ABSTRACT
OBJECTIVE: The aim of this study was to characterize the demographic, behavioural, clinical and immunogenetic determinants of HIV-1 superinfection in a high-risk cohort of MSM. DESIGN: A retrospective cohort study of prospectively followed MSM. METHODS: Ninety-eight MSM with acute or early HIV-1 monoinfection were followed for a median of 15.6 months. Demographic and human leukocyte antigen (HLA) genotype data were collected at enrolment. Sexual behaviour, clinical and the infection status (monoinfection or superinfection) data were recorded at each visit (at enrolment and thereafter at a median of 4.2-month intervals). HIV-1 superinfection risk was determined by Cox regression and Kaplan-Meier survival analysis. RESULTS: Ten individuals (10.2%) had superinfection during follow-up. Cox regression did not show significantly increased superinfection risk for individuals with an increased amount of condomless anal intercourse, lower CD4 T-cell count or higher viral load, but higher number of sexual contacts demonstrated a trend towards significance [hazard ratio, 4.74; 95% confidence interval (95% CI), 0.87-25.97; Pâ=â0.073]. HLA-A*29 (hazard ratio, 4.10; 95% CI, 0.88-14.76; Pâ=â0.069), HLA-B*35 (hazard ratio, 4.64; 95% CI, 1.33-18.17; Pâ=â0.017), HLA-C*04 (hazard ratio, 5.30; 95% CI, 1.51-20.77; Pâ=â0.010), HLA-C*16 (hazard ratio, 4.05; 95% CI, 0.87-14.62; Pâ=â0.071), HLA-DRB1*07 (hazard ratio, 3.29; 95% CI, 0.94-12.90; Pâ=â0.062) and HLA-DRB1*08 (hazard ratio, 15.37; 95% CI, 2.11-79.80; Pâ=â0.011) were associated with an increased risk of superinfection at αâ=â0.10, whereas HLA-DRB1*11 was associated with decreased superinfection risk (hazard ratio, 0.13; 95% CI, 0.00-1.03; Pâ=â0.054). CONCLUSION: HLA genes may, in part, elucidate the genetic basis of differential superinfection risk, and provide important information for the development of efficient prevention and treatment strategies of HIV-1 superinfection.
Subject(s)
Alleles , HIV Infections/epidemiology , HIV Infections/immunology , HLA Antigens/genetics , Superinfection/epidemiology , Adult , Follow-Up Studies , Homosexuality, Male , Humans , Male , Middle Aged , Prospective Studies , Risk Assessment , Young AdultABSTRACT
The article contains raw and analyzed data related to the research article "Neuronal ceroid lipofuscinosis genes, CLN2, CLN3, CLN5 are spatially and temporally co-expressed in a developing mouse brain" (Fabritius et al., 2014) [1]. The processed data gives an understanding of the development of the cell types that are mostly affected by defective function of CLN proteins, timing of expression of CLN1, CLN2, CLN3 and CLN5 genes in a murine model. The data shows relationship between the expression pattern of these genes during neural development. Immunohistochemistry was used to identify known interneuronal markers for neurotransmission and cell proliferation: parvalbumin, somatostatin subpopulations of interneurons. Non-radioactive in-situ hybridization detected CLN5 mRNA in the hippocampus. Throughout the development strong expression of CLN genes were identified in the germinal epithelium and in ventricle regions, cortex, hippocampus, and cerebellum. This provides supportive evidence that CLN1, CLN2, CLN3 and CLN5 genes may be involved in synaptic pruning.
ABSTRACT
Mitochondrial dysfunction has recently been implicated as an underlying factor to several common neurodegenerative diseases, including Parkinson's disease, Alzheimer's and amyotrophic lateral sclerosis (ALS). Valosin containing protein (VCP)-associated multisystem proteinopathy is a new hereditary disorder associated with inclusion body myopathy, Paget disease of bone (PDB), frontotemporal dementia (FTD) and ALS. VCP has been implicated in several transduction pathways including autophagy, apoptosis and the PINK1/Parkin cascade of mitophagy. In this report, we characterized VCP patient and mouse fibroblasts/myoblasts to examine their mitochondrial dynamics and bioenergetics. Using the Seahorse XF-24 technology, we discovered decreased spare respiratory capacity (measurement of extra ATP that can be produced by oxidative phosphorylation in stressful conditions) and increased ECAR levels (measurement of glycolysis), and proton leak in VCP human fibroblasts compared with age- and sex-matched unaffected first degree relatives. We found decreased levels of ATP and membrane potential, but higher mitochondrial enzyme complexes II+III and complex IV activities in the patient VCP myoblasts when compared to the values of the control cell lines. These results suggest that mutations in VCP affect the mitochondria's ability to produce ATP, thereby resulting in a compensatory increase in the cells' mitochondrial complex activity levels. Thus, this novel in vitro model may be useful in understanding the pathophysiology and discovering new drug targets of mitochondrial dynamics and physiology to modify the clinical phenotype in VCP and related multisystem proteinopathies (MSP).
Subject(s)
Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/metabolism , Energy Metabolism , Mitochondria/physiology , Neurodegenerative Diseases/pathology , Proteostasis Deficiencies/pathology , Adenosine Triphosphate/analysis , Animals , Disease Models, Animal , Electron Transport Chain Complex Proteins/analysis , Fibroblasts/metabolism , Humans , Membrane Potential, Mitochondrial , Mice , Mitochondria/enzymology , Mitochondria/metabolism , Myoblasts/metabolism , Valosin Containing ProteinABSTRACT
Dominant mutations in the valosin-containing protein (VCP) gene cause inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia, which is characterized by progressive muscle weakness, dysfunction in bone remodeling, and frontotemporal dementia. More recently, VCP has been linked to 2% of familial amyotrophic lateral sclerosis cases. VCP plays a significant role in a plethora of cellular functions including membrane fusion, transcription activation, nuclear envelope reconstruction, postmitotic organelle reassembly, and cell cycle control. To elucidate the pathological mechanisms underlying the VCP disease progression, we have previously generated a VCP(R155H/+) mouse model with the R155H mutation. Histological analyses of mutant muscle showed vacuolization of myofibrils, centrally located nuclei, and disorganized muscle fibers. Global expression profiling of VCP(R155H/+) mice using gene annotations by DAVID identified key dysregulated signaling pathways including genes involved in the physiological system development and function, diseases and disorders, and molecular and cellular functions. There were a total of 212 significantly dysregulated genes, several of which are involved in the regulation of proteasomal function and NF-κB signaling cascade. Findings of the gene expression study were validated by using quantitative reverse transcriptase polymerase chain reaction analyses to test genes involved in various signaling cascades. This investigation reveals the importance of the VCP(R155H/+) mouse model in the understanding of cellular and molecular mechanisms causing VCP-associated neurodegenerative diseases and in the discovery of novel therapeutic advancements and strategies for patients suffering with these debilitating disorders.
Subject(s)
Adenosine Triphosphatases/genetics , Amino Acid Substitution , Cell Cycle Proteins/genetics , Gene Expression Profiling , Gene Knock-In Techniques , Nerve Degeneration/genetics , Animals , Disease Models, Animal , Gene Expression Regulation , Gene Regulatory Networks , Humans , Mice , Muscles/pathology , Nerve Degeneration/pathology , Oligonucleotide Array Sequence Analysis , Real-Time Polymerase Chain Reaction , Reproducibility of Results , Valosin Containing ProteinABSTRACT
INTRODUCTION: Mutations in the valosin-containing protein (VCP) gene cause hereditary inclusion body myopathy (IBM) associated with Paget disease of bone (PDB), and frontotemporal dementia (FTD). More recently, these mutations have been linked to 2% of familial amyotrophic lateral sclerosis (ALS) cases. A knock-in mouse model offers the opportunity to study VCP-associated pathogenesis. METHODS: The VCP(R155H/+) knock-in mouse model was assessed for muscle strength and immunohistochemical, Western blot, apoptosis, autophagy, and microPET/CT imaging analyses. RESULTS: VCP(R155H/+) mice developed significant progressive muscle weakness, and the quadriceps and brain developed progressive cytoplasmic accumulation of TDP-43, ubiquitin-positive inclusion bodies, and increased LC3-II staining. MicroCT analyses revealed Paget-like lesions at the ends of long bones. Spinal cord demonstrated neurodegenerative changes, ubiquitin, and TDP-43 pathology of motor neurons. CONCLUSIONS: VCP(R155H/+) knock-in mice represent an excellent preclinical model for understanding VCP-associated disease mechanisms and future treatments.
Subject(s)
Adenosine Triphosphatases/genetics , Cell Cycle Proteins/genetics , Disease Models, Animal , Frontotemporal Dementia/genetics , Myositis, Inclusion Body/genetics , Osteitis Deformans/genetics , Adenosine Triphosphatases/metabolism , Animals , Brain/metabolism , Brain/pathology , Cell Cycle Proteins/metabolism , Disease Progression , Frontotemporal Dementia/metabolism , Frontotemporal Dementia/pathology , Mice , Mice, Transgenic , Motor Neurons/metabolism , Motor Neurons/pathology , Myositis, Inclusion Body/metabolism , Myositis, Inclusion Body/pathology , Osteitis Deformans/metabolism , Osteitis Deformans/pathology , Spinal Cord/metabolism , Spinal Cord/pathology , Valosin Containing ProteinABSTRACT
Inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia (IBMPFD) is an autosomal dominant disorder caused by mutations in the Valosin-containing protein (VCP) gene on chromosome 9p12-13. Patients demonstrate limb girdle muscle weakness, which eventually progresses to involve respiratory muscles, and death from respiratory and cardiac failure. This is the first investigation to analyze key molecular mediators and signaling cascades in skeletal muscle causing myopathy by global gene microarray in hopes of understanding the dysregulated genes and molecular mechanisms underlying IBMPFD and the hope of finding novel therapeutic targets. We determined expression profiles using Human Genome Array microarray technology in Vastus lateralis muscles from patients and their first-degree relatives. We analyzed gene annotations by Database for Annotation, Visualization and Integration Discovery and identified differentially dysregulated genes with roles in several novel biological pathways, including regulation of actin cytoskeleton, ErbB signaling, cancer, in addition to regulation of autophagy, and lysosomal signaling, known disrupted pathways in VCP disease. In this report, we present data from the first global microarray analyzing IBMPFD patient muscles and elucidating dysregulated pathways to further understand the pathogenesis of the disease and discover potential therapeutics.
Subject(s)
Gene Expression Profiling , Myositis, Inclusion Body/genetics , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Adult , Case-Control Studies , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Female , Gene Regulatory Networks/genetics , Humans , Male , Middle Aged , Oligonucleotide Array Sequence Analysis , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/genetics , Valosin Containing ProteinABSTRACT
CLN3 is an endosomal/lysosomal transmembrane protein mutated in classical juvenile onset neuronal ceroid lipofuscinosis, a fatal inherited neurodegenerative lysosomal storage disorder. The function of CLN3 in endosomal/lysosomal events has remained elusive due to poor understanding of its interactions in these compartments. It has previously been shown that the localisation of late endosomal/lysosomal compartments is disturbed in cells expressing the most common disease-associated CLN3 mutant, CLN3∆ex7-8 (c.462-677del). We report here that a protracted disease causing mutant, CLN3E295K, affects the properties of late endocytic compartments, since over-expression of the CLN3E295K mutant protein in HeLa cells induced relocalisation of Rab7 and a perinuclear clustering of late endosomes/lysosomes. In addition to the previously reported disturbances in the endocytic pathway, we now show that the anterograde transport of late endosomal/lysosomal compartments is affected in CLN3 deficiency. CLN3 interacted with motor components driving both plus and minus end microtubular trafficking: tubulin, dynactin, dynein and kinesin-2. Most importantly, CLN3 was found to interact directly with active, guanosine-5'-triphosphate (GTP)-bound Rab7 and with the Rab7-interacting lysosomal protein (RILP) that anchors the dynein motor. The data presented in this study provide novel insights into the role of CLN3 in late endosomal/lysosomal membrane transport.
Subject(s)
Endosomes/metabolism , Membrane Glycoproteins/metabolism , Molecular Chaperones/metabolism , Molecular Motor Proteins/metabolism , Neuronal Ceroid-Lipofuscinoses/metabolism , HeLa Cells , Humans , Lysosomes/metabolism , Membrane Glycoproteins/genetics , Molecular Chaperones/genetics , Mutation , rab GTP-Binding Proteins/metabolism , rab7 GTP-Binding ProteinsABSTRACT
Inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia (IBMPFD) is a progressive, fatal genetic disorder with variable penetrance, predominantly affecting three main tissue types: muscle (IBM), bone (PDB), and brain (FTD). IBMPFD is caused by mutations in the ubiquitously expressed valosin-containing protein (VCP) gene, a member of the AAA-ATPase superfamily. The majority of individuals who develop IBM have progressive proximal muscle weakness. Muscle biopsies reveal rimmed vacuoles and inclusions that are ubiquitin- and TAR DNA binding protein-43 (TDP-43)-positive using immunohistochemistry. PDB, seen in half the individuals, is caused by overactive osteoclasts and is associated clinically with pain, elevated serum alkaline phosphatase, and X-ray findings of coarse trabeculation and sclerotic lesions. FTD diagnosed at a mean age of 55 years in a third of individuals is characterized clinically by comprehension deficits, dysnomia, dyscalculia, and social unawareness. Ubiquitin- and TDP-43-positive neuronal inclusions are also found in the brain. Genotype-phenotype correlations are difficult with marked intra-familial and inter-familial variations being seen. Varied phenotypes within families include frontotemporal dementia, amyotrophic lateral sclerosis, Parkinsonism, myotonia, cataracts, and anal incompetence, among others. Cellular and animal models indicate pathogenetic disturbances in IBMPFD tissues including altered protein degradation, autophagy pathway alterations, apoptosis, and mitochondrial dysfunction. Currently, mouse and drosophila models carrying VCP mutations provide insights into the human IBMPFD pathology and are useful as tools for preclinical studies and testing of therapeutic strategies. In this review, we will explore the pathogenesis and clinical phenotype of IBMPFD caused by VCP mutations.
Subject(s)
Adenosine Triphosphatases/genetics , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/pathology , Cell Cycle Proteins/genetics , Frontotemporal Dementia/genetics , Frontotemporal Dementia/pathology , Myositis, Inclusion Body , Osteitis Deformans , Amyotrophic Lateral Sclerosis/physiopathology , Animals , Autophagy , DNA-Binding Proteins/genetics , Disease Models, Animal , Frontotemporal Dementia/physiopathology , Genetic Association Studies , Humans , Mutation , Myositis, Inclusion Body/genetics , Myositis, Inclusion Body/pathology , Myositis, Inclusion Body/physiopathology , Osteitis Deformans/genetics , Osteitis Deformans/pathology , Osteitis Deformans/physiopathology , Valosin Containing ProteinABSTRACT
Angelman syndrome (AS) is a severe neurological disorder caused by a deficiency of ubiquitin protein ligase E3A (UBE3A), but the pathophysiology of the disease remains unknown. We now report that in the brains of AS mice in which the maternal UBE3A allele is mutated (m-) and the paternal allele is potentially inactivated by imprinting (p+) (UBE3A m-\p+), the mitochondria are abnormal and exhibit a partial oxidative phosphorylation (OXPHOS) defect. Electron microscopy of the hippocampal region of the UBE3A m-\p+ mice (n=6) reveals small, dense mitochondria with altered cristae, relative to wild-type littermates (n=6) and reduced synaptic vesicle density. The specific activity of OXPHOS complex III is reduced in whole brain mitochondria in UBE3A m-\p+ (n=5) mice versus wild-type littermates (n=5). Therefore, mitochondrial dysfunction may contribute to the pathophysiology of Angelman syndrome.
Subject(s)
Angelman Syndrome/enzymology , CA1 Region, Hippocampal/enzymology , Disease Models, Animal , Mitochondria/enzymology , Neurons/enzymology , Ubiquitin-Protein Ligases/deficiency , Angelman Syndrome/genetics , Angelman Syndrome/pathology , Animals , CA1 Region, Hippocampal/pathology , Female , Genotype , Male , Mice , Mice, Knockout , Mice, Transgenic , Mitochondria/genetics , Mitochondria/pathology , Neurons/pathology , Neurons/physiology , Purkinje Cells/enzymology , Purkinje Cells/pathology , Synaptic Vesicles/genetics , Synaptic Vesicles/pathology , Ubiquitin-Protein Ligases/biosynthesis , Ubiquitin-Protein Ligases/geneticsABSTRACT
Dominant mutations in the valosin containing protein (VCP) gene cause inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia (IBMPFD). We have generated a knock-in mouse model with the common R155H mutation. Mice demonstrate progressive muscle weakness starting approximately at the age of 6 months. Histology of mutant muscle showed progressive vacuolization of myofibrils and centrally located nuclei, and immunostaining shows progressive cytoplasmic accumulation of TDP-43 and ubiquitin-positive inclusion bodies in quadriceps myofibrils and brain. Increased LC3-II staining of muscle sections representing increased number of autophagosomes suggested impaired autophagy. Increased apoptosis was demonstrated by elevated caspase-3 activity and increased TUNEL-positive nuclei. X-ray microtomography (uCT) images show radiolucency of distal femurs and proximal tibiae in knock-in mice and uCT morphometrics shows decreased trabecular pattern and increased cortical wall thickness. Bone histology and bone marrow derived macrophage cultures in these mice revealed increased osteoclastogenesis observed by TRAP staining suggestive of Paget bone disease. The VCP(R155H/+) knock-in mice replicate the muscle, bone and brain pathology of inclusion body myopathy, thus representing a useful model for preclinical studies.
Subject(s)
Adenosine Triphosphatases/physiology , Cell Cycle Proteins/physiology , Myositis, Inclusion Body/physiopathology , Osteitis Deformans/pathology , Animals , In Situ Nick-End Labeling , Mice , Mice, Transgenic , Valosin Containing ProteinABSTRACT
VCP (VCP/p97) is a ubiquitously expressed member of the AAA(+)-ATPase family of chaperone-like proteins that regulates numerous cellular processes including chromatin decondensation, homotypic membrane fusion and ubiquitin-dependent protein degradation by the proteasome. Mutations in VCP cause a multisystem degenerative disease consisting of inclusion body myopathy, Paget disease of bone, and frontotemporal dementia (IBMPFD). Here we show that VCP is essential for autophagosome maturation. We generated cells stably expressing dual-tagged LC3 (mCherry-EGFP-LC3) which permit monitoring of autophagosome maturation. We determined that VCP deficiency by RNAi-mediated knockdown or overexpression of dominant-negative VCP results in significant accumulation of immature autophagic vesicles, some of which are abnormally large, acidified and exhibit cathepsin B activity. Furthermore, expression of disease-associated VCP mutants (R155H and A232E) also causes this autophagy defect. VCP was found to be essential to autophagosome maturation under basal conditions and in cells challenged by proteasome inhibition, but not in cells challenged by starvation, suggesting that VCP might be selectively required for autophagic degradation of ubiquitinated substrates. Indeed, a high percentage of the accumulated autophagic vesicles contain ubiquitin-positive contents, a feature that is not observed in autophagic vesicles that accumulate following starvation or treatment with Bafilomycin A. Finally, we show accumulation of numerous, large LAMP-1 and LAMP-2-positive vacuoles and accumulation of LC3-II in myoblasts derived from patients with IBMPFD. We conclude that VCP is essential for maturation of ubiquitin-containing autophagosomes and that defect in this function may contribute to IBMPFD pathogenesis.
Subject(s)
Adenosine Triphosphatases , Cell Cycle Proteins , Frontotemporal Dementia , Mutation , Myositis, Inclusion Body , Osteitis Deformans , Phagosomes/metabolism , Ubiquitin/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Animals , Cathepsin B/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cells, Cultured , Frontotemporal Dementia/genetics , Frontotemporal Dementia/physiopathology , Humans , Lysosomal-Associated Membrane Protein 1/genetics , Lysosomal-Associated Membrane Protein 1/metabolism , Lysosomal-Associated Membrane Protein 2/genetics , Lysosomal-Associated Membrane Protein 2/metabolism , Mice , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Myoblasts/cytology , Myoblasts/metabolism , Myositis, Inclusion Body/genetics , Myositis, Inclusion Body/physiopathology , Osteitis Deformans/genetics , Osteitis Deformans/physiopathology , Proteasome Endopeptidase Complex/metabolism , RNA Interference , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Syndrome , Vacuoles/metabolism , Valosin Containing ProteinABSTRACT
Inclusion body myopathy associated with Paget's disease and frontotemporal dementia (IBMPFD) is caused by mutations in the valosin containing protein (VCP) gene. The disease is associated with progressive proximal muscle weakness, inclusions and vacuoles in muscle fibers, malfunction in the bone remodeling process resulting in Paget's disease, and premature frontotemporal dementia. VCP is involved in several cellular processes related to the endoplasmic reticulum associated degradation of proteins. To understand the pathological mechanisms underlying the myopathy in IBMPFD, we have studied the cellular consequences of VCP mutations in human primary myoblasts. Our results revealed that patients' myoblasts accumulate large vacuoles. Lysosomal membrane proteins Lamp1 and Lamp2 show increased molecular weights in patients' myoblasts due to differential N-glycosylation. Additionally, mutant myoblasts show increased autophagy when cultured in the absence of nutrients, as well as defective cell fusion and increased apoptosis. Our results elucidate that VCP mutations result in disturbances in several cellular processes, which will help us in the understanding of the pathological mechanisms resulting in muscle weakness and other features of VCP associated disease.
Subject(s)
Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Autophagy/physiology , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Myoblasts/physiology , Myositis, Inclusion Body/metabolism , Adult , Apoptosis/genetics , Apoptosis/physiology , Caspase 3/metabolism , Cell Fusion/methods , Cells, Cultured , Down-Regulation/genetics , Down-Regulation/physiology , Female , Frontotemporal Dementia/complications , Humans , In Situ Nick-End Labeling/methods , Lysosomal-Associated Membrane Protein 2 , Lysosomal Membrane Proteins/genetics , Male , Microscopy, Electron, Transmission/methods , Middle Aged , Muscle Fibers, Skeletal/physiology , Mutation/genetics , Myoblasts/pathology , Myoblasts/ultrastructure , Myositis, Inclusion Body/pathology , Osteitis Deformans/complications , Valosin Containing ProteinABSTRACT
Inclusion body myopathy (IBM) associated with Paget disease of the bone (PDB) and frontotemporal dementia (FTD) (now called IBMPFD), is a progressive autosomal dominant disorder that was recently identified as being caused by mutations in the VCP (p97 or CDC48) gene which plays a key role in the ubiquitin-proteasome dependent degradation of cytosolic proteins and in the retro translocation of misfolded proteins from the endoplasmic reticulum into the cytoplasm. Approximately 90% of the affected persons in the study have myopathy or muscle weakness particularly of the shoulder and hip girdles, which can lead to loss of walking ability and even death by complications of respiratory and cardiac failure. About half of affected study participants have Paget disease of bone characterized by abnormal rates of bone growth that can result in bone pain, enlargement and fractures. Findings of premature FTD affecting behavior and personality are seen in a third of affected individuals. Within 20 IBMPFD families whose data was analyzed for this study, ten missense mutations have been identified, the majority of which are located in the N-terminal ubiquitin binding domain. Inclusions seen in the muscle, brain and heart in VCP disease contain ubiquitin, beta amyloid and TDP-43, also seen in other neurodegenerative disorders thus implicating common pathways in their pathogenesis.
Subject(s)
Adenosine Triphosphatases/genetics , Cell Cycle Proteins/genetics , Dementia/genetics , Frontal Lobe/physiopathology , Muscular Diseases/genetics , Mutation/genetics , Osteitis Deformans/genetics , Dementia/pathology , Genetic Predisposition to Disease , Humans , Muscular Diseases/pathology , Osteitis Deformans/pathology , Valosin Containing ProteinABSTRACT
Hydrolethalus syndrome (HLS) is an autosomal recessive lethal malformation syndrome characterized by multiple developmental defects of fetus. We have earlier mapped and restricted the HLS region to a critical 1 cM interval on 11q23-25. The linkage disequilibrium (LD) and haplotype analyses of single nucleotide polymorphism (SNP) markers helped to further restrict the HLS locus to 476 kb between genes PKNOX2 and DDX25. An HLS associated mutation was identified in a novel regional transcript (GenBank accession no. FLJ32915), referred to here as the HYLS1 gene. The identified A to G transition results in a D211G change in the 299 amino acid polypeptide with unknown function. The HYLS1 gene shows alternative splicing and the transcript is found in multiple tissues during fetal development. In situ hybridization shows spatial and temporal distributions of transcripts in good agreement with the tissue phenotype of HLS patients. Immunostaining of in vitro expressed polypeptides from wild-type (WT) cDNA revealed cytoplasmic staining, whereas mutant polypeptides became localized in distinct nuclear structures, implying a disturbed cellular localization of the mutant protein. The Drosophila melanogaster model confirmed these findings and provides evidence for the significance of the mutation both in vitro and in vivo.
Subject(s)
Abnormalities, Multiple/genetics , Genes, Lethal , Mutation, Missense , Proteins/genetics , Amino Acid Sequence , Animals , Animals, Genetically Modified , Base Sequence , COS Cells , Chlorocebus aethiops , DNA, Complementary , Drosophila melanogaster/genetics , Fetal Development , Genes, Recessive , Humans , In Situ Hybridization , Linkage Disequilibrium , Mice , Molecular Sequence Data , Proteins/chemistry , Proteins/metabolism , RNA, Messenger/genetics , Sequence Homology, Amino Acid , Subcellular Fractions/metabolismABSTRACT
Infantile Neuronal Ceroid Lipofuscinosis (INCL) results from mutations in the palmitoyl protein thioesterase (PPT1, CLN1) gene and is characterized by dramatic death of cortical neurons. We generated Ppt1Deltaex4 mice by a targeted deletion of exon 4 of the mouse Ppt1 gene. Similar to the clinical phenotype, the homozygous mutants show loss of vision from the age of 8 weeks, seizures after 4 months and paralysis of hind limbs at the age of 5 months. Autopsy revealed a dramatic loss of brain mass and histopathology demonstrated accumulation of autofluorescent granular osmiophilic deposits (GRODS), both characteristic of INCL. At 6 months, the homozygous Ppt1Deltaex4 mice showed a prominent loss of GABAergic interneurons in several brain areas. The transcript profiles of wild-type and mutant mouse brains revealed that most prominent alterations involved parts of the immune response, implicating alterations similar to those of the aging brain and neurodegeneration. These findings make the Ppt1Deltaex4 mouse an interesting model for the inflammation-associated death of interneurons.
Subject(s)
Cerebral Cortex/metabolism , Encephalitis/genetics , Interneurons/metabolism , Nerve Degeneration/genetics , Neuronal Ceroid-Lipofuscinoses/genetics , Thiolester Hydrolases/genetics , Animals , Animals, Newborn , Blindness, Cortical/genetics , Blindness, Cortical/metabolism , Blindness, Cortical/physiopathology , Cell Death/genetics , Cerebral Cortex/pathology , Cerebral Cortex/ultrastructure , Disease Models, Animal , Encephalitis/pathology , Encephalitis/physiopathology , Female , Gene Deletion , Gene Targeting , Inclusion Bodies/genetics , Inclusion Bodies/pathology , Inclusion Bodies/ultrastructure , Interneurons/pathology , Interneurons/ultrastructure , Male , Mice , Mice, Neurologic Mutants , Microscopy, Electron, Transmission , Mutation/genetics , Nerve Degeneration/pathology , Nerve Degeneration/physiopathology , Neuronal Ceroid-Lipofuscinoses/pathology , Neuronal Ceroid-Lipofuscinoses/physiopathology , Paralysis/genetics , Paralysis/metabolism , Paralysis/physiopathology , Phenotype , Seizures/genetics , Seizures/metabolism , Seizures/physiopathology , Viscera/metabolism , Viscera/pathology , Viscera/ultrastructure , gamma-Aminobutyric Acid/metabolismABSTRACT
Down syndrome (DS) is caused by trisomy for human chromosome 21 and is the most common genetic cause of mental retardation. The distal 10 Mb region of the long arm of the chromosome has been proposed to be associated with many of the abnormalities seen in DS. This region is often referred to as the Down syndrome critical region (DSCR). We report here the results of our analyses of the DSCR protein 2 (DSCR2). Results from transiently transfected COS-1 and HEK293 cells suggest that DSCR2 is synthesized as a 43 kDa precursor protein, from which the N-terminus is cleaved resulting in a polypeptide of 41 kDa. The polypeptide is modified by still uncharacterized co- or post-translational modifications increasing the predicted molecular weight of 32.8 kDa by about 10 kDa. Analyses of the only putative N-glycosylation site by in vitro mutagenesis excluded the possibility of the contribution of N-glycosylation to this increase in molecular weight. Further, the results of intracellular localization studies and membrane fractionation assays indicate that DSCR2 is targeted to a cytoplasmic compartment as a soluble form.
Subject(s)
Cytoplasm/metabolism , Down Syndrome/metabolism , Kidney/metabolism , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Muscle Proteins/chemistry , Muscle Proteins/metabolism , Subcellular Fractions/metabolism , Amino Acid Sequence , Animals , COS Cells , Chlorocebus aethiops , Down Syndrome/genetics , Humans , Membrane Proteins/genetics , Molecular Chaperones , Molecular Sequence Data , Molecular Weight , Muscle Proteins/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Solubility , Species SpecificityABSTRACT
Neuronal ceroid lipofuscinoses (NCL) comprise the most common group of childhood encephalopathies caused by mutations in eight genetic loci, CLN1-CLN8. Here, we have developed a novel mouse model for the human vLINCL (CLN5) by targeted deletion of exon 3 of the mouse Cln5 gene. The Cln5-/- mice showed loss of vision and accumulation of autofluorescent storage material in the central nervous system (CNS) and peripheral tissues without prominent brain atrophy. The ultrastructure of the storage material accurately replicated the abnormalities in human patients revealing mixture of lamellar profiles including fingerprint profiles as well as curvilinear and rectilinear bodies in electronmicroscopic analysis. Prominent loss of a subset of GABAergic interneurons in several brain areas was seen in the Cln5-/- mice. Transcript profiling of the brains of the Cln5-/- mice revealed altered expression in several genes involved in neurodegeneration, as well as in defense and immune response, typical of age-associated changes in the CNS. Downregulation of structural components of myelin was detected and this agrees well with the hypomyelination seen in the human vLINCL patients. In general, the progressive pathology of the Cln5-/- brain mimics the symptoms of the corresponding neurodegenerative disorder in man. Since the Cln5-/- mice do not exhibit significant brain atrophy, these mice could serve as models for studies on molecular processes associated with advanced aging.
Subject(s)
Aging , Brain/pathology , Disease Models, Animal , Membrane Proteins/physiology , Neuronal Ceroid-Lipofuscinoses/genetics , Animals , Base Sequence , Brain/enzymology , Brain/physiopathology , DNA Primers , Gene Expression Profiling , Humans , Immunohistochemistry , Lysosomal Membrane Proteins , Lysosomes/enzymology , Membrane Proteins/genetics , Mice , Mice, Knockout , Neuronal Ceroid-Lipofuscinoses/pathology , Reverse Transcriptase Polymerase Chain Reaction , gamma-Aminobutyric Acid/physiologyABSTRACT
Batten disease is a neurodegenerative disorder resulting from mutations in CLN3, a polytopic membrane protein, whose predominant intracellular destination in nonneuronal cells is the lysosome. The topology of CLN3 protein, its lysosomal targeting mechanism, and the development of Batten disease are poorly understood. We provide experimental evidence that both the N and C termini and one large loop domain of CLN3 face the cytoplasm. We have identified two lysosomal targeting motifs that mediate the sorting of CLN3 in transfected nonneuronal and neuronal cells: an unconventional motif in the long C-terminal cytosolic tail consisting of a methionine and a glycine separated by nine amino acids [M(X)9G], and a more conventional dileucine motif, located in the large cytosolic loop domain and preceded by an acidic patch. Each motif on its own was sufficient to mediate lysosomal targeting, but optimal efficiency required both. Interestingly, in primary neurons, CLN3 was prominently seen both in lysosomes in the cell body and in endosomes, containing early endosomal antigen-1 along neuronal processes. Because there are few lysosomes in axons and peripheral parts of dendrites, the presence of CLN3 in endosomes of neurons may be functionally important. Endosomal association of the protein was independent of the two lysosomal targeting motifs.
Subject(s)
Cell Membrane/metabolism , Endosomes/metabolism , Lysosomes/metabolism , Membrane Glycoproteins/metabolism , Molecular Chaperones/metabolism , Neurons/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Cells, Cultured , Cloning, Molecular , HeLa Cells , Hippocampus/metabolism , Humans , Molecular Sequence Data , RatsABSTRACT
GRACILE (growth retardation, aminoaciduria, cholestasis, iron overload, lactacidosis, and early death) syndrome is a recessively inherited lethal disease characterized by fetal growth retardation, lactic acidosis, aminoaciduria, cholestasis, and abnormalities in iron metabolism. We previously localized the causative gene to a 1.5-cM region on chromosome 2q33-37. In the present study, we report the molecular defect causing this metabolic disorder, by identifying a homozygous missense mutation that results in an S78G amino acid change in the BCS1L gene in Finnish patients with GRACILE syndrome, as well as five different mutations in three British infants. BCS1L, a mitochondrial inner-membrane protein, is a chaperone necessary for the assembly of mitochondrial respiratory chain complex III. Pulse-chase experiments performed in COS-1 cells indicated that the S78G amino acid change results in instability of the polypeptide, and yeast complementation studies revealed a functional defect in the mutated BCS1L protein. Four different mutations in the BCS1L gene have been reported elsewhere, in Turkish patients with a distinctly different phenotype. Interestingly, the British and Turkish patients had complex III deficiency, whereas in the Finnish patients with GRACILE syndrome complex III activity was within the normal range, implying that BCS1L has another cellular function that is uncharacterized but essential and is putatively involved in iron metabolism.
Subject(s)
Iron Overload/genetics , Metabolic Diseases/genetics , Proteins/genetics , ATPases Associated with Diverse Cellular Activities , Animals , Blotting, Northern , COS Cells , Electron Transport Complex III/metabolism , Female , Genetic Complementation Test , Humans , Infant , Infant, Newborn , Iron Overload/enzymology , Male , Metabolic Diseases/enzymology , Molecular Sequence Data , Point Mutation , Proteins/metabolism , Saccharomyces cerevisiae/genetics , Sequence Analysis, DNA , TransfectionABSTRACT
Neuronal ceroid lipofuscinoses (NCLs) are neurodegenerative storage diseases characterized by mental retardation, visual failure, and brain atrophy as well as accumulation of storage material in multiple cell types. The diseases are caused by mutations in the ubiquitously expressed genes, of which six are known. Herein, we report that three NCL disease forms with similar tissue pathology are connected at the molecular level: CLN5 polypeptides directly interact with the CLN2 and CLN3 proteins based on coimmunoprecipitation and in vitro binding assays. Furthermore, disease mutations in CLN5 abolished interaction with CLN2, while not affecting association with CLN3. The molecular characterization of CLN5 revealed that it was synthesized as four precursor forms, due to usage of alternative initiator methionines in translation. All forms were targeted to lysosomes and the longest form, translated from the first potential methionine, was associated with membranes. Interactions between CLN polypeptides were shown to occur with this longest, membrane-bound form of CLN5. Both intracellular targeting and posttranslational glycosylation of the polypeptides carrying human disease mutations were similar to wild-type CLN5.