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1.
Sci Rep ; 8(1): 272, 2018 01 05.
Article in English | MEDLINE | ID: mdl-29305592

ABSTRACT

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

2.
Eur J Neurol ; 25(1): 154-163, 2018 01.
Article in English | MEDLINE | ID: mdl-29029362

ABSTRACT

BACKGROUND AND PURPOSE: Mutations in the small heat-shock protein 22 gene (HSPB8) have been associated with Charcot-Marie-Tooth disease type 2L, distal hereditary motor neuropathy (dHMN) type IIa and, more recently, distal myopathy/myofibrillar myopathy (MFM) with protein aggregates and TDP-43 inclusions. The aim was to report a novel family with HSPB8K141E -related dHMN/MFM and to investigate, in a patient muscle biopsy, whether the presence of protein aggregates was paralleled by altered TDP-43 function. METHODS: We reviewed clinical and genetic data. We assessed TDP-43 expression by qPCR and alternative splicing of four previously validated direct TDP-43 target exons in four genes by reverse transcriptase-polymerase chain reaction. RESULTS: The triplets and their mother presented in the second to third decade of life with progressive weakness affecting distal and proximal lower limb and truncal muscles. Nerve conduction study showed a motor axonal neuropathy. The clinical features, moderately raised creatin kinase levels, selective pattern of muscle involvement on magnetic resonance imaging and pathological changes on muscle biopsy, including the presence of protein aggregates, supported the diagnosis of a contemporary primary muscle involvement. In affected muscle tissue we observed a consistent alteration of TDP-43-dependent splicing in three out of four TDP-43-target transcripts (POLDIP3, FNIP1 and BRD8), as well as a significant decrease of TDP-43 mRNA levels. CONCLUSIONS: Our study confirmed the role of mutated HSPB8 as a cause of a combined neuromuscular disorder encompassing dHMN and MFM with protein aggregates. We identified impaired RNA metabolism, secondary to TDP-43 loss of function, as a possible pathological mechanism of HSPB8K141E toxicity, leading to muscle and nerve degeneration.


Subject(s)
DNA-Binding Proteins/genetics , Heat-Shock Proteins/genetics , Hereditary Sensory and Motor Neuropathy/genetics , Protein Serine-Threonine Kinases/genetics , Adult , Age of Onset , Alternative Splicing , Biopsy , Disease Progression , Female , Hereditary Sensory and Motor Neuropathy/diagnostic imaging , Hereditary Sensory and Motor Neuropathy/pathology , Humans , Magnetic Resonance Imaging , Male , Middle Aged , Molecular Chaperones , Muscle, Skeletal/pathology , Neural Conduction , Pedigree , RNA/metabolism , TDP-43 Proteinopathies/genetics
3.
Sci Rep ; 7(1): 6723, 2017 07 27.
Article in English | MEDLINE | ID: mdl-28751743

ABSTRACT

Frontotemporal Dementia (FTD) is a neurodegenerative disorder mainly characterised by Tau or TDP43 inclusions. A co-autoimmune aetiology has been hypothesised. In this study, we aimed at defining the pathogenetic role of anti-AMPA GluA3 antibodies in FTD. Serum and cerebrospinal fluid (CSF) anti-GluA3 antibody dosage was carried out and the effect of CSF with and without anti-GluA3 antibodies was tested in rat hippocampal neuronal primary cultures and in differentiated neurons from human induced pluripotent stem cells (hiPSCs). TDP43 and Tau expression in hiPSCs exposed to CSF was assayed. Forty-one out of 175 screened FTD sera were positive for the presence of anti-GluA3 antibodies (23.4%). FTD patients with anti-GluA3 antibodies more often presented presenile onset, behavioural variant FTD with bitemporal atrophy. Incubation of rat hippocampal neuronal primary cultures with CSF with anti-GluA3 antibodies led to a decrease of GluA3 subunit synaptic localization of the AMPA receptor (AMPAR) and loss of dendritic spines. These results were confirmed in differentiated neurons from hiPSCs, with a significant reduction of the GluA3 subunit in the postsynaptic fraction along with increased levels of neuronal Tau. In conclusion, autoimmune mechanism might represent a new potentially treatable target in FTD and might open new lights in the disease underpinnings.


Subject(s)
Autoantibodies/cerebrospinal fluid , Autoimmunity , DNA-Binding Proteins/immunology , Frontotemporal Dementia/immunology , Hippocampus/immunology , Neurons/immunology , Receptors, AMPA/antagonists & inhibitors , Aged , Animals , Autoantibodies/pharmacology , COS Cells , Case-Control Studies , Cell Differentiation/drug effects , Chlorocebus aethiops , DNA-Binding Proteins/genetics , Embryo, Mammalian , Female , Frontotemporal Dementia/cerebrospinal fluid , Frontotemporal Dementia/diagnosis , Frontotemporal Dementia/genetics , Gene Expression , Hippocampus/pathology , Humans , Induced Pluripotent Stem Cells/drug effects , Male , Middle Aged , Neurons/drug effects , Neurons/pathology , Primary Cell Culture , Rats , Receptors, AMPA/genetics , Receptors, AMPA/immunology , tau Proteins/genetics , tau Proteins/immunology
4.
Acta Neuropathol Commun ; 4(1): 52, 2016 05 18.
Article in English | MEDLINE | ID: mdl-27193329

ABSTRACT

Niemann-Pick type C (NPC) disease is a lysosomal storage disorder characterized by the occurrence of visceral and neurological symptoms. At present, the molecular mechanisms causing neurodegeneration in this disease are unknown. Here we report the altered expression and/or mislocalization of the TAR-DNA binding protein 43 (TDP-43) in both NPC mouse and in a human neuronal model of the disease. We also report the neuropathologic study of a NPC patient's brain, showing that while TDP-43 is below immunohistochemical detection in nuclei of cerebellar Purkinje cells, it has a predominant localization in the cytoplasm of these cells. From a functional point of view, the TDP-43 mislocalization, that occurs in a human experimental neuronal model system, is associated with specific alterations in TDP-43 controlled genes. Most interestingly, treatment with N-Acetyl-cysteine (NAC) or beta-cyclodextrin (CD) can partially restore TDP-43 nuclear localization. Taken together, the results of these studies extend the role of TDP-43 beyond the Amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD)/Alzheimer disease (AD) spectrum. These findings may open novel research/therapeutic avenues for a better understanding of both NPC disease and the TDP-43 proteinopathy disease mechanism.


Subject(s)
DNA-Binding Proteins/metabolism , Niemann-Pick Disease, Type C/metabolism , Acetylcysteine/pharmacology , Animals , Brain/metabolism , Brain/pathology , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Cell Nucleus/pathology , Cells, Cultured , Disease Models, Animal , Fibroblasts/drug effects , Fibroblasts/metabolism , Fibroblasts/pathology , Humans , Intracellular Signaling Peptides and Proteins , Male , Mice, Inbred BALB C , Mice, Transgenic , Middle Aged , Neurons/drug effects , Neurons/metabolism , Neurons/pathology , Neuroprotective Agents/pharmacology , Niemann-Pick C1 Protein , Niemann-Pick Disease, Type C/drug therapy , Niemann-Pick Disease, Type C/genetics , Niemann-Pick Disease, Type C/pathology , Proteins/genetics , Proteins/metabolism , Spinal Cord/metabolism , Spinal Cord/pathology , beta-Cyclodextrins/pharmacology
5.
Hum Mutat ; 30(11): E974-83, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19655382

ABSTRACT

It has been recently demonstrated that the 43-kDa transactive response (TAR)-DNA-binding protein (TARDBP) is the neuropathological hallmark of Frontotemporal Dementia (FTD) with ubiquitin-positive and tau-negative inclusions. Large series of FTD patients without motor neuron disease have been previously analysed, but no TARDBP mutation was identified. The aim of the present study was to evaluate whether TARDBP gene mutations may be associated with FTD. We report that a pathogenetic TARDBP mutation is causative of behavioural variant FTD (bvFTD). An aged woman in her seventies initially started to present apathy and depression associated with impairment in executive functions. The diagnosis of bvFTD (apathetic syndrome) was accomplished by three-year follow-up, and structural and functional neuroimaging. By five-years after onset, extensive electrophysiological investigations excluded subclinical motor neuron disease. In this patient, a single base substitution c.800A>G of TARDBP gene was identified. This mutation, already described as causative of ALS, predicted the amino acidic change arginine to serine at position 267 (N267S). In silico analysis demonstrated that this substitution generates a new phosphorylation site, and western blot analysis on lymphoblastoid cells reported a decrease of protein expression in N267S mutation carrier. Our study suggests that TARDBP mutations can be pathogenetic of bvFTD without motor neuron disease. TARDBP screening needs to be considered in FTD cases.


Subject(s)
DNA-Binding Proteins/genetics , Frontotemporal Dementia/genetics , Aged , Female , Frontotemporal Dementia/diagnosis , Humans
6.
Biochim Biophys Acta ; 1487(2-3): 155-62, 2000 Sep 27.
Article in English | MEDLINE | ID: mdl-11018468

ABSTRACT

Lysosomal acid lipase (LAL) is essential for the intracellular degradation of cholesteryl esters (CE) and triacylglycerols (TG) that are delivered to lysosomes by low density lipoprotein (LDL) receptor mediated endocytosis. We have analysed the difference in the catalytic properties and substrate specificity of human and mouse LALs. LAL activities were measured in human and mouse fibroblasts and in HeLa cells transiently expressing wild-type or site-directed mutant LALs of the two species using the T7 vaccinia system. Cholesteryl esterase and triacylglycerol lipase activities were determined in cellular homogenates with a phospholipid/detergent vesicle assay, an assay frequently used to diagnose human LAL deficiency syndromes, and with LDL particles, a more physiological substrate. Characterisation of human and mouse LAL using these two assays demonstrated marked differences in their TG and CE hydrolysing activities. Compared to human LAL mouse LAL showed a much lower cholesteryl esterase activity in both assays used. The difference was more pronounced in the vesicle assay. The lower cholesteryl esterase activity of mouse LAL did not affect the LDL-CE degradation in intact fibroblasts. The analysis of site-directed mutants suggests a role of the non-conserved cysteine residue at position 240 in cholesteryl esterase activity in human LAL. Our results show a significant difference between human and mouse LAL in their specificity toward cholesteryl esters. The low cholesteryl esterase activity does not result in reduced LDL-cholesterol ester degradation in mouse fibroblasts in situ. In addition, this work emphasises the importance of the physical state of substrates in studies of the specificity and properties of lipolytic enzymes.


Subject(s)
Cholesterol Esters/metabolism , Lipase/metabolism , Lysosomes/enzymology , Animals , Cells, Cultured , Cholesterol, LDL/metabolism , Fibroblasts , HeLa Cells , Humans , Lipase/chemistry , Mice , Plasmids , Sterol Esterase/metabolism , Substrate Specificity
7.
J Biol Chem ; 275(28): 21041-7, 2000 Jul 14.
Article in English | MEDLINE | ID: mdl-10766763

ABSTRACT

In monosymptomatic forms of cystic fibrosis such as congenital bilateral absence of vas deferens, variations in the TG(m) and T(n) polymorphic repeats at the 3' end of intron 8 of the cystic fibrosis transmembrane regulator (CFTR) gene are associated with the alternative splicing of exon 9, which results in a nonfunctional CFTR protein. Using a minigene model system, we have previously shown a direct relationship between the TG(m)T(n) polymorphism and exon 9 splicing. We have now evaluated the role of splicing factors in the regulation of the alternative splicing of this exon. Serine-arginine-rich proteins and the heterogeneous nuclear ribonucleoprotein A1 induced exon skipping in the human gene but not in its mouse counterpart. The effect of these proteins on exon 9 exclusion was strictly dependent on the composition of the TG(m) and T(n) polymorphic repeats. The comparative and functional analysis of the human and mouse CFTR genes showed that a region of about 150 nucleotides, present only in the human intron 9, mediates the exon 9 splicing inhibition in association with exonic regulatory elements. This region, defined as the CFTR exon 9 intronic splicing silencer, is a target for serine-arginine-rich protein interactions. Thus, the nonevolutionary conserved CFTR exon 9 alternative splicing is modulated by the TG(m) and T(n) polymorphism at the 3' splice region, enhancer and silencer exonic elements, and the intronic splicing silencer in the proximal 5' intronic region. Tissue levels and individual variability of splicing factors would determine the penetrance of the TG(m)T(n) locus in monosymptomatic forms of cystic fibrosis.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Cystic Fibrosis/genetics , Exons , Introns , Animals , Base Sequence , Conserved Sequence , Gene Amplification , Genes, Synthetic , Humans , Mice , Mutagenesis, Site-Directed , Polymorphism, Genetic , Protein Multimerization , Recombinant Proteins/metabolism , Repetitive Sequences, Nucleic Acid , Restriction Mapping
8.
J Lipid Res ; 39(7): 1382-8, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9684740

ABSTRACT

Deficiency of lysosomal acid lipase (LAL) leads to either Wolman disease (WD) or the more benign cholesteryl ester storage disease (CESD). To identify the molecular basis of the different phenotypes we have characterised the LAL gene mutations in three new patients with LAL deficiency. A patient with WD was homozygote for a null allele Y303X. The other two patients, with CESD, presented either homozygosity for T267I or compound heterozygosity consisting of Q64R and an exon 8 donor splice site substitution (G-->A in position -1). The mutants T267I and Q64R and the previously reported L273S, G66V, and H274Y CESD substitutions, overexpressed in stable clones, were found to be fully glycosylated and show an enzymatic activity of 3-8% of that of normal LAL. On the other hand, the delta254-277 mutant protein derived from exon 8 skipping and the Y303X protein were totally inactive. By transient transfection of hybrid minigene constructs, the CESD G-->A (-1) substitution resulted in partial exon inclusion, thus allowing the production of a small amount of normal LAL mRNA and hence of a functional enzyme. In contrast, a G-->A substitution observed in WD at position + 1 of the same exon 8 donor site resulted in complete exon skipping and the sole production of an inactive delta254-277 protein. In conclusion, LAL genotypes determine the level of residual enzymatic activity, thus explaining the severity of the phenotype.


Subject(s)
Cholesterol Ester Storage Disease/genetics , Lipase/genetics , Point Mutation , Sequence Deletion , Wolman Disease/genetics , Amino Acid Substitution , Base Sequence , Child , Cholesterol Ester Storage Disease/enzymology , Exons , Female , Genetic Variation , Humans , Infant , Lysosomes/enzymology , Male , Phenotype , Polymerase Chain Reaction , Wolman Disease/enzymology
9.
Biochem J ; 326 ( Pt 1): 265-9, 1997 Aug 15.
Article in English | MEDLINE | ID: mdl-9337878

ABSTRACT

Human lysosomal acid lipase (LAL) catalyses the deacylation of triacylglycerol and cholesteryl esters in the acidic lysosomal compartment. Treatment of LAL with the reducing agent dithiothreitol affected the triacylglycerol and cholesteryl esterase activities differentially, suggesting the involvement of cysteine residues in determining substrate specificity. To identify the residues involved, human LAL cDNA, under the control of the T7 promoter and tagged with a herpes simplex virus coding epitope, was specifically mutated in order to introduce single amino acid substitutions of each of the six cysteine residues of mature LAL. All Cys-227 mutants showed selectively decreased activity towards cholesteryl oleate, while preserving that towards trioleylglycerol. Substitutions of Cys-236, Cys-240 and Cys-244 affected catalysis towards the two substrates to a variable degree, depending on the side chain of the amino acid introduced. The replacement of Cys-41 or Cys-188 did not result in the preferential cleavage of either one of the two substrates. These data indicate that Cys-227, Cys-236, Cys-240 and Cys-244 play a crucial role in determining LAL substrate specificity. We propose that these cysteine residues are involved in the hydrolysis of cholesteryl ester by affecting selectively the access of this substrate to the catalytic active site. In addition, the fact that the catalytic activity is never completely abolished in cysteine mutants demonstrates that LAL is not a thiol enzyme.


Subject(s)
Cysteine/physiology , Lipase/physiology , Sterol Esterase/metabolism , Cysteine/genetics , Dithiothreitol/pharmacology , Enzyme Activation/drug effects , HeLa Cells , Humans , Lipase/chemistry , Lipase/drug effects , Lysosomes/enzymology , Mutagenesis, Site-Directed , Reducing Agents/pharmacology , Substrate Specificity/drug effects , Substrate Specificity/genetics
10.
FEBS Lett ; 397(1): 79-82, 1996 Nov 11.
Article in English | MEDLINE | ID: mdl-8941718

ABSTRACT

Human lysosomal acid lipase (LAL), when expressed in HeLa cells using the Vaccinia T7 expression system, showed four major molecular forms ranging from 42 to 54 kDa. Treatment with endoglycosidase H resulted in a 42 kDa protein, indicating that the molecular weight variations were due to N-glycosylation. A missense substitution, L273S, previously detected in a patient with cholesteryl ester storage disease (CESD), produced catalytically inactive LAL showing a largest molecular mass form of 56 kDa instead of 54 kDa. Analysis of the amino acid sequence in the close proximity of the mutation (NMS- NML) indicated that the L273S mutation creates an additional N-glycosylation consensus (N-X-S/T) in this region. Two site directed mutants disrupting this consensus, QMS and QML, when expressed in HeLa cells, did not show the 56 kDa form but the normal 54 kDa band whereas deglycosylation always resulted in the major 42 kDa form, as observed with normal LAL and the L273S mutant. These data confirmed that an additional N-glycosylation at N271 was responsible for the 56 kDa form of the protein produced from the L273S allele. Furthermore, deglycosylation of normal LAL reduced the acid hydrolase activity towards both tri-oleyl glycerol and cholesteryl oleate by 50%, strongly suggesting that N-linked carbohydrate residues are important for optimal catalytic activity.


Subject(s)
Lipase/chemistry , Lysosomes/enzymology , Amino Acid Sequence , Catalysis , Glycosylation , HeLa Cells , Hexosaminidases/metabolism , Humans , Lipase/genetics , Lipase/metabolism , Molecular Weight , Mutagenesis, Site-Directed , Mutation , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sterol Esterase/metabolism , Transfection
11.
Hum Mol Genet ; 5(10): 1611-7, 1996 Oct.
Article in English | MEDLINE | ID: mdl-8894696

ABSTRACT

Lysosomal acid lipase (LAL) gene mutations were identified in three patients with cholesteryl ester storage disease (CESD). Direct sequencing of genomic DNA revealed that: patient 1 was a compound heterozygote for a P181L mutation and an A to G3' splice site substitution that causes skipping of exon 7, with a loss of 49 amino acids from LAL (delta 205-253); patient 2 was a compound heterozygote for a G66V mutation and a 5' splice site mutation (G to A) that leads to skipping of exon 8 (delta 254-277); and patient 3 was a compound heterozygote for a L273S mutation and an unidentified null allele. Furthermore, patients 2 and 3 showed a novel G-2A polymorphism that could be detected by an Xbal restriction fragment length polymorphism. All these mutants and a previously reported H274Y allele were expressed in vitro in HeLa cells using the vaccinia T7 expression system. The resulting recombinant proteins were inactive towards cholesteryl oleate and trioleylglycerol, demonstrating the direct involvement of these mutations in the pathogenesis of CESD. Immunoblotting of normal LAL expressed in HeLa cells revealed four major molecular forms, at least two of high molecular mass (54 and 50-51 kDa) and two of low molecular mass (42 and 43 kDa). L273S and P181L substitutions and delta 254-277 were shown to result in altered LAL molecular forms, some of which suggest that post-translational processing may interfere with the catalytic activity of LAL.


Subject(s)
Cholesterol Ester Storage Disease/genetics , Lipase/genetics , Child , Child, Preschool , Cholesterol Ester Storage Disease/enzymology , HeLa Cells , Heterozygote , Humans , Lipase/biosynthesis , Male , Mutation , Polymorphism, Genetic , Sequence Analysis
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