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

Behavioral and Other Phenotypes in a Cytoplasmic Dynein Light Intermediate Chain 1 Mutant Mouse

The cytoplasmic dynein complex is fundamentally important to all eukaryotic cells for transporting a variety of essential cargoes along microtubules within the cell. This complex also plays more specialized roles in neurons. The complex consists of 11 types of protein that interact with each other and with external adaptors, regulators and cargoes. Despite the importance of the cytoplasmic dynein complex, we know comparatively little of the roles of each component protein, and in mammals few mutants exist that allow us to explore the effects of defects in dynein-controlled processes in the context of the whole organism. Here we have taken a genotype-driven approach in mouse (Mus musculus) to analyze the role of one subunit, the dynein light intermediate chain 1 (Dync1li1). We find that, surprisingly, an N235Y point mutation in this protein results in altered neuronal development, as shown from in vivo studies in the developing cortex, and analyses of electrophysiological function. Moreover, mutant mice display increased anxiety, thus linking dynein functions to a behavioral phenotype in mammals for the first time. These results demonstrate the important role that dynein-controlled processes play in the correct development and function of the mammalian nervous system

Guidelines for the use and interpretation of assays for monitoring autophagy (2nd edition)

In 2008 we published the first set of guidelines for standardiz- ing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new tech- nologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in differ- ent organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes..

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

Acknowledgements We are indebted to Jim Humphries, JennyCorrigan, LizDarley, Elizabeth Joynson, Natalie Walters, Sara Wells and the whole necropsy, histology, genotyping and MLC ward 6 teams at MRC Harwell for excellent technical assistance. We thank the staff of the WTSI Illumina Bespoke Team for the RNA-seq data, the Sanger Mouse Genetics Project for the initial mouse characterization and Dr David Adams for critical reading of the manuscript. We also thank KOMP for the mouse embryonic stem cells carrying the knockout first promoter-less allele (tm1a(KOMP)Wtsi) within Zfp016. Conflict of Interest statement. None declared. Funding This work was funded by the UK Medical Research Council (MRC) to A.A.-A. and a Motor Neurone Disease Association (MNDA) project grant to A.A.-A. and EMCF. D.L.H.B. is a Wellcome Trust Senior Clinical Scientist Fellow and P.F. is a MRC/MNDA Lady Edith Wolfson Clinician Scientist Fellow. Funding to pay the Open Access publication charges for this article was provided by the MRC grant number: MC_UP_A390_1106.Peer reviewedPublisher PD

A nonsense mutation in mouse tardbp affects TDP43 alternative splicing activity and causes limb-clasping and body tone defects.

Mutations in TARDBP, encoding Tar DNA binding protein-43 (TDP43), cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Attempts to model TDP43 dysfunction in mice have used knockouts or transgenic overexpressors, which have revealed the difficulties of manipulating TDP43, whose level is tightly controlled by auto-regulation. In a complementary approach, to create useful mouse models for the dissection of TDP43 function and pathology, we have identified a nonsense mutation in the endogenous mouse Tardbp gene through screening an N-ethyl-N-nitrosourea (ENU) mutant mouse archive. The mutation is predicted to cause a Q101X truncation in TDP43. We have characterised Tardbp(Q101X) mice to investigate this mutation in perturbing TDP43 biology at endogenous expression levels. We found the Tardbp(Q101X) mutation is homozygous embryonic lethal, highlighting the importance of TDP43 in early development. Heterozygotes (Tardbp(+/Q101X) ) have abnormal levels of mutant transcript, but we find no evidence of the truncated protein and mice have similar full-length TDP43 protein levels as wildtype littermates. Nevertheless, Tardbp(+/Q101X) mice have abnormal alternative splicing of downstream gene targets, and limb-clasp and body tone phenotypes. Thus the nonsense mutation in Tardbp causes a mild loss-of-function phenotype and behavioural assessment suggests underlying neurological abnormalities. Due to the role of TDP43 in ALS, we investigated potential interactions with another known causative gene, mutant superoxide dismutase 1 (SOD1). Tardbp(+/Q101X) mice were crossed with the SOD1(G93Adl) transgenic mouse model of ALS. Behavioural and physiological assessment did not reveal modifying effects on the progression of ALS-like symptoms in the double mutant progeny from this cross. In summary, the Tardbp(Q101X) mutant mice are a useful tool for the dissection of TDP43 protein regulation, effects on splicing, embryonic development and neuromuscular phenotypes. These mice are freely available to the community

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

Loss of function mutations in the 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 have 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 recordings also revealed abnormal EEG 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 and GLUA4 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

Rilmenidine attenuates toxicity of polyglutamine expansions in a mouse model of Huntington's disease

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by a polyglutamine expansion in huntingtin. There are no treatments that are known to slow the neurodegeneration caused by this mutation. Mutant huntingtin causes disease via a toxic gain-of-function mechanism and has the propensity to aggregate and form intraneuronal inclusions. One therapeutic approach for HD is to enhance the degradation of the mutant protein. We have shown that this can be achieved by upregulating autophagy, using the drug rapamycin. In order to find safer ways of inducing autophagy for clinical purposes, we previously screened United States Food and Drug Administration-approved drugs for their autophagy-stimulating potential. This screen suggested that rilmenidine, a well tolerated, safe, centrally acting anti-hypertensive drug, could induce autophagy in cell culture via a pathway that was independent of the mammalian target of rapamycin. Here we have shown that rilmenidine induces autophagy in mice and in primary neuronal culture. Rilmenidine administration attenuated the signs of disease in a HD mouse model and reduced levels of the mutant huntingtin fragment. As rilmenidine has a long safety record and is designed for chronic use, our data suggests that it should be considered for the treatment of HD and related conditions

A Spontaneous Mutation of the Rat Themis Gene Leads to Impaired Function of Regulatory T Cells Linked to Inflammatory Bowel Disease

Spontaneous or chemically induced germline mutations, which lead to Mendelian phenotypes, are powerful tools to discover new genes and their functions. Here, we report an autosomal recessive mutation that occurred spontaneously in a Brown-Norway (BN) rat colony and was identified as causing marked T cell lymphopenia. This mutation was stabilized in a new rat strain, named BNm for “BN mutated.” In BNm rats, we found that the T cell lymphopenia originated in the thymus, was intrinsic to CD4 T lymphocytes, and was associated with the development of an inflammatory bowel disease. Furthermore, we demonstrate that the suppressive activity of both peripheral and thymic CD4+ CD25bright regulatory T cells (Treg) is defective in BNm rats. Complementation of mutant animals with BN Treg decreases disease incidence and severity, thus suggesting that the impaired Treg function is involved in the development of inflammatory bowel disease in BNm rats. Moreover, the cytokine profile of effector CD4 T cells is skewed toward Th2 and Th17 phenotypes in BNm rats. Linkage analysis and genetic dissection of the CD4 T cell lymphopenia in rats issued from BNm×DA crosses allowed the localization of the mutation on chromosome 1, within a 1.5 megabase interval. Gene expression and sequencing studies identified a frameshift mutation caused by a four-nucleotide insertion in the Themis gene, leading to its disruption. This result is the first to link Themis to the suppressive function of Treg and to suggest that, in Themis-deficient animals, defect of this function is involved in intestinal inflammation. Thus, this study highlights the importance of Themis as a new target gene that could participate in the pathogenesis of immune diseases characterized by chronic inflammation resulting from a defect in the Treg compartment

Oxr1 Is Essential for Protection against Oxidative Stress-Induced Neurodegeneration

Oxidative stress is a common etiological feature of neurological disorders, although the pathways that govern defence against reactive oxygen species (ROS) in neurodegeneration remain unclear. We have identified the role of oxidation resistance 1 (Oxr1) as a vital protein that controls the sensitivity of neuronal cells to oxidative stress; mice lacking Oxr1 display cerebellar neurodegeneration, and neurons are less susceptible to exogenous stress when the gene is over-expressed. A conserved short isoform of Oxr1 is also sufficient to confer this neuroprotective property both in vitro and in vivo. In addition, biochemical assays indicate that Oxr1 itself is susceptible to cysteine-mediated oxidation. Finally we show up-regulation of Oxr1 in both human and pre-symptomatic mouse models of amyotrophic lateral sclerosis, indicating that Oxr1 is potentially a novel neuroprotective factor in neurodegenerative disease

Novel mutations in human and mouse SCN4A implicate AMPK in myotonia and periodic paralysis

Mutations in the skeletal muscle channel (SCN4A), encoding the Nav1.4 voltage-gated sodium channel, are causative of a variety of muscle channelopathies, including non-dystrophic myotonias and periodic paralysis. The effects of many of these mutations on channel function have been characterized both in vitro and in vivo. However, little is known about the consequences of SCN4A mutations downstream from their impact on the electrophysiology of the Nav1.4 channel. Here we report the discovery of a novel SCN4A mutation (c.1762A>G; p.I588V) in a patient with myotonia and periodic paralysis, located within the S1 segment of the second domain of the Nav1.4 channel. Using N-ethyl-N-nitrosourea mutagenesis, we generated and characterized a mouse model (named draggen), carrying the equivalent point mutation (c.1744A>G; p.I582V) to that found in the patient with periodic paralysis and myotonia. Draggen mice have myotonia and suffer from intermittent hind-limb immobility attacks. In-depth characterization of draggen mice uncovered novel systemic metabolic abnormalities in Scn4a mouse models and provided novel insights into disease mechanisms. We discovered metabolic alterations leading to lean mice, as well as abnormal AMP-activated protein kinase activation, which were associated with the immobility attacks and may provide a novel potential therapeutic target