell autonomous effect on barrier function through a loss of nuclear TDP-43. We further examined two models of Tardbp deletion in ECs, and found that the loss of TDP-43 throughout the endothelium led to systemic endothelial activation and permeability. Deletion specifically within the brain endothelium acutely increased BBB permeability, and eventually led to hallmarks of FTD, including fibrin deposition, microglial and astrocyte activation, and behavioral defects. Together, these data show that TDP-43 dysfunction specifically within brain ECs would contribute to the BBB defects observed early in the progression of ALS/FTLD.

Promoting validation and cross-phylogenetic integration in model organism research.

Model organism (MO) research provides a basic understanding of biology and disease due to the evolutionary conservation of the molecular and cellular language of life. MOs have been used to identify and understand the function of orthologous genes, proteins, cells and tissues involved in biological processes, to develop and evaluate techniques and methods, and to perform whole-organism-based chemical screens to test drug efficacy and toxicity. However, a growing richness of datasets and the rising power of computation raise an important question: How do we maximize the value of MOs? In-depth discussions in over 50 virtual presentations organized by the National Institutes of Health across more than 10 weeks yielded important suggestions for improving the rigor, validation, reproducibility and translatability of MO research. The effort clarified challenges and opportunities for developing and integrating tools and resources. Maintenance of critical existing infrastructure and the implementation of suggested improvements will play important roles in maintaining productivity and facilitating the validation of animal models of human biology and disease.

Stride-level analysis of mouse open field behavior using deep-learning-based pose estimation.

Gait and posture are often perturbed in many neurological, neuromuscular, and neuropsychiatric conditions. Rodents provide a tractable model for elucidating disease mechanisms and interventions. Here, we develop a neural-network-based assay that adopts the commonly used open field apparatus for mouse gait and posture analysis. We quantitate both with high precision across 62 strains of mice. We characterize four mutants with known gait deficits and demonstrate that multiple autism spectrum disorder (ASD) models show gait and posture deficits, implying this is a general feature of ASD. Mouse gait and posture measures are highly heritable and fall into three distinct classes. We conduct a genome-wide association study to define the genetic architecture of stride-level mouse movement in the open field. We provide a method for gait and posture extraction from the open field and one of the largest laboratory mouse gait and posture data resources for the research community.

The NIH Somatic Cell Genome Editing program.

The move from reading to writing the human genome offers new opportunities to improve human health. The United States National Institutes of Health (NIH) Somatic Cell Genome Editing (SCGE) Consortium aims to accelerate the development of safer and more-effective methods to edit the genomes of disease-relevant somatic cells in patients, even in tissues that are difficult to reach. Here we discuss the consortium's plans to develop and benchmark approaches to induce and measure genome modifications, and to define downstream functional consequences of genome editing within human cells. Central to this effort is a rigorous and innovative approach that requires validation of the technology through third-party testing in small and large animals. New genome editors, delivery technologies and methods for tracking edited cells in vivo, as well as newly developed animal models and human biological systems, will be assembled-along with validated datasets-into an SCGE Toolkit, which will be disseminated widely to the biomedical research community. We visualize this toolkit-and the knowledge generated by its applications-as a means to accelerate the clinical development of new therapies for a wide range of conditions.

Impaired prenatal motor axon development necessitates early therapeutic intervention in severe SMA.

Gene replacement and pre-mRNA splicing modifier therapies represent breakthrough gene targeting treatments for the neuromuscular disease spinal muscular atrophy (SMA), but mechanisms underlying variable efficacy of treatment are incompletely understood. Our examination of severe infantile onset human SMA tissues obtained at expedited autopsy revealed persistence of developmentally immature motor neuron axons, many of which are actively degenerating. We identified similar features in a mouse model of severe SMA, in which impaired radial growth and Schwann cell ensheathment of motor axons began during embryogenesis and resulted in reduced acquisition of myelinated axons that impeded motor axon function neonatally. Axons that failed to ensheath degenerated rapidly postnatally, specifically releasing neurofilament light chain protein into the blood. Genetic restoration of survival motor neuron protein (SMN) expression in mouse motor neurons, but not in Schwann cells or muscle, improved SMA motor axon development and maintenance. Treatment with small-molecule SMN2 splice modifiers beginning immediately after birth in mice increased radial growth of the already myelinated axons, but in utero treatment was required to restore axonal growth and associated maturation, prevent subsequent neonatal axon degeneration, and enhance motor axon function. Together, these data reveal a cellular basis for the fulminant neonatal worsening of patients with infantile onset SMA and identify a temporal window for more effective treatment. These findings suggest that minimizing treatment delay is critical to achieve optimal therapeutic efficacy.

A platform for experimental precision medicine: The extended BXD mouse family.

The challenge of precision medicine is to model complex interactions among DNA variants, phenotypes, development, environments, and treatments. We address this challenge by expanding the BXD family of mice to 140 fully isogenic strains, creating a uniquely powerful model for precision medicine. This family segregates for 6 million common DNA variants-a level that exceeds many human populations. Because each member can be replicated, heritable traits can be mapped with high power and precision. Current BXD phenomes are unsurpassed in coverage and include much omics data and thousands of quantitative traits. BXDs can be extended by a single-generation cross to as many as 19,460 isogenic F1 progeny, and this extended BXD family is an effective platform for testing causal modeling and for predictive validation. BXDs are a unique core resource for the field of experimental precision medicine.

A Drosophila screen identifies NKCC1 as a modifier of NGLY1 deficiency.

N-Glycanase 1 (NGLY1) is a cytoplasmic deglycosylating enzyme. Loss-of-function mutations in the NGLY1 gene cause NGLY1 deficiency, which is characterized by developmental delay, seizures, and a lack of sweat and tears. To model the phenotypic variability observed among patients, we crossed a Drosophila model of NGLY1 deficiency onto a panel of genetically diverse strains. The resulting progeny showed a phenotypic spectrum from 0 to 100% lethality. Association analysis on the lethality phenotype, as well as an evolutionary rate covariation analysis, generated lists of modifying genes, providing insight into NGLY1 function and disease. The top association hit was Ncc69 (human NKCC1/2), a conserved ion transporter. Analyses in NGLY1-/- mouse cells demonstrated that NKCC1 has an altered average molecular weight and reduced function. The misregulation of this ion transporter may explain the observed defects in secretory epithelium function in NGLY1 deficiency patients.

Reduced GABAergic Neuron Excitability, Altered Synaptic Connectivity, and Seizures in a KCNT1 Gain-of-Function Mouse Model of Childhood Epilepsy.

Gain-of-function (GOF) variants in K+ channels cause severe childhood epilepsies, but there are no mechanisms to explain how increased K+ currents lead to network hyperexcitability. Here, we introduce a human Na+-activated K+ (KNa) channel variant (KCNT1-Y796H) into mice and, using a multiplatform approach, find motor cortex hyperexcitability and early-onset seizures, phenotypes strikingly similar to those of human patients. Although the variant increases KNa currents in cortical excitatory and inhibitory neurons, there is an increase in the KNa current across subthreshold voltages only in inhibitory neurons, particularly in those with non-fast-spiking properties, resulting in inhibitory-neuron-specific impairments in excitability and action potential (AP) generation. We further observe evidence of synaptic rewiring, including increases in homotypic synaptic connectivity, accompanied by network hyperexcitability and hypersynchronicity. These findings support inhibitory-neuron-specific mechanisms in mediating the epileptogenic effects of KCNT1 channel GOF, offering cell-type-specific currents and effects as promising targets for therapeutic intervention.

Regulation of BMP4/Dpp retrotranslocation and signaling by deglycosylation.

During endoplasmic reticulum-associated degradation (ERAD), the cytoplasmic enzyme N-glycanase 1 (NGLY1) is proposed to remove N-glycans from misfolded N-glycoproteins after their retrotranslocation from the ER to the cytosol. We previously reported that NGLY1 regulates Drosophila BMP signaling in a tissue-specific manner (Galeone et al., 2017). Here, we establish the Drosophila Dpp and its mouse ortholog BMP4 as biologically relevant targets of NGLY1 and find, unexpectedly, that NGLY1-mediated deglycosylation of misfolded BMP4 is required for its retrotranslocation. Accumulation of misfolded BMP4 in the ER results in ER stress and prompts the ER recruitment of NGLY1. The ER-associated NGLY1 then deglycosylates misfolded BMP4 molecules to promote their retrotranslocation and proteasomal degradation, thereby allowing properly-folded BMP4 molecules to proceed through the secretory pathway and activate signaling in other cells. Our study redefines the role of NGLY1 during ERAD and suggests that impaired BMP4 signaling might underlie some of the NGLY1 deficiency patient phenotypes.

Modelling and treating GRIN2A developmental and epileptic encephalopathy in mice.

NMDA receptors play crucial roles in excitatory synaptic transmission. Rare variants in GRIN2A encoding the GluN2A subunit are associated with a spectrum of disorders, ranging from mild speech and language delay to intractable neurodevelopmental disorders, including but not limited to developmental and epileptic encephalopathy. A de novo missense variant, p.Ser644Gly, was identified in a child with this disorder, and Grin2a knock-in mice were generated to model and extend understanding of this intractable childhood disease. Homozygous and heterozygous mutant mice exhibited altered hippocampal morphology at 2 weeks of age, and all homozygotes exhibited lethal tonic-clonic seizures by mid-third week. Heterozygous adults displayed susceptibility to induced generalized seizures, hyperactivity, repetitive and reduced anxiety behaviours, plus several unexpected features, including significant resistance to electrically-induced limbic seizures and to pentylenetetrazole induced tonic-clonic seizures. Multielectrode recordings of neuronal networks revealed hyperexcitability and altered bursting and synchronicity. In heterologous cells, mutant receptors had enhanced NMDA receptor agonist potency and slow deactivation following rapid removal of glutamate, as occurs at synapses. NMDA receptor-mediated synaptic currents in heterozygous hippocampal slices also showed a prolonged deactivation time course. Standard anti-epileptic drug monotherapy was ineffective in the patient. Introduction of NMDA receptor antagonists was correlated with a decrease in seizure burden. Chronic treatment of homozygous mouse pups with NMDA receptor antagonists significantly delayed the onset of lethal seizures but did not prevent them. These studies illustrate the power of using multiple experimental modalities to model and test therapies for severe neurodevelopmental disorders, while revealing significant biological complexities associated with GRIN2A developmental and epileptic encephalopathy.

Mitochondrial damage and senescence phenotype of cells derived from a novel frataxin G127V point mutation mouse model of Friedreich's ataxia.

Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused by reduced expression of the mitochondrial protein frataxin (FXN). Most FRDA patients are homozygous for large expansions of GAA repeat sequences in intron 1 of FXN, whereas a fraction of patients are compound heterozygotes, with a missense or nonsense mutation in one FXN allele and expanded GAAs in the other. A prevalent missense mutation among FRDA patients changes a glycine at position 130 to valine (G130V). Herein, we report generation of the first mouse model harboring an Fxn point mutation. Changing the evolutionarily conserved glycine 127 in mouse Fxn to valine results in a failure-to-thrive phenotype in homozygous animals and a substantially reduced number of offspring. Like G130V in FRDA, the G127V mutation results in a dramatic decrease of Fxn protein without affecting transcript synthesis or splicing. FxnG127V mouse embryonic fibroblasts exhibit significantly reduced proliferation and increased cell senescence. These defects are evident in early passage cells and are exacerbated at later passages. Furthermore, increased frequency of mitochondrial DNA lesions and fragmentation are accompanied by marked amplification of mitochondrial DNA in FxnG127V cells. Bioenergetics analyses demonstrate higher sensitivity and reduced cellular respiration of FxnG127V cells upon alteration of fatty acid availability. Importantly, substitution of FxnWT with FxnG127V is compatible with life, and cellular proliferation defects can be rescued by mitigation of oxidative stress via hypoxia or induction of the NRF2 pathway. We propose FxnG127V cells as a simple and robust model for testing therapeutic approaches for FRDA.

TRPV4 Antagonism Prevents Mechanically Induced Myotonia.

OBJECTIVE: Myotonia is caused by involuntary firing of skeletal muscle action potentials and causes debilitating stiffness. Current treatments are insufficiently efficacious and associated with side effects. Myotonia can be triggered by voluntary movement (electrically induced myotonia) or percussion (mechanically induced myotonia). Whether distinct molecular mechanisms underlie these triggers is unknown. Our goal was to identify ion channels involved in mechanically induced myotonia and to evaluate block of the channels involved as a novel approach to therapy. METHODS: We developed a novel system to enable study of mechanically induced myotonia using both genetic and pharmacologic mouse models of myotonia congenita. We extended ex vivo studies of excitability to in vivo studies of muscle stiffness. RESULTS: As previous work suggests activation of transient receptor potential vanilloid 4 (TRPV4) channels by mechanical stimuli in muscle, we examined the role of this cation channel. Mechanically induced myotonia was markedly suppressed in TRPV4-null muscles and in muscles treated with TRPV4 small molecule antagonists. The suppression of mechanically induced myotonia occurred without altering intrinsic muscle excitability, such that myotonia triggered by firing of action potentials (electrically induced myotonia) was unaffected. When injected intraperitoneally, TRPV4 antagonists lessened the severity of myotonia in vivo by approximately 80%. INTERPRETATION: These data demonstrate that there are distinct molecular mechanisms triggering electrically induced and mechanically induced myotonia. Our data indicates that activation of TRPV4 during muscle contraction plays an important role in triggering myotonia in vivo. Elimination of mechanically induced myotonia by TRPV4 inhibition offers a new approach to treating myotonia. ANN NEUROL 2020;88:297-308.

The Loss of TBK1 Kinase Activity in Motor Neurons or in All Cell Types Differentially Impacts ALS Disease Progression in SOD1 Mice.

DNA sequence variants in the TBK1 gene associate with or cause sporadic or familial amyotrophic lateral sclerosis (ALS). Here we show that mice bearing human ALS-associated TBK1 missense loss-of-function mutations, or mice in which the Tbk1 gene is selectively deleted in motor neurons, do not display a neurodegenerative disease phenotype. However, loss of TBK1 function in motor neurons of the SOD1G93A mouse model of ALS impairs autophagy, increases SOD1 aggregation, and accelerates early disease onset without affecting lifespan. By contrast, point mutations that decrease TBK1 kinase activity in all cells also accelerate disease onset but extend the lifespan of SOD1 mice. This difference correlates with the failure to activate high levels of expression of interferon-inducible genes in glia. We conclude that loss of TBK1 kinase activity impacts ALS disease progression through distinct pathways in different spinal cord cell types and further implicate the importance of glia in neurodegeneration.

Dominant mutations of the Notch ligand Jagged1 cause peripheral neuropathy.

Notch signaling is a highly conserved intercellular pathway with tightly regulated and pleiotropic roles in normal tissue development and homeostasis. Dysregulated Notch signaling has also been implicated in human disease, including multiple forms of cancer, and represents an emerging therapeutic target. Successful development of such therapeutics requires a detailed understanding of potential on-target toxicities. Here, we identify autosomal dominant mutations of the canonical Notch ligand Jagged1 (or JAG1) as a cause of peripheral nerve disease in 2 unrelated families with the hereditary axonal neuropathy Charcot-Marie-Tooth disease type 2 (CMT2). Affected individuals in both families exhibited severe vocal fold paresis, a rare feature of peripheral nerve disease that can be life-threatening. Our studies of mutant protein posttranslational modification and localization indicated that the mutations (p.Ser577Arg, p.Ser650Pro) impair protein glycosylation and reduce JAG1 cell surface expression. Mice harboring heterozygous CMT2-associated mutations exhibited mild peripheral neuropathy, and homozygous expression resulted in embryonic lethality by midgestation. Together, our findings highlight a critical role for JAG1 in maintaining peripheral nerve integrity, particularly in the recurrent laryngeal nerve, and provide a basis for the evaluation of peripheral neuropathy as part of the clinical development of Notch pathway-modulating therapeutics.