Publications by authors named "Abraham Acevedo-Arozena"

35 Publications

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

Autophagy 2021 Jan 8;17(1):1-382. Epub 2021 Feb 8.

University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.

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 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.
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http://dx.doi.org/10.1080/15548627.2020.1797280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996087PMC
January 2021

Uses for humanised mouse models in precision medicine for neurodegenerative disease.

Mamm Genome 2019 08 15;30(7-8):173-191. Epub 2019 Jun 15.

Mammalian Genetics Unit, MRC Harwell Institute, Oxfordshire, OX11 0RD, UK.

Neurodegenerative disease encompasses a wide range of disorders afflicting the central and peripheral nervous systems and is a major unmet biomedical need of our time. There are very limited treatments, and no cures, for most of these diseases, including Alzheimer's Disease, Parkinson's Disease, Huntington Disease, and Motor Neuron Diseases. Mouse and other animal models provide hope by analysing them to understand pathogenic mechanisms, to identify drug targets, and to develop gene therapies and stem cell therapies. However, despite many decades of research, virtually no new treatments have reached the clinic. Increasingly, it is apparent that human heterogeneity within clinically defined neurodegenerative disorders, and between patients with the same genetic mutations, significantly impacts disease presentation and, potentially, therapeutic efficacy. Therefore, stratifying patients according to genetics, lifestyle, disease presentation, ethnicity, and other parameters may hold the key to bringing effective therapies from the bench to the clinic. Here, we discuss genetic and cellular humanised mouse models, and how they help in defining the genetic and environmental parameters associated with neurodegenerative disease, and so help in developing effective precision medicine strategies for future healthcare.
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http://dx.doi.org/10.1007/s00335-019-09807-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6759662PMC
August 2019

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

Am J Transplant 2019 12 8;19(12):3240-3249. Epub 2019 Jul 8.

Hospital Universitario de Canarias, Instituto de Tecnologías Biomédicas (ITB), University of La Laguna, Tenerife, Spain.

The mechanisms of tacrolimus-induced β 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 β 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 β 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.
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http://dx.doi.org/10.1111/ajt.15483DOI Listing
December 2019

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

Blood 2019 07 31;134(3):277-290. Epub 2019 May 31.

INSERM Unité Mixte de Recherche 1163, Laboratory of Genome Dynamics in the Immune System, Equipe Labellisée Ligue contre le cancer, Paris, France.

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 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.
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http://dx.doi.org/10.1182/blood.2018893404DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6754720PMC
July 2019

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

Dis Model Mech 2019 02 22;12(2). Epub 2019 Feb 22.

MRC Harwell Institute, Harwell Campus, Oxfordshire OX11 0RD, UK

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 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 null mutant. The study revealed several neurological defects that mirrored those seen in human patients. We established that mice lacking suffered from a broad spectrum of early-onset motor deficits with no progressive, age-related deterioration. Moreover, 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 mice. Parallel investigations into disease aetiology identified a specific deficiency in AMPA receptor levels in the brain of 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 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 , 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.
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http://dx.doi.org/10.1242/dmm.036806DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6398485PMC
February 2019

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

Dis Model Mech 2019 01 2;12(1). Epub 2019 Jan 2.

Unidad de Investigación Hospital Universitario de Canarias, Fundación Canaria de Investigación Sanitaria and Instituto de Tecnologías Biomédicas (ITB), La Laguna, 38320 Tenerife, Spain

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.
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http://dx.doi.org/10.1242/dmm.037424DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6361152PMC
January 2019

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

J Exp Neurosci 2018 30;12:1179069518809059. Epub 2018 Oct 30.

Unidad de Investigación, Hospital Universitario de Canarias, Fundación Canaria de Investigación Sanitaria e Instituto de Tecnologías Biomédicas, La Laguna, Spain.

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 , a skeletal muscle-specific sodium channel gene, is a modifier of the disease. Mutations in enhance HD disease progression and weight loss by accelerating muscle waste and cachexia, increasing skeletal muscle activity and energy demands. At the molecular level, mutations activate AMP-activated protein kinase (AMPK), leading to a fibre switch towards more oxidative types. These adaptations seen in HD; 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.
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http://dx.doi.org/10.1177/1179069518809059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6210627PMC
October 2018

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

EMBO J 2018 06 15;37(11). Epub 2018 May 15.

UCL Institute of Neurology and Francis Crick Institute, London, UK.

TDP-43 (encoded by the gene ) is an RNA binding protein central to the pathogenesis of amyotrophic lateral sclerosis (ALS). However, how mutations trigger pathogenesis remains unknown. Here, we use novel mouse mutants carrying point mutations in endogenous to dissect TDP-43 function at physiological levels both and 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. , this gain-of-function mutation in endogenous 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.
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http://dx.doi.org/10.15252/embj.201798684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983119PMC
June 2018

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

Hum Mol Genet 2018 05;27(10):1723-1731

Mammalian Genetics Unit, Harwell Institute, Medical Research Council, Oxfordshire, UK.

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.
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http://dx.doi.org/10.1093/hmg/ddy077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5932560PMC
May 2018

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

Exp Neurol 2018 01 19;299(Pt A):137-147. Epub 2017 Oct 19.

Departamento de Ciencias Médicas Básicas, Universidad de La Laguna, Spain; Instituto de Tecnologías Biomédicas (CIBICAN), Spain. Electronic address:

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 D and D receptor (DR/DR) agonists. Since autophagy plays a central role in the degradation of misfolded proteins, and striatal neurons express DR and DR, DR/DR 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 DR/DR 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 DR expression is very low, and behavioral and biochemical effects in the striatum were prevented by a DR antagonist. The findings indicate that PPX protects striatal neurons by promoting the clearance of soluble mHTT through a DR-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.
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http://dx.doi.org/10.1016/j.expneurol.2017.10.019DOI Listing
January 2018

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

Brain 2017 Nov;140(11):2797-2805

Department of Neurodegenerative Disease, UCL Institute of Neurology, Queen Square, London WC1N 3BG, UK.

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.
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http://dx.doi.org/10.1093/brain/awx248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5841203PMC
November 2017

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

Nat Commun 2016 08 18;7:12444. Epub 2016 Aug 18.

The Nuffield Laboratory of Ophthalmology &NIHR Oxford Biomedical Research Centre, University of Oxford, Oxford OX3 9DU, UK.

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.
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http://dx.doi.org/10.1038/ncomms12444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4992138PMC
August 2016

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

Front Behav Neurosci 2016 10;10:106. Epub 2016 Jun 10.

Actual Analytics LtdEdinburgh, UK; School of Informatics, University of EdinburghEdinburgh, UK.

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.
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http://dx.doi.org/10.3389/fnbeh.2016.00106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4901040PMC
July 2016

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

Am J Hum Genet 2016 06 26;98(6):1249-1255. Epub 2016 May 26.

Mammalian Genetics Unit, Medical Research Council Harwell, Oxfordshire OX11 ORD, UK.

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.
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http://dx.doi.org/10.1016/j.ajhg.2016.04.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4908178PMC
June 2016

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

Hum Mol Genet 2016 Jan 24;25(2):291-307. Epub 2015 Nov 24.

MRC Mammalian Genetics Unit, Harwell, Oxfordshire OX11 0RD, UK,

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.
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http://dx.doi.org/10.1093/hmg/ddv471DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4706115PMC
January 2016

A novel SOD1-ALS mutation separates central and peripheral effects of mutant SOD1 toxicity.

Hum Mol Genet 2015 Apr 2;24(7):1883-97. Epub 2014 Dec 2.

MRC Mammalian Genetics Unit, Harwell, Oxfordshire OX11 0RD, UK,

Transgenic mouse models expressing mutant superoxide dismutase 1 (SOD1) have been critical in furthering our understanding of amyotrophic lateral sclerosis (ALS). However, such models generally overexpress the mutant protein, which may give rise to phenotypes not directly relevant to the disorder. Here, we have analysed a novel mouse model that has a point mutation in the endogenous mouse Sod1 gene; this mutation is identical to a pathological change in human familial ALS (fALS) which results in a D83G change in SOD1 protein. Homozgous Sod1(D83G/D83G) mice develop progressive degeneration of lower (LMN) and upper motor neurons, likely due to the same unknown toxic gain of function as occurs in human fALS cases, but intriguingly LMN cell death appears to stop in early adulthood and the mice do not become paralyzed. The D83 residue coordinates zinc binding, and the D83G mutation results in loss of dismutase activity and SOD1 protein instability. As a result, Sod1(D83G/D83G) mice also phenocopy the distal axonopathy and hepatocellular carcinoma found in Sod1 null mice (Sod1(-/-)). These unique mice allow us to further our understanding of ALS by separating the central motor neuron body degeneration and the peripheral effects from a fALS mutation expressed at endogenous levels.
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http://dx.doi.org/10.1093/hmg/ddu605DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4355022PMC
April 2015

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

Brain 2014 Dec 27;137(Pt 12):3171-85. Epub 2014 Oct 27.

1 MRC Mammalian Genetics Unit, Harwell, Oxfordshire, UK.

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.
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http://dx.doi.org/10.1093/brain/awu292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4240299PMC
December 2014

Reducing Igf-1r levels leads to paradoxical and sexually dimorphic effects in HD mice.

PLoS One 2014 20;9(8):e105595. Epub 2014 Aug 20.

MRC Mammalian Genetics Unit, Harwell, Oxfordshire, United Kingdom.

Many of the neurodegenerative diseases that afflict people in later life are associated with the formation of protein aggregates. These so-called "proteinopathies" include Alzheimer's disease (AD) and Huntington's disease (HD). The insulin/insulin-like growth factor signalling (IIS) pathway has been proposed to modulate such diseases in model organisms, as well as the general ageing process. In this pathway, insulin-like growth factor binds to insulin-like growth factor receptors, such as the insulin-like growth factor 1 receptor (IGF-1R). Heterozygous deletion of Igf-1r has been shown to lead to increased lifespan in mice. Reducing the activity of this pathway had benefits in a HD C. elegans model, and some of these may be attributed to the expected inhibition of mTOR activity resulting in an increase in autophagy, which would enhance mutant huntingtin clearance. Thus, we tested if heterozygous deletion of Igf-1r would lead to benefits in HD related phenotypes in the mouse. Surprisingly, reducing Igf-1r levels led to some beneficial effects in HD females, but also led to some detrimental effects in HD males. Interestingly, Igf-1r deficiency had no discernible effects on downstream mTOR signalling in HD mice. These results do not support a broad beneficial effect of diminishing the IIS pathway in HD pathology in a mammalian system.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0105595PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4139380PMC
November 2015

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

PLoS One 2014 21;9(1):e85962. Epub 2014 Jan 21.

MRC Mammalian Genetics Unit, Harwell, Oxfordshire, United Kingdom ; Department of Neurodegenerative Diseases and MRC Centre for Neuromuscular Diseases, UCL Institute of Neurology, London, United Kingdom.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0085962PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3897576PMC
October 2014

Widespread RNA metabolism impairment in sporadic inclusion body myositis TDP43-proteinopathy.

Neurobiol Aging 2014 Jun 30;35(6):1491-8. Epub 2013 Dec 30.

MRC Centre for Neuromuscular Disease, UCL Institute of Neurology, London, UK; Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK. Electronic address:

TDP43 protein mislocalization is a hallmark of the neurodegenerative diseases amyotrophic lateral sclerosis and frontotemporal dementia, and mutations in the gene encoding TDP43 cause both disorders, further highlighting its role in disease pathogenesis. TDP43 is a heterogenous ribonucleoprotein, therefore suggesting that alterations in RNA metabolism play a role in these disorders, although direct evidence in patients is lacking. Sporadic inclusion body myositis (sIBM) is the most common acquired myopathy occurring in adults aged older than 50 years and abnormal cytoplasmic accumulations of TDP43 have been consistently described in sIBM myofibers. Here, we exploit high quality RNA from frozen sIBM muscle biopsies for transcriptomic studies on TDP43-proteinopathy patient tissue. Surprisingly, we found widespread sIBM-specific changes in the RNA metabolism pathways themselves. Consistent with this finding, we describe novel RNA binding proteins to mislocalize in the cytoplasm of sIBM myofibers and splicing changes in MAPT, a gene previously shown to play a role in sIBM. Our data indicate widespread alterations of RNA metabolism are a novel aspect of disease pathogenesis in sIBM. These findings also document an association, in TDP43-proteinopathy patients, between heterogenous ribonucleoprotein pathology and RNA metabolism alterations and carry importance for neurodegenerative diseases, such as amyotrophic lateral sclerosis and frontotemporal dementia.
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http://dx.doi.org/10.1016/j.neurobiolaging.2013.12.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3988933PMC
June 2014

IGF-1 receptor antagonism inhibits autophagy.

Hum Mol Genet 2013 Nov 25;22(22):4528-44. Epub 2013 Jun 25.

Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Wellcome/MRC Building, Addenbrooke's Hospital, Hills Road, Cambridge CB2 0XY, UK.

Inhibition of the insulin/insulin-like growth factor signalling pathway increases lifespan and protects against neurodegeneration in model organisms, and has been considered as a potential therapeutic target. This pathway is upstream of mTORC1, a negative regulator of autophagy. Thus, we expected autophagy to be activated by insulin-like growth factor-1 (IGF-1) inhibition, which could account for many of its beneficial effects. Paradoxically, we found that IGF-1 inhibition attenuates autophagosome formation. The reduced amount of autophagosomes present in IGF-1R depleted cells can be, at least in part, explained by a reduced formation of autophagosomal precursors at the plasma membrane. In particular, IGF-1R depletion inhibits mTORC2, which, in turn, reduces the activity of protein kinase C (PKCα/β). This perturbs the actin cytoskeleton dynamics and decreases the rate of clathrin-dependent endocytosis, which impacts autophagosome precursor formation. Finally, with important implications for human diseases, we demonstrate that pharmacological inhibition of the IGF-1R signalling cascade reduces autophagy also in zebrafish and mice models. The novel link we describe here has important consequences for the interpretation of genetic experiments in mammalian systems and for evaluating the potential of targeting the IGF-1R receptor or modulating its signalling through the downstream pathway for therapeutic purposes under clinically relevant conditions, such as neurodegenerative diseases, where autophagy stimulation is considered beneficial.
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http://dx.doi.org/10.1093/hmg/ddt300DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3889807PMC
November 2013

Otitis media in the Tgif knockout mouse implicates TGFβ signalling in chronic middle ear inflammatory disease.

Hum Mol Genet 2013 Jul 3;22(13):2553-65. Epub 2013 Mar 3.

MRC Mammalian Genetics Unit, Harwell, UK

Otitis media with effusion (OME) is the most common cause of hearing loss in children and tympanostomy to alleviate the condition remains the commonest surgical intervention in children in the developed world. Chronic and recurrent forms of OM are known to have a very significant genetic component, however, until recently little was known of the underlying genes involved. The identification of mouse models of chronic OM has indicated a role of transforming growth factor beta (TGFβ) signalling and its impact on responses to hypoxia in the inflamed middle ear. We have, therefore, investigated the role of TGFβ signalling and identified and characterized a new model of chronic OM carrying a mutation in the gene for transforming growth interacting factor 1 (Tgif1). Tgif1 homozygous mutant mice have significantly raised auditory thresholds due to a conductive deafness arising from a chronic effusion starting at around 3 weeks of age. The OM is accompanied by a significant thickening of the middle ear mucosa lining, expansion of mucin-secreting goblet cell populations and raised levels of vascular endothelial growth factor, TNF-α and IL-1β in ear fluids. We also identified downstream effects on TGFβ signalling in middle ear epithelia at the time of development of chronic OM. Both phosphorylated SMAD2 and p21 levels were lowered in the homozygous mutant, demonstrating a suppression of the TGFβ pathway. The identification and characterization of the Tgif mutant supports the role of TGFβ signalling in the development of chronic OM and provides an important candidate gene for genetic studies in the human population.
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http://dx.doi.org/10.1093/hmg/ddt103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3674796PMC
July 2013

Guidelines for the use and interpretation of assays for monitoring autophagy.

Autophagy 2012 Apr;8(4):445-544

Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA.

In 2008 we published the first set of guidelines for standardizing 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 technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different 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. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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http://dx.doi.org/10.4161/auto.19496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3404883PMC
April 2012

α-Synuclein levels affect autophagosome numbers in vivo and modulate Huntington disease pathology.

Autophagy 2012 Mar 24;8(3):431-2. Epub 2012 Feb 24.

MRC Mammalian Genetics Unit, Harwell, Oxfordshire, UK.

Huntington and Parkinson diseases (HD and PD) are two major neurodegenerative disorders pathologically characterized by the accumulation of the aggregate-prone proteins mutant huntingtin (in HD) and α-synuclein (in PD). Mutant huntingtin is an autophagy substrate and autophagy modulators affect HD pathology both in vitro and in vivo. In vitro, α-synuclein levels are able to modulate autophagy: α-synuclein overexpression inhibits autophagy, whereas downregulation promotes autophagy. Here, we review our recent studies showing that α-synuclein levels modulate mutant huntingtin toxicity in mouse models. This phenotypic modification is accompanied by the in vivo modulation of autophagosome numbers in mouse brains from both control and HD mice expressing different levels of α-synuclein.
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http://dx.doi.org/10.4161/auto.19259DOI Listing
March 2012

α-Synuclein levels modulate Huntington's disease in mice.

Hum Mol Genet 2012 Feb 18;21(3):485-94. Epub 2011 Oct 18.

MRC Mammalian Genetics Unit, Harwell, Oxfordshire OX11 0RD, UK.

α-Synuclein and mutant huntingtin are the major constituents of the intracellular aggregates that characterize the pathology of Parkinson's disease (PD) and Huntington's disease (HD), respectively. α-Synuclein is likely to be a major contributor to PD, since overexpression of this protein resulting from genetic triplication is sufficient to cause human forms of PD. We have previously demonstrated that wild-type α-synuclein overexpression impairs macroautophagy in mammalian cells and in transgenic mice. Overexpression of human wild-type α-synuclein in cells and Drosophila models of HD worsens the disease phenotype. Here, we examined whether α-synuclein overexpression also worsens the HD phenotype in a mammalian system using two widely used N-terminal HD mouse models (R6/1 and N171-82Q). We also tested the effects of α-synuclein deletion in the same N-terminal HD mouse models, as well as assessed the effects of α-synuclein deletion on macroautophagy in mouse brains. We show that overexpression of wild-type α-synuclein in both mouse models of HD enhances the onset of tremors and has some influence on the rate of weight loss. On the other hand, α-synuclein deletion in both HD models increases autophagosome numbers and this is associated with a delayed onset of tremors and weight loss, two of the most prominent endophenotypes of the HD-like disease in mice. We have therefore established a functional link between these two aggregate-prone proteins in mammals and provide further support for the model that wild-type α-synuclein negatively regulates autophagy even at physiological levels.
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http://dx.doi.org/10.1093/hmg/ddr477DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3259010PMC
February 2012

SOD1 and TDP-43 animal models of amyotrophic lateral sclerosis: recent advances in understanding disease toward the development of clinical treatments.

Mamm Genome 2011 Aug 26;22(7-8):420-48. Epub 2011 Jun 26.

MRC Mammalian Genetics Unit, Harwell, Oxfordshire, UK.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease with no cure. Breakthroughs in understanding ALS pathogenesis came with the discovery of dominant mutations in the superoxide dismutase 1 gene (SOD1) and other genes, including the gene encoding transactivating response element DNA binding protein-43 (TDP-43). This has led to the creation of animal models to further our understanding of the disease and identify a number of ALS-causing mechanisms, including mitochondrial dysfunction, protein misfolding and aggregation, oxidative damage, neuronal excitotoxicity, non-cell autonomous effects and neuroinflammation, axonal transport defects, neurotrophin depletion, effects from extracellular mutant SOD1, and aberrant RNA processing. Here we summarise the SOD1 and TDP-43 animal models created to date, report on recent findings supporting the potential mechanisms of ALS pathogenesis, and correlate this understanding with current developments in the clinic.
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http://dx.doi.org/10.1007/s00335-011-9339-1DOI Listing
August 2011

A comprehensive assessment of the SOD1G93A low-copy transgenic mouse, which models human amyotrophic lateral sclerosis.

Dis Model Mech 2011 Sep 2;4(5):686-700. Epub 2011 May 2.

MRC Mammalian Genetics Unit, Harwell, OX11 0RD, UK.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder that results in the death of motor neurons in the brain and spinal cord. The disorder generally strikes in mid-life, relentlessly leading to paralysis and death, typically 3-5 years after diagnosis. No effective treatments are available. Up to 10% of ALS is familial, usually autosomal dominant. Several causative genes are known and, of these, mutant superoxide dismutase 1 (SOD1) is by far the most frequently found, accounting for up to 20% of familial ALS. A range of human mutant SOD1 transgenic mouse strains has been produced, and these largely successfully model the human disease. Of these, the most widely used is the SOD1 mouse, which expresses a human SOD1 transgene with a causative G93A mutation. This mouse model is excellent for many purposes but carries up to 25 copies of the transgene and produces a great excess of SOD1 protein, which might affect our interpretation of disease processes. A variant of this strain carries a deletion of the transgene array such that the copy number is dropped to eight to ten mutant SOD1 genes. This 'deleted' 'low-copy' mouse undergoes a slower course of disease, over many months. Here we have carried out a comprehensive analysis of phenotype, including nerve and muscle physiology and histology, to add to our knowledge of this 'deleted' strain and give baseline data for future studies. We find differences in phenotype that arise from genetic background and sex, and we quantify the loss of nerve and muscle function over time. The slowly progressive pathology observed in this mouse strain could provide us with a more appropriate model for studying early-stage pathological processes in ALS and aid the development of therapies for early-stage treatments.
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http://dx.doi.org/10.1242/dmm.007237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3180233PMC
September 2011

Behavioral and other phenotypes in a cytoplasmic Dynein light intermediate chain 1 mutant mouse.

J Neurosci 2011 Apr;31(14):5483-94

Department of Neurodegenerative Disease, Medical Research Council Centre for Neuromuscular Diseases, University College London Institute of Neurology, London WC1N 3BG, United Kingdom.

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.
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http://dx.doi.org/10.1523/JNEUROSCI.5244-10.2011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3096546PMC
April 2011
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