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1.
Int J Mol Sci ; 24(18)2023 Sep 18.
Article in English | MEDLINE | ID: mdl-37762529

ABSTRACT

While cells in the human body function in an environment where the blood supply constantly delivers nutrients and removes waste, cells in conventional tissue culture well platforms are grown with a static pool of media above them and often lack maturity, limiting their utility to study cell biology in health and disease. In contrast, organ-chip microfluidic systems allow the growth of cells under constant flow, more akin to the in vivo situation. Here, we differentiated human induced pluripotent stem cells into dopamine neurons and assessed cellular properties in conventional multi-well cultures and organ-chips. We show that organ-chip cultures, compared to multi-well cultures, provide an overall greater proportion and homogeneity of dopaminergic neurons as well as increased levels of maturation markers. These organ-chips are an ideal platform to study mature dopamine neurons to better understand their biology in health and ultimately in neurological disorders.


Subject(s)
Dopaminergic Neurons , Induced Pluripotent Stem Cells , Humans , Cell Differentiation , Cells, Cultured , Organ Culture Techniques
2.
J Peripher Nerv Syst ; 28(3): 329-340, 2023 09.
Article in English | MEDLINE | ID: mdl-37220142

ABSTRACT

BACKGROUND AND AIMS: Mitofusin 1 (MFN1) and MFN2 are outer mitochondrial membrane fusogenic proteins regulating mitochondrial network morphology. MFN2 mutations cause Charcot-Marie-Tooth type 2A (CMT2A), an axonal neuropathy characterized by mitochondrial fusion defects, which in the case of a GTPase domain mutant, were rescued following wild-type MFN1/2 (MFN1/2WT ) overexpression. In this study, we compared the therapeutic efficiency between MFN1WT and MFN2WT overexpression in correcting mitochondrial defects induced by the novel MFN2K357T mutation located in the highly conserved R3 region. METHODS: Constructs expressing either MFN2K357T , MFN2WT , or MFN1WT under the ubiquitous chicken ß-actin hybrid (CBh) promoter were generated. Flag or myc tag was used for their detection. Differentiated SH-SY5Y cells were single transfected with MFN1WT , MFN2WT , or MFN2K357T , as well as double transfected with MFN2K357T /MFN2WT or MFN2K357T /MFN1WT . RESULTS: SH-SY5Y cells transfected with MFN2K357T exhibited severe perinuclear mitochondrial clustering with axon-like processes devoid of mitochondria. Single transfection with MFN1WT resulted in a more interconnected mitochondrial network than transfection with MFN2WT , accompanied by mitochondrial clusters. Double transfection of MFN2K357T with either MFN1WT or MFN2WT resolved the mutant-induced mitochondrial clusters and led to detectable mitochondria throughout the axon-like processes. MFN1WT showed higher efficacy than MFN2WT in rescuing these defects. INTERPRETATION: These results further demonstrate the higher potential of MFN1WT over MFN2WT overexpression to rescue CMT2A-induced mitochondrial network abnormalities due to mutations outside the GTPase domain. This higher phenotypic rescue conferred by MFN1WT , possibly due to its higher mitochondrial fusogenic ability, may be applied to different CMT2A cases regardless of the MFN2 mutation type.


Subject(s)
Charcot-Marie-Tooth Disease , Neuroblastoma , Humans , Mitochondrial Dynamics , Neuroblastoma/metabolism , Mitochondria/genetics , Mitochondria/metabolism , Mutation , GTP Phosphohydrolases/genetics , Mitochondrial Proteins/genetics , Charcot-Marie-Tooth Disease/genetics
3.
Stem Cell Reports ; 18(8): 1629-1642, 2023 08 08.
Article in English | MEDLINE | ID: mdl-37084724

ABSTRACT

Human induced pluripotent stem cells (iPSCs) are a renewable cell source that can be differentiated into neural progenitor cells (iNPCs) and transduced with glial cell line-derived neurotrophic factor (iNPC-GDNFs). The goal of the current study is to characterize iNPC-GDNFs and test their therapeutic potential and safety. Single-nuclei RNA-seq show iNPC-GDNFs express NPC markers. iNPC-GDNFs delivered into the subretinal space of the Royal College of Surgeons rodent model of retinal degeneration preserve photoreceptors and visual function. Additionally, iNPC-GDNF transplants in the spinal cord of SOD1G93A amyotrophic lateral sclerosis (ALS) rats preserve motor neurons. Finally, iNPC-GDNF transplants in the spinal cord of athymic nude rats survive and produce GDNF for 9 months, with no signs of tumor formation or continual cell proliferation. iNPC-GDNFs survive long-term, are safe, and provide neuroprotection in models of both retinal degeneration and ALS, indicating their potential as a combined cell and gene therapy for various neurodegenerative diseases.


Subject(s)
Amyotrophic Lateral Sclerosis , Induced Pluripotent Stem Cells , Retinal Degeneration , Humans , Rats , Animals , Amyotrophic Lateral Sclerosis/pathology , Induced Pluripotent Stem Cells/pathology , Rodentia , Retinal Degeneration/therapy , Retinal Degeneration/pathology , Glial Cell Line-Derived Neurotrophic Factor/genetics , Astrocytes/pathology , Disease Models, Animal
4.
Neuron ; 109(14): 2275-2291.e8, 2021 07 21.
Article in English | MEDLINE | ID: mdl-34133945

ABSTRACT

C9orf72 repeat expansions cause inherited amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD) and result in both loss of C9orf72 protein expression and production of potentially toxic RNA and dipeptide repeat proteins. In addition to ALS/FTD, C9orf72 repeat expansions have been reported in a broad array of neurodegenerative syndromes, including Alzheimer's disease. Here we show that C9orf72 deficiency promotes a change in the homeostatic signature in microglia and a transition to an inflammatory state characterized by an enhanced type I IFN signature. Furthermore, C9orf72-depleted microglia trigger age-dependent neuronal defects, in particular enhanced cortical synaptic pruning, leading to altered learning and memory behaviors in mice. Interestingly, C9orf72-deficient microglia promote enhanced synapse loss and neuronal deficits in a mouse model of amyloid accumulation while paradoxically improving plaque clearance. These findings suggest that altered microglial function due to decreased C9orf72 expression directly contributes to neurodegeneration in repeat expansion carriers independent of gain-of-function toxicities.


Subject(s)
Aging/metabolism , Amyloid/metabolism , C9orf72 Protein/metabolism , Microglia/metabolism , Synapses/metabolism , Aging/genetics , Aging/pathology , Amyloid/genetics , Animals , C9orf72 Protein/genetics , DNA Repeat Expansion , Disease Models, Animal , Lysosomes/metabolism , Mice , Mice, Knockout , Synapses/pathology
6.
Nature ; 585(7823): 96-101, 2020 09.
Article in English | MEDLINE | ID: mdl-32814898

ABSTRACT

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative disorders that overlap in their clinical presentation, pathology and genetic origin. Autoimmune disorders are also overrepresented in both ALS and FTD, but this remains an unexplained epidemiologic observation1-3. Expansions of a hexanucleotide repeat (GGGGCC) in the C9orf72 gene are the most common cause of familial ALS and FTD (C9-ALS/FTD), and lead to both repeat-containing RNA and dipeptide accumulation, coupled with decreased C9orf72 protein expression in brain and peripheral blood cells4-6. Here we show in mice that loss of C9orf72 from myeloid cells alone is sufficient to recapitulate the age-dependent lymphoid hypertrophy and autoinflammation seen in animals with a complete knockout of C9orf72. Dendritic cells isolated from C9orf72-/- mice show marked early activation of the type I interferon response, and C9orf72-/- myeloid cells are selectively hyperresponsive to activators of the stimulator of interferon genes (STING) protein-a key regulator of the innate immune response to cytosolic DNA. Degradation of STING through the autolysosomal pathway is diminished in C9orf72-/- myeloid cells, and blocking STING suppresses hyperactive type I interferon responses in C9orf72-/- immune cells as well as splenomegaly and inflammation in C9orf72-/- mice. Moreover, mice lacking one or both copies of C9orf72 are more susceptible to experimental autoimmune encephalitis, mirroring the susceptibility to autoimmune diseases seen in people with C9-ALS/FTD. Finally, blood-derived macrophages, whole blood and brain tissue from patients with C9-ALS/FTD all show an elevated type I interferon signature compared with samples from people with sporadic ALS/FTD; this increased interferon response can be suppressed with a STING inhibitor. Collectively, our results suggest that patients with C9-ALS/FTD have an altered immunophenotype because their reduced levels of C9orf72 cannot suppress the inflammation mediated by the induction of type I interferons by STING.


Subject(s)
C9orf72 Protein/genetics , C9orf72 Protein/metabolism , Inflammation/metabolism , Inflammation/prevention & control , Membrane Proteins/metabolism , Myeloid Cells/metabolism , Aging/immunology , Amyotrophic Lateral Sclerosis/genetics , Animals , C9orf72 Protein/deficiency , Dendritic Cells/cytology , Dendritic Cells/immunology , Encephalomyelitis, Autoimmune, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/immunology , Female , Humans , Inflammation/genetics , Inflammation/immunology , Interferon Type I/biosynthesis , Interferon Type I/immunology , Membrane Proteins/antagonists & inhibitors , Mice , Myeloid Cells/immunology , Neoplasms/immunology , T-Lymphocytes/cytology , T-Lymphocytes/immunology
7.
Nat Biomed Eng ; 3(7): 571-582, 2019 07.
Article in English | MEDLINE | ID: mdl-30962586

ABSTRACT

Patient-specific human-induced pluripotent stem cells (hiPSCs) hold great promise for the modelling of genetic disorders. However, these cells display wide intra- and interindividual variations in gene expression, which makes distinguishing true-positive and false-positive phenotypes challenging. Data from hiPSC phenotypes and human embryonic stem cells (hESCs) harbouring the same disease mutation are also lacking. Here, we report a comparison of the molecular, cellular and functional characteristics of three congruent patient-specific cell types-hiPSCs, hESCs and direct-lineage-converted cells-derived from currently available differentiation and direct-reprogramming technologies for use in the modelling of Charcot-Marie-Tooth 1A, a human genetic Schwann-cell disorder featuring a 1.4 Mb chromosomal duplication. We find that the chemokines C-X-C motif ligand chemokine-1 (CXCL1) and macrophage chemoattractant protein-1 (MCP1) are commonly upregulated in all three congruent models and in clinical patient samples. The development of congruent models of a single genetic disease using somatic cells from a common patient will facilitate the search for convergent phenotypes.


Subject(s)
Chemokine CCL2/genetics , Chemokine CXCL1/genetics , Genetic Diseases, Inborn , Induced Pluripotent Stem Cells/metabolism , Schwann Cells/metabolism , Adult , Animals , CRISPR-Cas Systems , Cell Differentiation/genetics , Cell Line , Cell Lineage/genetics , Cells, Cultured , Cellular Reprogramming , Chemokine CCL2/metabolism , Chemokine CXCL1/metabolism , Chemokines , Embryonic Stem Cells/pathology , Female , Gene Editing , Gene Expression , Gene Expression Profiling , Genetic Predisposition to Disease/genetics , Human Genetics , Humans , Induced Pluripotent Stem Cells/pathology , Male , Mice , Mice, Inbred NOD , Myelin Proteins/genetics , Myelin Proteins/metabolism , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , Phenotype , Rats , SOXE Transcription Factors/genetics , SOXE Transcription Factors/metabolism , Schwann Cells/pathology , Transplantation
8.
J Clin Invest ; 129(4): 1756-1771, 2019 03 18.
Article in English | MEDLINE | ID: mdl-30882371

ABSTRACT

Mitofusin-2 (MFN2) is a mitochondrial outer-membrane protein that plays a pivotal role in mitochondrial dynamics in most tissues, yet mutations in MFN2, which cause Charcot-Marie-Tooth disease type 2A (CMT2A), primarily affect the nervous system. We generated a transgenic mouse model of CMT2A that developed severe early onset vision loss and neurological deficits, axonal degeneration without cell body loss, and cytoplasmic and axonal accumulations of fragmented mitochondria. While mitochondrial aggregates were labeled for mitophagy, mutant MFN2 did not inhibit Parkin-mediated degradation, but instead had a dominant negative effect on mitochondrial fusion only when MFN1 was at low levels, as occurs in neurons. Finally, using a transgenic approach, we found that augmenting the level of MFN1 in the nervous system in vivo rescued all phenotypes in mutant MFN2R94Q-expressing mice. These data demonstrate that the MFN1/MFN2 ratio is a key determinant of tissue specificity in CMT2A and indicate that augmentation of MFN1 in the nervous system is a viable therapeutic strategy for the disease.


Subject(s)
Axons/metabolism , Charcot-Marie-Tooth Disease/metabolism , GTP Phosphohydrolases/metabolism , Animals , Axons/pathology , Charcot-Marie-Tooth Disease/genetics , Charcot-Marie-Tooth Disease/pathology , Charcot-Marie-Tooth Disease/prevention & control , Disease Models, Animal , GTP Phosphohydrolases/genetics , Mice , Mice, Transgenic , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism
9.
Ann Clin Transl Neurol ; 5(2): 186-200, 2018 02.
Article in English | MEDLINE | ID: mdl-29468179

ABSTRACT

Objective: To investigate transplantation of rat Schwann cells or human iPSC-derived neural crest cells and derivatives into models of acquired and inherited peripheral myelin damage. Methods: Primary cultured rat Schwann cells labeled with a fluorescent protein for monitoring at various times after transplantation. Human-induced pluripotent stem cells (iPSCs) were differentiated into neural crest stem cells, and subsequently toward a Schwann cell lineage via two different protocols. Cell types were characterized using flow cytometry, immunocytochemistry, and transcriptomics. Rat Schwann cells and human iPSC derivatives were transplanted into (1) nude rats pretreated with lysolecithin to induce demyelination or (2) a transgenic rat model of dysmyelination due to PMP22 overexpression. Results: Rat Schwann cells transplanted into sciatic nerves with either toxic demyelination or genetic dysmyelination engrafted successfully, and migrated longitudinally for relatively long distances, with more limited axial migration. Transplanted Schwann cells engaged existing axons and displaced dysfunctional Schwann cells to form normal-appearing myelin. Human iPSC-derived neural crest stem cells and their derivatives shared similar engraftment and migration characteristics to rat Schwann cells after transplantation, but did not further differentiate into Schwann cells or form myelin. Interpretation: These results indicate that cultured Schwann cells surgically delivered to peripheral nerve can engraft and form myelin in either acquired or inherited myelin injury, as proof of concept for pursuing cell therapy for diseases of peripheral nerve. However, lack of reliable technology for generating human iPSC-derived Schwann cells for transplantation therapy remains a barrier in the field.

10.
Neuron ; 88(5): 892-901, 2015 Dec 02.
Article in English | MEDLINE | ID: mdl-26637796

ABSTRACT

Noncoding expansions of a hexanucleotide repeat (GGGGCC) in the C9orf72 gene are the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia. Here we report transgenic mice carrying a bacterial artificial chromosome (BAC) containing the full human C9orf72 gene with either a normal allele (15 repeats) or disease-associated expansion (∼100-1,000 repeats; C9-BACexp). C9-BACexp mice displayed pathologic features seen in C9orf72 expansion patients, including widespread RNA foci and repeat-associated non-ATG (RAN) translated dipeptides, which were suppressed by antisense oligonucleotides targeting human C9orf72. Nucleolin distribution was altered, supporting that either C9orf72 transcripts or RAN dipeptides promote nucleolar dysfunction. Despite early and widespread production of RNA foci and RAN dipeptides in C9-BACexp mice, behavioral abnormalities and neurodegeneration were not observed even at advanced ages, supporting the hypothesis that RNA foci and RAN dipeptides occur presymptomatically and are not sufficient to drive neurodegeneration in mice at levels seen in patients.


Subject(s)
Amyotrophic Lateral Sclerosis/pathology , Brain/pathology , DNA Repeat Expansion/genetics , Frontotemporal Dementia/pathology , Proteins/genetics , Spinal Cord/pathology , Age Factors , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/physiopathology , Animals , Brain/metabolism , C9orf72 Protein , Cells, Cultured , Chromosomes, Artificial, Bacterial/genetics , Chromosomes, Artificial, Bacterial/metabolism , Disease Models, Animal , Frontotemporal Dementia/genetics , Frontotemporal Dementia/physiopathology , Glutamic Acid/pharmacology , Humans , Mice , Mice, Transgenic , Motor Activity/genetics , Muscle Strength/genetics , Neuromuscular Junction/genetics , Neuromuscular Junction/pathology , Neurons/drug effects , Psychomotor Performance/physiology , Spinal Cord/metabolism
11.
Exp Neurol ; 273: 24-35, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26222336

ABSTRACT

Transactive response DNA-binding protein-43 (TDP-43) is a multifunctional nucleic acid binding protein present in ubiquitinated inclusions in tissues of patients with amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar degeneration (FTLD). The ALS-associated mutations in the glycine-rich C-terminal domain of TDP-43 established a causal link between TDP-43 and disease, and conferred both loss- and gain-of-function properties in neurons. Since it has not been established whether these intra-neuronal changes are sufficient to cause ALS or whether non-cell autonomous neuronal-glial signaling could be involved, we investigated the extracellular effects of TDP-43 proteins on microglial activation and motoneuron toxicity. Wild-type, truncated 25kD C-terminal fragments, or mutant forms of TDP-43 all activated microglia and upregulated NOX2, TNF-α, and IL-1ß, with WT forms being significantly less effective in activating microglia. This response to TDP-43 was mediated by its interaction with the microglial surface CD14 receptor and subsequent stimulation of the NF-κB and AP-1 pathways, as well as the intracellular inflammasome. At the cell surface, CD14 blocking antibodies suppressed microglial NF-κB activation and proinflammatory cytokine production mediated by TDP-43. Intracellularly, the NLRP3 inflammasome was induced and functional caspase-1 was produced augmenting the release of mature IL-1ß. Further, TDP-43-mediated activation of microglia caused a proinflammatory cascade that was toxic to motoneurons. In the absence of microglia, TDP-43 was not toxic to motoneurons. The ability of TDP-43 to promote CD14-mediated activation of microglial NF-κB and AP-1 pathways, as well as the NLRP3 inflammasome, suggests the involvement of a non-cell autonomous proinflammatory signaling that enhances motoneuron injury, and may offer novel therapeutic targets in ALS.


Subject(s)
Carrier Proteins/metabolism , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/pharmacology , Microglia/metabolism , NF-kappa B/metabolism , Animals , Animals, Newborn , Antibodies/pharmacology , Caspase 1/metabolism , DNA-Binding Proteins/genetics , Embryo, Mammalian , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Lipopolysaccharide Receptors/immunology , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred C57BL , Motor Neurons/drug effects , Motor Neurons/metabolism , Mutation/genetics , NADPH Oxidase 2 , NADPH Oxidases/genetics , NADPH Oxidases/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein , RNA, Messenger/metabolism , Retinoids/pharmacology , Spinal Cord/cytology , Transfection
12.
Hum Mol Genet ; 23(1): 157-70, 2014 Jan 01.
Article in English | MEDLINE | ID: mdl-23962724

ABSTRACT

TDP-43 aggregation in the cytoplasm or nucleus is a key feature of the pathology of amyotrophic lateral sclerosis and frontotemporal dementia and is observed in numerous other neurodegenerative diseases, including Alzheimer's disease. Despite this fact, the inciting events leading to TDP-43 aggregation remain unclear. We observed that endogenous TDP-43 undergoes reversible aggregation in the nucleus after the heat shock and that this behavior is mediated by the C-terminal prion domain. Substitution of the prion domain from TIA-1 or an authentic yeast prion domain from RNQ1 into TDP-43 can completely recapitulate heat shock-induced aggregation. TDP-43 is constitutively bound to members of the Hsp40/Hsp70 family, and we found that heat shock-induced TDP-43 aggregation is mediated by the availability of these chaperones interacting with the inherently disordered C-terminal prion domain. Finally, we observed that the aggregation of TDP-43 during heat shock led to decreased binding to hnRNPA1, and a change in TDP-43 RNA-binding partners suggesting that TDP-43 aggregation alters its function in response to misfolded protein stress. These findings indicate that TDP-43 shares properties with physiologic prions from yeast, in that self-aggregation is mediated by a Q/N-rich disordered domain, is modulated by chaperone proteins and leads to altered function of the protein. Furthermore, they indicate that TDP-43 aggregation is regulated by chaperone availability, explaining the recurrent observation of TDP-43 aggregates in degenerative diseases of both the brain and muscle where protein homeostasis is disrupted.


Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , HSP40 Heat-Shock Proteins/physiology , HSP70 Heat-Shock Proteins/physiology , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/metabolism , Prions/chemistry , Amino Acid Motifs , Animals , Brain/metabolism , COS Cells , Cell Nucleus/metabolism , Chlorocebus aethiops , Cytoplasm/metabolism , HEK293 Cells , HeLa Cells , Heat-Shock Response , Heterogeneous Nuclear Ribonucleoprotein A1 , Humans , Muscles/metabolism , Prions/metabolism , Protein Folding
13.
Sci Transl Med ; 5(208): 208ra149, 2013 Oct 23.
Article in English | MEDLINE | ID: mdl-24154603

ABSTRACT

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative condition characterized by loss of motor neurons in the brain and spinal cord. Expansions of a hexanucleotide repeat (GGGGCC) in the noncoding region of the C9ORF72 gene are the most common cause of the familial form of ALS (C9-ALS), as well as frontotemporal lobar degeneration and other neurological diseases. How the repeat expansion causes disease remains unclear, with both loss of function (haploinsufficiency) and gain of function (either toxic RNA or protein products) proposed. We report a cellular model of C9-ALS with motor neurons differentiated from induced pluripotent stem cells (iPSCs) derived from ALS patients carrying the C9ORF72 repeat expansion. No significant loss of C9ORF72 expression was observed, and knockdown of the transcript was not toxic to cultured human motor neurons. Transcription of the repeat was increased, leading to accumulation of GGGGCC repeat-containing RNA foci selectively in C9-ALS iPSC-derived motor neurons. Repeat-containing RNA foci colocalized with hnRNPA1 and Pur-α, suggesting that they may be able to alter RNA metabolism. C9-ALS motor neurons showed altered expression of genes involved in membrane excitability including DPP6, and demonstrated a diminished capacity to fire continuous spikes upon depolarization compared to control motor neurons. Antisense oligonucleotides targeting the C9ORF72 transcript suppressed RNA foci formation and reversed gene expression alterations in C9-ALS motor neurons. These data show that patient-derived motor neurons can be used to delineate pathogenic events in ALS.


Subject(s)
Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/pathology , DNA Repeat Expansion/genetics , Induced Pluripotent Stem Cells/pathology , Motor Neurons/metabolism , Motor Neurons/pathology , Proteins/genetics , RNA/metabolism , C9orf72 Protein , Exons/genetics , Gene Knockdown Techniques , Humans , Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/metabolism , Motor Neurons/drug effects , Oligonucleotides, Antisense/pharmacology , RNA/biosynthesis , RNA/genetics , Transcription, Genetic/drug effects
14.
Ann Neurol ; 71(3): 407-16, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22334415

ABSTRACT

OBJECTIVE: To identify the causative gene in an autosomal dominant limb-girdle muscular dystrophy (LGMD) with skeletal muscle vacuoles. METHODS: Exome sequencing was used to identify candidate mutations in the studied pedigree. Genome-wide linkage was then used to narrow the list of candidates to a single disease-associated mutation. Additional pedigrees with dominant or sporadic myopathy were screened for mutations in the same gene (DNAJB6) using exome sequencing. Skeletal muscle from affected patients was evaluated with histochemistry and immunohistochemical stains for dystrophy-related proteins, SMI-31, TDP43, and DNAJB6. RESULTS: Exome analysis in 3 affected individuals from a family with dominant LGMD and vacuolar pathology identified novel candidate mutations in 22 genes. Linkage analysis excluded all variants except a Phe93Leu mutation in the G/F domain of the DNAJB6 gene, which resides within the LGMD locus at 7q36. Analysis of exome sequencing data from other pedigrees with dominant myopathy identified a second G/F domain mutation (Pro96Arg) in DNAJB6. Affected muscle showed mild dystrophic changes, vacuoles, and abnormal aggregation of proteins, including TDP-43 and DNAJB6 itself. INTERPRETATION: Mutations within the G/F domain of DNAJB6 are a novel cause of dominantly-inherited myopathy. DNAJB6 is a member of the HSP40/DNAJ family of molecular co-chaperones tasked with protecting client proteins from irreversible aggregation during protein synthesis or during times of cellular stress. The abnormal accumulation of several proteins in patient muscle, including DNAJB6 itself, suggest that DNAJB6 function is compromised by the identified G/F domain mutations.


Subject(s)
Exome/genetics , Genes, Dominant , HSP40 Heat-Shock Proteins/genetics , Molecular Chaperones/genetics , Muscular Diseases/genetics , Muscular Dystrophies, Limb-Girdle/genetics , Mutation/genetics , Nerve Tissue Proteins/genetics , Sequence Analysis, DNA , Adolescent , Adult , Amino Acid Sequence , Arginine/genetics , Female , Genome-Wide Association Study/methods , Humans , Male , Middle Aged , Molecular Sequence Data , Muscular Diseases/diagnosis , Muscular Dystrophies, Limb-Girdle/diagnosis , Pedigree , Proline/genetics , Protein Structure, Tertiary/genetics , Sequence Analysis, DNA/methods , Young Adult
15.
J Biol Chem ; 285(34): 26304-14, 2010 Aug 20.
Article in English | MEDLINE | ID: mdl-20554523

ABSTRACT

The identification of pathologic TDP-43 aggregates in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration, followed by the discovery of dominantly inherited point mutations in TDP-43 in familial ALS, have been critical insights into the mechanism of these untreatable neurodegenerative diseases. However, the biochemical basis of TDP-43 aggregation and the mechanism of how mutations in TDP-43 lead to disease remain enigmatic. In efforts to understand how TDP-43 alters its cellular localization in response to proteotoxic stress, we found that TDP-43 is sequestered into polyglutamine aggregates. Furthermore, we found that binding to polyglutamine aggregates requires a previously uncharacterized glutamine/asparagine (Q/N)-rich region in the C-terminal domain of TDP-43. Sequestration into polyglutamine aggregates causes TDP-43 to be cleared from the nucleus and become detergent-insoluble. Finally, we observed that sequestration into polyglutamine aggregates led to loss of TDP-43-mediated splicing in the nucleus and that polyglutamine toxicity could be partially rescued by increasing expression of TDP-43. These data indicate pathologic sequestration into polyglutamine aggregates, and loss of nuclear TDP-43 function may play an unexpected role in polyglutamine disease pathogenesis. Furthermore, as Q/N domains have a strong tendency to self-aggregate and in some cases can function as prions, the identification of a Q/N domain in TDP-43 has important implications for the mechanism of pathologic aggregation of TDP-43 in ALS and other neurodegenerative diseases.


Subject(s)
Amyotrophic Lateral Sclerosis/etiology , DNA-Binding Proteins/metabolism , Peptides/metabolism , Asparagine , Cell Line , Cell Nucleus/chemistry , DNA-Binding Proteins/genetics , Glutamine , Humans , Multiprotein Complexes , Protein Multimerization
16.
Proc Natl Acad Sci U S A ; 106(44): 18809-14, 2009 Nov 03.
Article in English | MEDLINE | ID: mdl-19833869

ABSTRACT

Frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are neurodegenerative diseases that show considerable clinical and pathologic overlap, with no effective treatments available. Mutations in the RNA binding protein TDP-43 were recently identified in patients with familial amyotrophic lateral sclerosis (ALS), and TDP-43 aggregates are found in both ALS and FTLD-U (FTLD with ubiquitin aggregates), suggesting a common underlying mechanism. We report that mice expressing a mutant form of human TDP-43 develop a progressive and fatal neurodegenerative disease reminiscent of both ALS and FTLD-U. Despite universal transgene expression throughout the nervous system, pathologic aggregates of ubiquitinated proteins accumulate only in specific neuronal populations, including layer 5 pyramidal neurons in frontal cortex, as well as spinal motor neurons, recapitulating the phenomenon of selective vulnerability seen in patients with FTLD-U and ALS. Surprisingly, cytoplasmic TDP-43 aggregates are not present, and hence are not required for TDP-43-induced neurodegeneration. These results indicate that the cellular and molecular substrates for selective vulnerability in FTLD-U and ALS are shared between mice and humans, and suggest that altered DNA/RNA-binding protein function, rather than toxic aggregation, is central to TDP-43-related neurodegeneration.


Subject(s)
Amyotrophic Lateral Sclerosis/complications , Amyotrophic Lateral Sclerosis/pathology , DNA-Binding Proteins/genetics , Frontotemporal Lobar Degeneration/complications , Frontotemporal Lobar Degeneration/pathology , Mutation/genetics , Amyotrophic Lateral Sclerosis/physiopathology , Animals , Brain/pathology , Brain/physiopathology , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Disease Models, Animal , Frontotemporal Lobar Degeneration/physiopathology , Gait/physiology , Humans , Mice , Mice, Transgenic , Survival Analysis
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