TDP-43-M323K causes abnormal brain development and progressive cognitive and motor deficits associated with mislocalised and increased levels of TDP-43.

TDP-43 pathology is found in several neurodegenerative disorders, collectively referred to as "TDP-43 proteinopathies". Aggregates of TDP-43 are present in the brains and spinal cords of >97% of amyotrophic lateral sclerosis (ALS), and in brains of ∼50% of frontotemporal dementia (FTD) patients. While mutations in the TDP-43 gene (TARDBP) are usually associated with ALS, many clinical reports have linked these mutations to cognitive impairments and/or FTD, but also to other neurodegenerative disorders including Parkinsonism (PD) or progressive supranuclear palsy (PSP). TDP-43 is a ubiquitously expressed, highly conserved RNA-binding protein that is involved in many cellular processes, mainly RNA metabolism. To investigate systemic pathological mechanisms in TDP-43 proteinopathies, aiming to capture the pleiotropic effects of TDP-43 mutations, we have further characterised a mouse model carrying a point mutation (M323K) within the endogenous Tardbp gene. Homozygous mutant mice developed cognitive and behavioural deficits as early as 3 months of age. This was coupled with significant brain structural abnormalities, mainly in the cortex, hippocampus, and white matter fibres, together with progressive cortical interneuron degeneration and neuroinflammation. At the motor level, progressive phenotypes appeared around 6 months of age. Thus, cognitive phenotypes appeared to be of a developmental origin with a mild associated progressive neurodegeneration, while the motor and neuromuscular phenotypes seemed neurodegenerative, underlined by a progressive loss of upper and lower motor neurons as well as distal denervation. This is accompanied by progressive elevated TDP-43 protein and mRNA levels in cortex and spinal cord of homozygous mutant mice from 3 months of age, together with increased cytoplasmic TDP-43 mislocalisation in cortex, hippocampus, hypothalamus, and spinal cord at 12 months of age. In conclusion, we find that Tardbp M323K homozygous mutant mice model many aspects of human TDP-43 proteinopathies, evidencing a dual role for TDP-43 in brain morphogenesis as well as in the maintenance of the motor system, making them an ideal in vivo model system to study the complex biology of TDP-43.

Pridopidine Promotes Synaptogenesis and Reduces Spatial Memory Deficits in the Alzheimer's Disease APP/PS1 Mouse Model.

Sigma-1 receptor agonists have recently gained a great deal of interest due to their anti-amnesic, neuroprotective, and neurorestorative properties. Compounds such as PRE-084 or pridopidine (ACR16) are being studied as a potential treatment against cognitive decline associated with neurodegenerative disease, also to include Alzheimer's disease. Here, we performed in vitro experiments using primary neuronal cell cultures from rats to evaluate the abilities of ACR16 and PRE-084 to induce new synapses and spines formation, analyzing the expression of the possible genes and proteins involved. We additionally examined their neuroprotective properties against neuronal death mediated by oxidative stress and excitotoxicity. Both ACR16 and PRE-084 exhibited a concentration-dependent neuroprotective effect against NMDA- and H2O2-related toxicity, in addition to promoting the formation of new synapses and dendritic spines. However, only ACR16 generated dendritic spines involved in new synapse establishment, maintaining a more expanded activation of MAPK/ERK and PI3K/Akt signaling cascades. Consequently, ACR16 was also evaluated in vivo, and a dose of 1.5 mg/kg/day was administered intraperitoneally in APP/PS1 mice before performing the Morris water maze. ACR16 diminished the spatial learning and memory deficits observed in APP/PS1 transgenic mice via PI3K/Akt pathway activation. These data point to ACR16 as a pharmacological tool to prevent synapse loss and memory deficits associated with Alzheimer's disease, due to its neuroprotective properties against oxidative stress and excitotoxicity, as well as the promotion of new synapses and spines through a mechanism that involves AKT and ERK signaling pathways.

Erratum: Generation and analysis of innovative genomically humanized knockin SOD1, TARDBP (TDP-43), and FUS mouse models.

[This corrects the article DOI: 10.1016/j.isci.2021.103463.].

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Generation and analysis of innovative genomically humanized knockin SOD1, TARDBP (TDP-43), and FUS mouse models.

Amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) is a fatal neurodegenerative disorder, and continued innovation is needed for improved understanding and for developing therapeutics. We have created next-generation genomically humanized knockin mouse models, by replacing the mouse genomic region of Sod1, Tardbp (TDP-43), and Fus, with their human orthologs, preserving human protein biochemistry and splicing with exons and introns intact. We establish a new standard of large knockin allele quality control, demonstrating the utility of indirect capture for enrichment of a genomic region of interest followed by Oxford Nanopore sequencing. Extensive analysis shows that homozygous humanized animals only express human protein at endogenous levels. Characterization of humanized FUS animals showed that they are phenotypically normal throughout their lifespan. These humanized strains are vital for preclinical assessment of interventions and serve as templates for the addition of coding or non-coding human ALS/FTD mutations to dissect disease pathomechanisms, in a physiological context.

EFL1 mutations impair eIF6 release to cause Shwachman-Diamond syndrome.

Shwachman-Diamond syndrome (SDS) is a recessive disorder typified by bone marrow failure and predisposition to hematological malignancies. SDS is predominantly caused by deficiency of the allosteric regulator Shwachman-Bodian-Diamond syndrome that cooperates with elongation factor-like GTPase 1 (EFL1) to catalyze release of the ribosome antiassociation factor eIF6 and activate translation. Here, we report biallelic mutations in EFL1 in 3 unrelated individuals with clinical features of SDS. Cellular defects in these individuals include impaired ribosomal subunit joining and attenuated global protein translation as a consequence of defective eIF6 eviction. In mice, Efl1 deficiency recapitulates key aspects of the SDS phenotype. By identifying biallelic EFL1 mutations in SDS, we define this leukemia predisposition disorder as a ribosomopathy that is caused by corruption of a fundamental, conserved mechanism, which licenses entry of the large ribosomal subunit into translation.

Inhibition of the mTOR pathway: A new mechanism of ß cell toxicity induced by tacrolimus.

The mechanisms of tacrolimus-induced ß cell toxicity are unknown. Tacrolimus (TAC) and rapamycin (Rapa) both bind to FK506-binding protein 12 (FKBP12). Also, both molecular structures are similar. Because of this similarity, we hypothesized that TAC can also inhibit the mTOR signalling, constituting a possible mechanism of ß cell toxicity. Thus, we studied the effect of TAC and Rapa over the mTOR pathway, v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA), and insulin secretion and content in INS-1 ß cells treated with or without glucose and palmitate and in islets from lean or obese rats. TAC and Rapa inhibited the mTOR pathway as reflected by lower levels of phospho-mTOR, phospo-p70S6K, and phospo-S6. The effect of Rapa was larger than TAC. Both drugs reduced the levels of MafA, insulin secretion, and content although these effects were larger with TAC. The changes on MafA and insulin metabolism were observed in cells on glucose and palmitate, in obese animals, and were absent in cells on maintenance medium or in lean animals. In silico docking and immunoprecipitation experiments confirmed that TAC can form a stable noncovalent interaction with FKBP12-mTOR. Thus, the mTOR inhibition may be a mechanism contributing to the diabetogenic effect of TAC.

Transgenic and physiological mouse models give insights into different aspects of amyotrophic lateral sclerosis.

A wide range of genetic mouse models is available to help researchers dissect human disease mechanisms. Each type of model has its own distinctive characteristics arising from the nature of the introduced mutation, as well as from the specific changes to the gene of interest. Here, we review the current range of mouse models with mutations in genes causative for the human neurodegenerative disease amyotrophic lateral sclerosis. We focus on the two main types of available mutants: transgenic mice and those that express mutant genes at physiological levels from gene targeting or from chemical mutagenesis. We compare the phenotypes for genes in which the two classes of model exist, to illustrate what they can teach us about different aspects of the disease, noting that informative models may not necessarily mimic the full trajectory of the human condition. Transgenic models can greatly overexpress mutant or wild-type proteins, giving us insight into protein deposition mechanisms, whereas models expressing mutant genes at physiological levels may develop slowly progressing phenotypes but illustrate early-stage disease processes. Although no mouse models fully recapitulate the human condition, almost all help researchers to understand normal and abnormal biological processes, providing that the individual characteristics of each model type, and how these may affect the interpretation of the data generated from each model, are considered and appreciated.

Loss of Frrs1l disrupts synaptic AMPA receptor function, and results in neurodevelopmental, motor, cognitive and electrographical abnormalities.

Loss-of-function mutations in a human AMPA receptor-associated protein, ferric chelate reductase 1-like (FRRS1L), are associated with a devastating neurological condition incorporating choreoathetosis, cognitive deficits and epileptic encephalopathies. Furthermore, evidence from overexpression and ex vivo studies has implicated FRRS1L in AMPA receptor biogenesis, suggesting that changes in glutamatergic signalling might underlie the disorder. Here, we investigated the neurological and neurobehavioural correlates of the disorder using a mouse Frrs1l null mutant. The study revealed several neurological defects that mirrored those seen in human patients. We established that mice lacking Frrs1l suffered from a broad spectrum of early-onset motor deficits with no progressive, age-related deterioration. Moreover, Frrs1l-/- mice were hyperactive, irrespective of test environment, exhibited working memory deficits and displayed significant sleep fragmentation. Longitudinal electroencephalographic (EEG) recordings also revealed abnormal EEG results in Frrs1l-/- mice. Parallel investigations into disease aetiology identified a specific deficiency in AMPA receptor levels in the brain of Frrs1l-/- mice, while the general levels of several other synaptic components remained unchanged, with no obvious alterations in the number of synapses. Furthermore, we established that Frrsl1 deletion results in an increased proportion of immature AMPA receptors, indicated by incomplete glycosylation of GLUA2 (also known as GRIA2) and GLUA4 (also known as GRIA4) AMPA receptor proteins. This incomplete maturation leads to cytoplasmic retention and a reduction of those specific AMPA receptor levels in the postsynaptic membrane. Overall, this study determines, for the first time in vivo, how loss of FRRS1L function can affect glutamatergic signalling, and provides mechanistic insight into the development and progression of a human hyperkinetic disorder.This article has an associated First Person interview with the first author of the paper.

Skeletal Muscle Modulates Huntington's Disease Pathogenesis in Mice: Role of Physical Exercise.

Huntington's disease (HD) is a monogenic fatal neurodegenerative disorder. However, there is increasing evidence that HD is a pleiotropic systemic disorder. In particular, skeletal muscle metabolism is greatly affected in HD, which in turn can have a major impact on whole-body metabolism and energetic balance. Throughout an unbiased mutagenesis approach in HD mice, we have found that Scn4a, a skeletal muscle-specific sodium channel gene, is a modifier of the disease. Mutations in Scn4a enhance HD disease progression and weight loss by accelerating muscle waste and cachexia, increasing skeletal muscle activity and energy demands. At the molecular level, Scn4a mutations activate AMP-activated protein kinase (AMPK), leading to a fibre switch towards more oxidative types. These adaptations seen in HD; Scn4a double mutant muscles are similar to those observed in healthy individuals after endurance exercise training regimes. This prompted us to assess the effects of an endurance exercise regime in HD mice, independently showing that skeletal muscle adaptations leading to the activation of AMPK are detrimental for HD pathogenesis. Although it is undeniable that physical exercise can lead to many health benefits, our work shows that, at least under certain situations such as in HD, an endurance exercise routine could be a detrimental therapeutic option.

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.

A genetic modifier suggests that endurance exercise exacerbates Huntington's disease.

Polyglutamine expansions in the huntingtin gene cause Huntington's disease (HD). Huntingtin is ubiquitously expressed, leading to pathological alterations also in peripheral organs. Variations in the length of the polyglutamine tract explain up to 70% of the age-at-onset variance, with the rest of the variance attributed to genetic and environmental modifiers. To identify novel disease modifiers, we performed an unbiased mutagenesis screen on an HD mouse model, identifying a mutation in the skeletal muscle voltage-gated sodium channel (Scn4a, termed 'draggen' mutation) as a novel disease enhancer. Double mutant mice (HD; Scn4aDgn/+) had decreased survival, weight loss and muscle atrophy. Expression patterns show that the main tissue affected is skeletal muscle. Intriguingly, muscles from HD; Scn4aDgn/+ mice showed adaptive changes similar to those found in endurance exercise, including AMPK activation, fibre type switching and upregulation of mitochondrial biogenesis. Therefore, we evaluated the effects of endurance training on HD mice. Crucially, this training regime also led to detrimental effects on HD mice. Overall, these results reveal a novel role for skeletal muscle in modulating systemic HD pathogenesis, suggesting that some forms of physical exercise could be deleterious in neurodegeneration.

Pramipexole reduces soluble mutant huntingtin and protects striatal neurons through dopamine D3 receptors in a genetic model of Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disorder caused by abnormal expansion of the polyglutamine tract in the huntingtin protein (HTT). The toxicity of mutant HTT (mHTT) is associated with intermediate mHTT soluble oligomers that subsequently form intranuclear inclusions. Thus, interventions promoting the clearance of soluble mHTT are regarded as neuroprotective. Striatal neurons are particularly vulnerable in HD. Their degeneration underlies motor symptoms and striatal atrophy, the anatomical hallmark of HD. Recent studies indicate that autophagy may be activated by dopamine D2 and D3 receptor (D2R/D3R) agonists. Since autophagy plays a central role in the degradation of misfolded proteins, and striatal neurons express D2R and D3R, D2R/D3R agonists may promote the clearance of mHTT in striatal neurons. Here, this hypothesis was tested by treating 8-week old R6/1 mice with the D2R/D3R agonist pramipexole for 4weeks. Pramipexole reduced striatal levels of soluble mHTT and increased the size of intranuclear inclusions in R6/1 mice. Furthermore, striatal DARPP-32 levels and motor functions were recovered. These effects were accompanied by an increase in LC3-II and a decrease in p62 in the striatum. Tollip, a selective adaptor of ubiquitinated polyQ proteins to LC3, was also reduced in the striata of R6/1mice but not in their wild-type littermates. No changes were detected in the cerebral cortex where D3R expression is very low, and behavioral and biochemical effects in the striatum were prevented by a D3R antagonist. The findings indicate that PPX protects striatal neurons by promoting the clearance of soluble mHTT through a D3R-mediated mechanism. The evidence of autophagy markers suggests that autophagy is activated, although it is not efficient at removing all mHTT recruited by the autophagic machinery as indicated by the increase in the size of intranuclear inclusions.

Humanized mutant FUS drives progressive motor neuron degeneration without aggregation in 'FUSDelta14' knockin mice.

Mutations in FUS are causative for amyotrophic lateral sclerosis with a dominant mode of inheritance. In trying to model FUS-amyotrophic lateral sclerosis (ALS) in mouse it is clear that FUS is dosage-sensitive and effects arise from overexpression per se in transgenic strains. Novel models are required that maintain physiological levels of FUS expression and that recapitulate the human disease-with progressive loss of motor neurons in heterozygous animals. Here, we describe a new humanized FUS-ALS mouse with a frameshift mutation, which fulfils both criteria: the FUS Delta14 mouse. Heterozygous animals express mutant humanized FUS protein at physiological levels and have adult onset progressive motor neuron loss and denervation of neuromuscular junctions. Additionally, we generated a novel antibody to the unique human frameshift peptide epitope, allowing specific identification of mutant FUS only. Using our new FUSDelta14 ALS mouse-antibody system we show that neurodegeneration occurs in the absence of FUS protein aggregation. FUS mislocalization increases as disease progresses, and mutant FUS accumulates at the rough endoplasmic reticulum. Further, transcriptomic analyses show progressive changes in ribosomal protein levels and mitochondrial function as early disease stages are initiated. Thus, our new physiological mouse model has provided novel insight into the early pathogenesis of FUS-ALS.

Novel gene function revealed by mouse mutagenesis screens for models of age-related disease.

Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss.

Analysis of Individual Mouse Activity in Group Housed Animals of Different Inbred Strains using a Novel Automated Home Cage Analysis System.

Central nervous system disorders such as autism as well as the range of neurodegenerative diseases such as Huntington's disease are commonly investigated using genetically altered mouse models. The current system for characterizing these mice usually involves removing the animals from their home-cage environment and placing them into novel environments where they undergo a battery of tests measuring a range of behavioral and physical phenotypes. These tests are often only conducted for short periods of times in social isolation. However, human manifestations of such disorders are often characterized by multiple phenotypes, presented over long periods of time and leading to significant social impacts. Here, we have developed a system which will allow the automated monitoring of individual mice housed socially in the cage they are reared and housed in, within established social groups and over long periods of time. We demonstrate that the system accurately reports individual locomotor behavior within the group and that the measurements taken can provide unique insights into the effects of genetic background on individual and group behavior not previously recognized.

Loss-of-Function Mutations in FRRS1L Lead to an Epileptic-Dyskinetic Encephalopathy.

Glutamatergic neurotransmission governs excitatory signaling in the mammalian brain, and abnormalities of glutamate signaling have been shown to contribute to both epilepsy and hyperkinetic movement disorders. The etiology of many severe childhood movement disorders and epilepsies remains uncharacterized. We describe a neurological disorder with epilepsy and prominent choreoathetosis caused by biallelic pathogenic variants in FRRS1L, which encodes an AMPA receptor outer-core protein. Loss of FRRS1L function attenuates AMPA-mediated currents, implicating chronic abnormalities of glutamatergic neurotransmission in this monogenic neurological disease of childhood.

Deficiency of the zinc finger protein ZFP106 causes motor and sensory neurodegeneration.

Zinc finger motifs are distributed amongst many eukaryotic protein families, directing nucleic acid-protein and protein-protein interactions. Zinc finger protein 106 (ZFP106) has previously been associated with roles in immune response, muscle differentiation, testes development and DNA damage, although little is known about its specific function. To further investigate the function of ZFP106, we performed an in-depth characterization of Zfp106 deficient mice (Zfp106(-/-)), and we report a novel role for ZFP106 in motor and sensory neuronal maintenance and survival. Zfp106(-/-) mice develop severe motor abnormalities, major deficits in muscle strength and histopathological changes in muscle. Intriguingly, despite being highly expressed throughout the central nervous system, Zfp106(-/-) mice undergo selective motor and sensory neuronal and axonal degeneration specific to the spinal cord and peripheral nervous system. Neurodegeneration does not occur during development of Zfp106(-/-) mice, suggesting that ZFP106 is likely required for the maintenance of mature peripheral motor and sensory neurons. Analysis of embryonic Zfp106(-/-) motor neurons revealed deficits in mitochondrial function, with an inhibition of Complex I within the mitochondrial electron transport chain. Our results highlight a vital role for ZFP106 in sensory and motor neuron maintenance and reveal a novel player in mitochondrial dysfunction and neurodegeneration.