RESUMO
The endoplasmic reticulum (ER) forms contacts with the lysosomal compartment, regulating lysosome positioning and motility. The movements of lysosomes are controlled by the attachment of molecular motors to their surface. However, the molecular mechanisms by which ER controls lysosome dynamics are still elusive. Here, using mouse brain extracts and mouse embryonic fibroblasts, we demonstrate that spatacsin is an ER-resident protein regulating the formation of tubular lysosomes, which are highly dynamic. Screening for spatacsin partners required for tubular lysosome formation showed spatacsin to act by regulating protein degradation. We demonstrate that spatacsin promotes the degradation of its partner AP5Z1, which regulates the relative amount of spastizin and AP5Z1 at lysosomes. Spastizin and AP5Z1 contribute to regulate tubular lysosome formation, as well as their trafficking by interacting with anterograde and retrograde motor proteins, kinesin KIF13A and dynein/dynactin subunit p150Glued, respectively. Ultimately, investigations in polarized mouse cortical neurons in culture demonstrated that spatacsin-regulated degradation of AP5Z1 controls the directionality of lysosomes trafficking. Collectively, our results identify spatacsin as a protein regulating the directionality of lysosome trafficking.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular , Fibroblastos , Proteínas , Animais , Camundongos , Dineínas/metabolismo , Fibroblastos/metabolismo , Lisossomos/metabolismo , Neurônios/metabolismo , Proteínas/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismoRESUMO
Biallelic variants in the SPG11 gene account for the most common form of autosomal recessive hereditary spastic paraplegia characterized by motor and cognitive impairment, with currently no therapeutic option. We previously observed in a Spg11 knockout mouse that neurodegeneration is associated with accumulation of gangliosides in lysosomes. To test whether a substrate reduction therapy could be a therapeutic option, we downregulated the key enzyme involved in ganglioside biosynthesis using an AAV-PHP.eB viral vector expressing a miRNA targeting St3gal5. Downregulation of St3gal5 in Spg11 knockout mice prevented the accumulation of gangliosides, delayed the onset of motor and cognitive symptoms, and prevented the upregulation of serum levels of neurofilament light chain, a biomarker widely used in neurodegenerative diseases. Importantly, similar results were observed when Spg11 knockout mice were administrated venglustat, a pharmacological inhibitor of glucosylceramide synthase expected to decrease ganglioside synthesis. Downregulation of St3gal5 or venglustat administration in Spg11 knockout mice strongly decreased the formation of axonal spheroids, previously associated with impaired trafficking. Venglustat had similar effect on cultured human SPG11 neurons. In conclusion, this work identifies the first disease-modifying therapeutic strategy in SPG11, and provides data supporting its relevance for therapeutic testing in SPG11 patients.
Assuntos
Gangliosídeos , Camundongos Knockout , Paraplegia Espástica Hereditária , Animais , Gangliosídeos/metabolismo , Camundongos , Paraplegia Espástica Hereditária/genética , Paraplegia Espástica Hereditária/metabolismo , Humanos , Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/genética , Proteínas/genética , Proteínas/metabolismo , Sialiltransferases/genética , Sialiltransferases/deficiência , Neurônios/metabolismo , Camundongos Endogâmicos C57BL , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Proteínas de NeurofilamentosRESUMO
Neurofilament light chain (NfL) is a marker of brain atrophy and predictor of disease progression in rare diseases such as Huntington Disease, but also in more common neurological disorders such as Alzheimer's disease. The aim of this study was to measure NfL longitudinally in autosomal dominant spinocerebellar ataxias (SCAs) and establish correlation with clinical and imaging parameters. We enrolled 62 pathological expansions carriers (17 SCA1, 13 SCA2, 19 SCA3, and 13 SCA7) and 19 age-matched controls in a prospective biomarker study between 2011 and 2015 and followed for 24 months at the Paris Brain Institute. We performed neurological examination, brain 3 T MRI and plasma NfL measurements using an ultrasensitive single-molecule array at baseline and at the two-year follow-up visit. We evaluated NfL correlations with ages, CAG repeat sizes, clinical scores and volumetric brain MRIs. NfL levels were significantly higher in SCAs than controls at both time points (p < 0.001). Age-adjusted NfL levels were significantly correlated at baseline with clinical scores (p < 0.01). We identified optimal NfL cut-off concentrations to differentiate controls from carriers for each genotype (SCA1 16.87 pg/mL, SCA2, 19.1 pg/mL, SCA3 16.04 pg/mL, SCA7 16.67 pg/mL). For all SCAs, NfL concentration was stable over two years (p = 0.95) despite a clinical progression (p < 0.0001). Clinical progression between baseline and follow-up was associated with higher NfL concentrations at baseline (p = 0.04). Of note, all premanifest carriers with NfL levels close to cut off concentrations had signs of the disease at follow-up. For all SCAs, the higher the observed NfL, the lower the pons volume at baseline (p < 0.01) and follow-up (p = 0.02). Higher NfL levels at baseline in all SCAs predicted a decrease in cerebellar volume (p = 0.03). This result remained significant for SCA2 only among all genotypes (p = 0.02). Overall, plasma NfL levels at baseline in SCA expansion carriers predict cerebellar volume change and clinical score progression. NfL levels might help refine inclusion criteria for clinical trials in carriers with very subtle signs.
Assuntos
Cerebelo/diagnóstico por imagem , Proteínas de Neurofilamentos/sangue , Ataxias Espinocerebelares/sangue , Adulto , Atrofia , Estudos de Casos e Controles , Cerebelo/patologia , Progressão da Doença , Feminino , Humanos , Doença de Machado-Joseph/sangue , Doença de Machado-Joseph/diagnóstico por imagem , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Proteínas de Neurofilamentos/genética , Ataxias Espinocerebelares/diagnóstico , Ataxias Espinocerebelares/diagnóstico por imagem , Ataxias Espinocerebelares/genética , Expansão das Repetições de TrinucleotídeosRESUMO
Hereditary spastic paraplegias (HSPs) are clinically and genetically heterogeneous human neurodegenerative diseases. Amongst the identified genetic causes, mutations in genes encoding motor proteins such as kinesins have been involved in various HSP clinical isoforms. Mutations in KIF1C are responsible for autosomal recessive spastic paraplegia type 58 (SPG58) and spastic ataxia 2 (SPAX2). Bovines also develop neurodegenerative diseases, some of them having a genetic aetiology. Bovine progressive ataxia was first described in the Charolais breed in the early 1970s in England and further cases in this breed were subsequently reported worldwide. We can now report that progressive ataxia of Charolais cattle results from a homozygous single nucleotide polymorphism in the coding region of the KIF1C gene. In this study, we show that the mutation at the heterozygous state is associated with a better score for muscular development, explaining its balancing selection for several decades, and the resulting high frequency (13%) of the allele in the French Charolais breed. We demonstrate that the KIF1C bovine mutation leads to a functional knock-out, therefore mimicking mutations in humans affected by SPG58/SPAX2. The functional consequences of KIF1C loss of function in cattle were also histologically reevaluated. We showed by an immunochemistry approach that demyelinating plaques were due to altered oligodendrocyte membrane protrusion, and we highlight an abnormal accumulation of actin in the core of demyelinating plaques, which is normally concentrated at the leading edge of oligodendrocytes during axon wrapping. We also observed that the lesions were associated with abnormal extension of paranodal sections. Moreover, this model highlights the role of KIF1C protein in preserving the structural integrity and function of myelin, since the clinical signs and lesions arise in young-adult Charolais cattle. Finally, this model provides useful information for SPG58/SPAX2 disease and other demyelinating lesions.
Assuntos
Doenças dos Bovinos/genética , Bovinos/genética , Cinesinas/metabolismo , Bainha de Mielina/metabolismo , Degenerações Espinocerebelares/veterinária , Sequência de Aminoácidos , Animais , Doenças dos Bovinos/diagnóstico , Modelos Animais de Doenças , Feminino , Heterozigoto , Homozigoto , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/genética , Deficiência Intelectual/veterinária , Cinesinas/genética , Masculino , Espasticidade Muscular/diagnóstico , Espasticidade Muscular/genética , Espasticidade Muscular/veterinária , Mutação de Sentido Incorreto , Atrofia Óptica/diagnóstico , Atrofia Óptica/genética , Atrofia Óptica/veterinária , Polimorfismo de Nucleotídeo Único , Paraplegia Espástica Hereditária/diagnóstico , Paraplegia Espástica Hereditária/genética , Paraplegia Espástica Hereditária/veterinária , Ataxias Espinocerebelares/diagnóstico , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/veterinária , Degenerações Espinocerebelares/diagnóstico , Degenerações Espinocerebelares/genética , Sequenciamento Completo do GenomaRESUMO
Hereditary spastic paraplegia, SPG31, is a rare neurological disorder caused by mutations in REEP1 gene encoding the microtubule-interacting protein, REEP1. The mechanism by which REEP1-dependent processes are linked with the disease is unclear. REEP1 regulates the morphology and trafficking of various organelles via interaction with the microtubules. In this study, we collected primary fibroblasts from SPG31 patients to investigate their mitochondrial morphology. We observed that the mitochondrial morphology in patient cells was highly tubular compared with control cells. We provide evidence that these morphological alterations are caused by the inhibition of mitochondrial fission protein, DRP1, due to the hyperphosphorylation of its serine 637 residue. This hyperphosphorylation is caused by impaired interactions between REEP1 and mitochondrial phosphatase PGAM5. Genetically or pharmacologically induced decrease of DRP1-S637 phosphorylation restores mitochondrial morphology in patient cells. Furthermore, ectopic expression of REEP1 carrying pathological mutations in primary neuronal culture targets REEP1 to the mitochondria. Mutated REEP1 proteins sequester mitochondria to the perinuclear region of the neurons and therefore, hamper mitochondrial transport along the axon. Considering the established role of mitochondrial distribution and morphology in neuronal health, our results support the involvement of a mitochondrial dysfunction in SPG31 pathology.
Assuntos
Núcleo Celular , GTP Fosfo-Hidrolases , Proteínas Associadas aos Microtúbulos , Mitocôndrias , Proteínas Mitocondriais , Neurônios/metabolismo , Paraplegia Espástica Hereditária , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Células Cultivadas , Dinaminas , Feminino , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Humanos , Masculino , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Neurônios/patologia , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Fosforilação/genética , Paraplegia Espástica Hereditária/metabolismo , Paraplegia Espástica Hereditária/patologiaRESUMO
Hereditary spastic paraplegia (HSP) is an inherited disorder of the central nervous system mainly characterized by gradual spasticity and weakness of the lower limbs. SPG56 is a rare autosomal recessive early onset complicated form of HSP caused by mutations in CYP2U1. The CYP2U1 enzyme was shown to catalyze the hydroxylation of arachidonic acid. Here, we report two further SPG56 families carrying three novel CYP2U1 missense variants and the development of an in vitro biochemical assay to determine the pathogenicity of missense variants of uncertain clinical significance. We compared spectroscopic, enzymatic, and structural (from a 3D model) characteristics of the over expressed wild-type or mutated CYP2U1 in HEK293T cells. Our findings demonstrated that most of the tested missense variants in CYP2U1 were functionally inactive because of a loss of proper heme binding or destabilization of the protein structure. We also showed that functional data do not necessarily correlate with in silico predictions of variants pathogenicity, using different bioinformatic phenotype prediction tools. Our results therefore highlight the importance to use biological tools, such as the enzymatic test set up in this study, to evaluate the effects of newly identified variants in clinical settings.
Assuntos
Família 2 do Citocromo P450/genética , Família 2 do Citocromo P450/metabolismo , Mutação de Sentido Incorreto , Paraplegia Espástica Hereditária/enzimologia , Paraplegia Espástica Hereditária/genética , Alelos , Substituição de Aminoácidos , Família 2 do Citocromo P450/química , Análise Mutacional de DNA , Ativação Enzimática , Expressão Gênica , Estudos de Associação Genética , Células HEK293 , Humanos , Modelos Moleculares , Oxirredução , Fenótipo , Conformação Proteica , Paraplegia Espástica Hereditária/diagnósticoRESUMO
Mutations in SPG11 account for the most common form of autosomal recessive hereditary spastic paraplegia (HSP), characterized by a gait disorder associated with various brain alterations. Mutations in the same gene are also responsible for rare forms of Charcot-Marie-Tooth (CMT) disease and progressive juvenile-onset amyotrophic lateral sclerosis (ALS). To elucidate the physiopathological mechanisms underlying these human pathologies, we disrupted the Spg11 gene in mice by inserting stop codons in exon 32, mimicking the most frequent mutations found in patients. The Spg11 knockout mouse developed early-onset motor impairment and cognitive deficits. These behavioral deficits were associated with progressive brain atrophy with the loss of neurons in the primary motor cortex, cerebellum and hippocampus, as well as with accumulation of dystrophic axons in the corticospinal tract. Spinal motor neurons also degenerated and this was accompanied by fragmentation of neuromuscular junctions and muscle atrophy. This new Spg11 knockout mouse therefore recapitulates the full range of symptoms associated with SPG11 mutations observed in HSP, ALS and CMT patients. Examination of the cellular alterations observed in this model suggests that the loss of spatacsin leads to the accumulation of lipids in lysosomes by perturbing their clearance from these organelles. Altogether, our results link lysosomal dysfunction and lipid metabolism to neurodegeneration and pinpoint a critical role of spatacsin in lipid turnover.
Assuntos
Metabolismo dos Lipídeos/fisiologia , Lisossomos/metabolismo , Doença dos Neurônios Motores/metabolismo , Proteínas/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Células Cultivadas , Transtornos Cognitivos/metabolismo , Transtornos Cognitivos/patologia , Modelos Animais de Doenças , Fibroblastos/metabolismo , Lisossomos/patologia , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/fisiologia , Doença dos Neurônios Motores/patologia , Neurônios/metabolismo , Neurônios/patologia , Fenótipo , Proteínas/genética , Medula Espinal/metabolismo , Medula Espinal/patologiaRESUMO
Adaptor proteins (AP 1-5) are heterotetrameric complexes that facilitate specialized cargo sorting in vesicular-mediated trafficking. Mutations in AP5Z1, encoding a subunit of the AP-5 complex, have been reported to cause hereditary spastic paraplegia (HSP), although their impact at the cellular level has not been assessed. Here we characterize three independent fibroblast lines derived from skin biopsies of patients harbouring nonsense mutations in AP5Z1 and presenting with spastic paraplegia accompanied by neuropathy, parkinsonism and/or cognitive impairment. In all three patient-derived lines, we show that there is complete loss of AP-5 ζ protein and a reduction in the associated AP-5 µ5 protein. Using ultrastructural analysis, we show that these patient-derived lines consistently exhibit abundant multilamellar structures that are positive for markers of endolysosomes and are filled with aberrant storage material organized as exaggerated multilamellar whorls, striated belts and 'fingerprint bodies'. This phenotype can be replicated in a HeLa cell culture model by siRNA knockdown of AP-5 ζ. The cellular phenotype bears striking resemblance to features described in a number of lysosomal storage diseases (LSDs). Collectively, these findings reveal an emerging picture of the role of AP-5 in endosomal and lysosomal homeostasis, illuminates a potential pathomechanism that is relevant to the role of AP-5 in neurons and expands the understanding of recessive HSPs. Moreover, the resulting accumulation of storage material in endolysosomes leads us to propose that AP-5 deficiency represents a new type of LSDs.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Endossomos/metabolismo , Doenças por Armazenamento dos Lisossomos/genética , Doenças por Armazenamento dos Lisossomos/metabolismo , Lisossomos/metabolismo , Mutação , Idoso , Feminino , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Técnicas de Silenciamento de Genes , Estudos de Associação Genética , Células HeLa , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Proteínas/genética , Proteínas/metabolismo , Interferência de RNARESUMO
Hereditary spastic paraplegias (HSPs) are clinically and genetically heterogeneous neurological conditions. Their main pathogenic mechanisms are thought to involve alterations in endomembrane trafficking, mitochondrial function, and lipid metabolism. With a combination of whole-genome mapping and exome sequencing, we identified three mutations in REEP2 in two families with HSP: a missense variant (c.107T>A [p.Val36Glu]) that segregated in the heterozygous state in a family with autosomal-dominant inheritance and a missense change (c.215T>A [p.Phe72Tyr]) that segregated in trans with a splice site mutation (c.105+3G>T) in a family with autosomal-recessive transmission. REEP2 belongs to a family of proteins that shape the endoplasmic reticulum, an organelle that was altered in fibroblasts from an affected subject. In vitro, the p.Val36Glu variant in the autosomal-dominant family had a dominant-negative effect; it inhibited the normal binding of wild-type REEP2 to membranes. The missense substitution p.Phe72Tyr, in the recessive family, decreased the affinity of the mutant protein for membranes that, together with the splice site mutation, is expected to cause complete loss of REEP2 function. Our findings illustrate how dominant and recessive inheritance can be explained by the effects and nature of mutations in the same gene. They have also important implications for genetic diagnosis and counseling in clinical practice because of the association of various modes of inheritance to this new clinico-genetic entity.
Assuntos
Proteínas de Membrana/genética , Paraplegia Espástica Hereditária/genética , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Mapeamento Cromossômico , Exoma , Feminino , Heterozigoto , Humanos , Masculino , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Linhagem , Fenótipo , Paraplegia Espástica Hereditária/patologiaRESUMO
The most common form of autosomal recessive hereditary spastic paraplegia is caused by mutations in the SPG11/KIAA1840 gene on chromosome 15q. The nature of the vast majority of SPG11 mutations found to date suggests a loss-of-function mechanism of the encoded protein, spatacsin. The SPG11 phenotype is, in most cases, characterized by a progressive spasticity with neuropathy, cognitive impairment and a thin corpus callosum on brain MRI. Full neuropathological characterization has not been reported to date despite the description of >100 SPG11 mutations. We describe here the clinical and pathological features observed in two unrelated females, members of genetically ascertained SPG11 families originating from Belgium and Italy, respectively. We confirm the presence of lesions of motor tracts in medulla oblongata and spinal cord associated with other lesions of the central nervous system. Interestingly, we report for the first time pathological hallmarks of SPG11 in neurons that include intracytoplasmic granular lysosome-like structures mainly in supratentorial areas, and others in subtentorial areas that are partially reminiscent of those observed in amyotrophic lateral sclerosis, such as ubiquitin and p62 aggregates, except that they are never labelled with anti-TDP-43 or anti-cystatin C. The neuropathological overlap with amyotrophic lateral sclerosis, associated with some shared clinical manifestations, opens up new fields of investigation in the physiopathological continuum of motor neuron degeneration.
Assuntos
Esclerose Lateral Amiotrófica/patologia , Neurônios Motores/patologia , Degeneração Neural/patologia , Paraplegia Espástica Hereditária/patologia , Adulto , Encéfalo/patologia , Feminino , Gânglios Espinais/patologia , Humanos , Lisossomos/ultraestrutura , Masculino , Bulbo/patologia , Pessoa de Meia-Idade , Mutação , Proteínas/genética , Paraplegia Espástica Hereditária/diagnóstico , Paraplegia Espástica Hereditária/diagnóstico por imagem , Medula Espinal/patologiaRESUMO
Hereditary spastic paraplegias (HSPs) form a heterogeneous group of neurological disorders. A whole-genome linkage mapping effort was made with three HSP-affected families from Spain, Portugal, and Tunisia and it allowed us to reduce the SPG26 locus interval from 34 to 9 Mb. Subsequently, a targeted capture was made to sequence the entire exome of affected individuals from these three families, as well as from two additional autosomal-recessive HSP-affected families of German and Brazilian origins. Five homozygous truncating (n = 3) and missense (n = 2) mutations were identified in B4GALNT1. After this finding, we analyzed the entire coding region of this gene in 65 additional cases, and three mutations were identified in two subjects. All mutated cases presented an early-onset spastic paraplegia, with frequent intellectual disability, cerebellar ataxia, and peripheral neuropathy as well as cortical atrophy and white matter hyperintensities on brain imaging. B4GALNT1 encodes ß-1,4-N-acetyl-galactosaminyl transferase 1 (B4GALNT1), involved in ganglioside biosynthesis. These findings confirm the increasing interest of lipid metabolism in HSPs. Interestingly, although the catabolism of gangliosides is implicated in a variety of neurological diseases, SPG26 is only the second human disease involving defects of their biosynthesis.
Assuntos
Disfunção Cognitiva/genética , Gangliosídeos/biossíntese , Paraplegia Espástica Hereditária/genética , Adolescente , Adulto , Idade de Início , Brasil , Ataxia Cerebelar/genética , Criança , Pré-Escolar , Mapeamento Cromossômico/métodos , Exoma , Feminino , Galactosiltransferases/genética , Galactosiltransferases/metabolismo , Gangliosídeos/genética , Predisposição Genética para Doença , Alemanha , Homozigoto , Humanos , Lactente , Metabolismo dos Lipídeos , Masculino , Mutação de Sentido Incorreto , Linhagem , Portugal , Espanha , Paraplegia Espástica Hereditária/metabolismo , Tunísia , Adulto JovemRESUMO
Hereditary spastic paraplegias are heterogeneous neurological disorders characterized by a pyramidal syndrome with symptoms predominantly affecting the lower limbs. Some limited pyramidal involvement also occurs in patients with an autosomal recessive neurocutaneous syndrome due to ALDH18A1 mutations. ALDH18A1 encodes delta-1-pyrroline-5-carboxylate synthase (P5CS), an enzyme that catalyses the first and common step of proline and ornithine biosynthesis from glutamate. Through exome sequencing and candidate gene screening, we report two families with autosomal recessive transmission of ALDH18A1 mutations, and predominant complex hereditary spastic paraplegia with marked cognitive impairment, without any cutaneous abnormality. More interestingly, we also identified monoallelic ALDH18A1 mutations segregating in three independent families with autosomal dominant pure or complex hereditary spastic paraplegia, as well as in two sporadic patients. Low levels of plasma ornithine, citrulline, arginine and proline in four individuals from two families suggested P5CS deficiency. Glutamine loading tests in two fibroblast cultures from two related affected subjects confirmed a metabolic block at the level of P5CS in vivo. Besides expanding the clinical spectrum of ALDH18A1-related pathology, we describe mutations segregating in an autosomal dominant pattern. The latter are associated with a potential trait biomarker; we therefore suggest including amino acid chromatography in the clinico-genetic work-up of hereditary spastic paraplegia, particularly in dominant cases, as the associated phenotype is not distinct from other causative genes.
Assuntos
Aldeído Desidrogenase/genética , Mutação/genética , Ornitina/genética , Ornitina/metabolismo , Paraplegia Espástica Hereditária/genética , Adolescente , Adulto , Arginina/metabolismo , Feminino , Ácido Glutâmico/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Paraplegia Espástica Hereditária/metabolismo , Adulto JovemRESUMO
Hereditary spastic paraplegia (HSP) is considered one of the most heterogeneous groups of neurological disorders, both clinically and genetically. The disease comprises pure and complex forms that clinically include slowly progressive lower-limb spasticity resulting from degeneration of the corticospinal tract. At least 48 loci accounting for these diseases have been mapped to date, and mutations have been identified in 22 genes, most of which play a role in intracellular trafficking. Here, we identified mutations in two functionally related genes (DDHD1 and CYP2U1) in individuals with autosomal-recessive forms of HSP by using either the classical positional cloning or a combination of whole-genome linkage mapping and next-generation sequencing. Interestingly, three subjects with CYP2U1 mutations presented with a thin corpus callosum, white-matter abnormalities, and/or calcification of the basal ganglia. These genes code for two enzymes involved in fatty-acid metabolism, and we have demonstrated in human cells that the HSP pathophysiology includes alteration of mitochondrial architecture and bioenergetics with increased oxidative stress. Our combined results focus attention on lipid metabolism as a critical HSP pathway with a deleterious impact on mitochondrial bioenergetic function.
Assuntos
Ácidos Graxos/metabolismo , Mitocôndrias/enzimologia , Mitocôndrias/genética , Paraplegia Espástica Hereditária/enzimologia , Paraplegia Espástica Hereditária/genética , Adolescente , Adulto , Criança , Pré-Escolar , Mapeamento Cromossômico , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Família 2 do Citocromo P450 , Feminino , Perfilação da Expressão Gênica , Genótipo , Humanos , Lactente , Recém-Nascido , Masculino , Mutação , Fenótipo , Fosfolipases/genética , Fosfolipases/metabolismo , Transporte Proteico , Adulto JovemRESUMO
We have previously demonstrated that neuroinflammation by the adaptive immune system acts as a robust and targetable disease amplifier in a mouse model of Spastic Paraplegia, type 11 (SPG11), a complicated form of Hereditary Spastic Paraplegia (HSP). While we identified an impact of neuroinflammation on distinct neuropathological changes and gait performance, neuropsychological features, typical and clinically highly relevant symptoms of complicated HSPs, were not addressed. Here we show that the corresponding SPG11 mouse model shows distinct behavioral abnormalities, particularly related to social behavior thus partially reflecting the neuropsychological changes in patients. We provide evidence that some behavioral abnormalities can be mitigated by genetic inactivation of the adaptive immune system. Translating this into a clinically applicable approach, we show that treatment with the established immunomodulators fingolimod or teriflunomide significantly attenuates distinct behavioral abnormalities, with the most striking effect on social behavior. This study links neuroinflammation to behavioral abnormalities in a mouse model of SPG11 and may thus pave the way for using immunomodulators as a treatment approach for SPG11 and possibly other complicated forms of HSP with neuropsychological involvement.
RESUMO
Generation of supramolecular architectures through controlled linking of suitable building blocks can offer new perspectives to medicine and applied technologies. Current linking strategies often rely on chemical methods that have limitations and cannot take full advantage of the recombinant technologies. Here we used SNARE proteins, namely, syntaxin, SNAP25, and synaptobrevin, which form stable tetrahelical complexes that drive fusion of intracellular membranes, as versatile tags for irreversible linking of recombinant and synthetic functional units. We show that SNARE tagging allows stepwise production of a functional modular medicinal toxin, namely, botulinum neurotoxin type A, commonly known as BOTOX. This toxin consists of three structurally independent units: Receptor-binding domain (Rbd), Translocation domain (Td), and the Light chain (Lc), the last being a proteolytic enzyme. Fusing the receptor-binding domain with synaptobrevin SNARE motif allowed delivery of the active part of botulinum neurotoxin (Lc-Td), tagged with SNAP25, into neurons. Our data show that SNARE-tagged toxin was able to cleave its intraneuronal molecular target and to inhibit release of neurotransmitters. The reassembled toxin provides a safer alternative to existing botulinum neurotoxin and may offer wider use of this popular research and medical tool. Finally, SNARE tagging allowed the Rbd portion of the toxin to be used to deliver quantum dots and other fluorescent markers into neurons, showing versatility of this unique tagging and self-assembly technique. Together, these results demonstrate that the SNARE tetrahelical coiled-coil allows controlled linking of various building blocks into multifunctional assemblies.
Assuntos
Toxinas Botulínicas Tipo A/química , Proteínas R-SNARE/química , Proteínas SNARE/química , Toxinas Botulínicas Tipo A/metabolismo , Proteínas R-SNARE/metabolismo , Proteínas SNARE/metabolismoRESUMO
Alpha-synuclein is a synaptic modulatory protein implicated in the pathogenesis of Parkinson disease. The precise functions of this small cytosolic protein are still under investigation. alpha-Synuclein has been proposed to regulate soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins involved in vesicle fusion. Interestingly, alpha-synuclein fails to interact with SNARE proteins in conventional protein-binding assays, thus suggesting an indirect mode of action. As the structural and functional properties of both alpha-synuclein and the SNARE proteins can be modified by arachidonic acid, a common lipid regulator, we analysed this possible tripartite link in detail. Here, we show that the ability of arachidonic acid to stimulate SNARE complex formation and exocytosis can be controlled by alpha-synuclein, both in vitro and in vivo. Alpha-synuclein sequesters arachidonic acid and thereby blocks the activation of SNAREs. Our data provide mechanistic insights into the action of alpha-synuclein in the modulation of neurotransmission.
Assuntos
Ácido Araquidônico/metabolismo , Exocitose/fisiologia , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo , alfa-Sinucleína/metabolismo , Animais , Células Cultivadas , Células Cromafins/citologia , Células Cromafins/metabolismo , Ácidos Graxos/metabolismo , Humanos , Camundongos , Camundongos Knockout , Células PC12 , Ratos , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/genética , Sinapses/metabolismo , Proteína 25 Associada a Sinaptossoma/genética , Proteína 25 Associada a Sinaptossoma/metabolismo , Sintaxina 1/genética , Sintaxina 1/metabolismo , alfa-Sinucleína/genéticaRESUMO
Growth of neurite processes from the cell body is the critical step in neuronal development and involves a large increase in cell membrane surface area. Arachidonic-acid-releasing phospholipases are highly enriched in nerve growth cones and have previously been implicated in neurite outgrowth. Cell membrane expansion is achieved through the fusion of transport organelles with the plasma membrane; however, the identity of the molecular target of arachidonic acid has remained elusive. Here we show that syntaxin 3 (STX3), a plasma membrane protein, has an important role in the growth of neurites, and also serves as a direct target for omega-6 arachidonic acid. By using syntaxin 3 in a screening assay, we determined that the dietary omega-3 linolenic and docosahexaenoic acids can efficiently substitute for arachidonic acid in activating syntaxin 3. Our findings provide a molecular basis for the previously established action of omega-3 and omega-6 polyunsaturated fatty acids in membrane expansion at the growth cones, and represent the first identification of a single effector molecule for these essential nutrients.
Assuntos
Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Ácidos Graxos Ômega-3/farmacologia , Ácidos Graxos Ômega-6/farmacologia , Proteínas Qa-SNARE/metabolismo , Animais , Ácido Araquidônico/metabolismo , Ácidos Graxos Ômega-3/metabolismo , Ácidos Graxos Ômega-6/metabolismo , Cones de Crescimento/efeitos dos fármacos , Fusão de Membrana/efeitos dos fármacos , Neuritos/efeitos dos fármacos , Células PC12 , Ratos , Proteínas SNARE/metabolismoRESUMO
Truncating mutations in the SPG11 and SPG15 genes cause complicated spastic paraplegia, severe neurological conditions due to loss of the functions of spatacsin and spastizin, respectively. We developed specific polyclonal anti-spatacsin (SPG11) and anti-spastizin (SPG15) antisera, which we then used to explore the intracellular and tissue localizations of these proteins. We observed expression of both proteins in human and rat central nervous system, which was particularly strong in cortical and spinal motor neurons as well as in retina. Both proteins were also expressed ubiquitously and strongly in embryos. In cultured cells, these two proteins had similar diffuse punctate, cytoplasmic and sometimes nuclear (spastizin) distributions. They partially co-localized with multiple organelles, particularly with protein-trafficking vesicles, endoplasmic reticulum and microtubules. Spastizin was also found at the mitochondria surface. This first study of the endogenous expression of spatacsin and spastizin shows similarities in their expression patterns that could account for their overlapping clinical phenotypes and involvement in a common protein complex.
Assuntos
Proteínas de Transporte/metabolismo , Citoplasma/metabolismo , Neurônios Motores/metabolismo , Proteínas/metabolismo , Animais , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Células Cultivadas , Citoplasma/genética , Humanos , Camundongos , Proteínas/genética , Ratos , Paraplegia Espástica Hereditária/genética , Paraplegia Espástica Hereditária/metabolismoRESUMO
Hereditary spastic paraplegias consist of a group of rare neurodegenerative diseases characterized by lower limb spasticity. These inherited Mendelian disorders show high genetic variability associated with wide clinical diversity. Pathophysiological investigations have suggested that mutations in genes affecting the same cellular pathway generally lead to similar clinical symptoms, highlighting the importance of genetic mutation in these diseases. However, phenotype-genotype correlations have failed to explain the observed large inter-individual variability linked to mutations in a single gene, suggesting that genetics alone is not sufficient to explain symptom diversity. The identification of biomarkers, such as neurofilament light chain, could fill the gap and predict disease evolution.
Assuntos
Paraplegia Espástica Hereditária , Humanos , Mutação/genética , Fenótipo , Paraplegia Espástica Hereditária/genéticaRESUMO
Pharmacological targeting of neuroinflammation in distinct models of genetically mediated disorders of the central nervous system (CNS) has been shown to attenuate disease outcome significantly. These include mouse models mimicking distinct subtypes of neuronal ceroid lipofuscinoses (NCL, CLN diseases) as well as hereditary spastic paraplegia type 2 (HSP/SPG2). We here show in a model of another, complicated HSP form (SPG11) that there is neuroinflammation in distinct compartments of the diseased CNS. Using a proof-of-principle experiment, we provide evidence that genetically targeting the adaptive immune system dampens disease progression including gait disturbance, demonstrating a pathogenic impact of neuroinflammation. Translating these studies into a clinically applicable approach, we show that the established immunomodulators fingolimod and teriflunomide significantly attenuate the neurodegenerative phenotype and improve gait performance in the SPG11 model, even when applied relatively late during disease progression. Particularly abnormalities in gait coordination, representing ataxia, could be attenuated, while features indicative of reduced strength during walking did not respond to treatment. Our study identifies neuroinflammation by the adaptive immune system as a robust and targetable disease amplifier in a mouse model of SPG11 and may thus pave the way for a translational approach in humans implicating approved immunomodulators.