The effect of mutant GBA1 on accumulation and aggregation of α-synuclein.

Gaucher disease (GD) patients and carriers of GD mutations have a higher propensity to develop Parkinson's disease (PD) in comparison to the non-GD population. This implies that mutant GBA1 allele is a predisposing factor for the development of PD. One of the major characteristics of PD is the presence of oligomeric α-synuclein-positive inclusions known as Lewy bodies in the dopaminergic neurons localized to the substantia nigra pars compacta. In the present study we tested whether presence of human mutant GCase leads to accumulation and aggregation of α-synuclein in two models: in SHSY5Y neuroblastoma cells endogenously expressing α-synuclein and stably transfected with human GCase variants, and in Drosophila melanogaster co-expressing normal human α-synuclein and mutant human GCase. Our results showed that heterologous expression of mutant, but not WT, human GCase in SHSY5Y cells, led to a significant stabilization of α-synuclein and to its aggregation. In parallel, there was also a significant stabilization of mutant, but not WT, GCase. Co-expression of human α-synuclein and human mutant GCase in the dopaminergic cells of flies initiated α-synuclein aggregation, earlier death of these cells and significantly shorter life span, compared with flies expressing α-synuclein or mutant GCase alone. Taken together, our results strongly indicate that human mutant GCase contributes to accumulation and aggregation of α-synuclein. In the fly, this aggregation leads to development of more severe parkinsonian signs in comparison to flies expressing either mutant GCase or α-synuclein alone.

NEK3-mediated SNAP29 phosphorylation modulates its membrane association and SNARE fusion dependent processes.

Intracellular membrane fusion depends on the presence of specific mediators, the vesicle (v-) and the target (t-) SNAREs (Soluble N-ethylmaleimide-sensitive factor, NSF, attachment protein SNAP receptors), whose interaction brings apposing membranes to close proximity and initiates their fusion. SNAP29 (synaptosomal-associated protein 29), a t-SNARE protein, is involved in multiple fusion events during intracellular transport and affects structure of organelles such as the Golgi apparatus and focal adhesions. Mutations in SNAP29 gene result in CEDNIK (Cerebral dysgenesis, neuropathy, ichthyosis and palmoplantar keratoderma) syndrome. In the present study, we show that NEK3 (NIMA-never in mitosis gene A-related kinase 3)-mediated serine 105 (S105) phosphorylation of SNAP29 directs its membrane association, without which cells present defective focal adhesion formation, impaired Golgi structure and attenuated cellular recycling. In contrast to a phosphorylation-defective serine 105 to alanine (S105A) mutant, wildtype SNAP29, partially rescued the abnormal morphology of a CEDNIK patient derived fibroblasts. Our results highlight the importance of NEK3-mediated S105 phosphorylation of SNAP29 for its membrane localization and for membrane fusion dependent processes.

EHD2 mediates trafficking from the plasma membrane by modulating Rac1 activity.

EHDs [EH (Eps15 homology)-domain-containing proteins] participate in different stages of endocytosis. EHD2 is a plasma-membrane-associated EHD which regulates trafficking from the plasma membrane and recycling. EHD2 has a role in nucleotide-dependent membrane remodelling and its ATP-binding domain is involved in dimerization, which creates a membrane-binding region. Nucleotide binding is important for association of EHD2 with the plasma membrane, since a nucleotide-free mutant (EHD2 T72A) failed to associate. To elucidate the possible function of EHD2 during endocytic trafficking, we attempted to unravel proteins that interact with EHD2, using the yeast two-hybrid system. A novel interaction was found between EHD2 and Nek3 [NIMA (never in mitosis in Aspergillus nidulans)-related kinase 3], a serine/threonine kinase. EHD2 was also found in association with Vav1, a Nek3-regulated GEF (guanine-nucleotide-exchange factor) for Rho GTPases. Since Vav1 regulates Rac1 activity and promotes actin polymerization, the impact of overexpression of EHD2 on Rac1 activity was tested. The results indicated that wt (wild-type) EHD2, but not its P-loop mutants, reduced Rac1 activity. The inhibitory effect of EHD2 overexpression was partially rescued by co-expression of Rac1 as measured using a cholera toxin trafficking assay. The results of the present study strongly indicate that EHD2 regulates trafficking from the plasma membrane by controlling Rac1 activity.

RIN2 deficiency results in macrocephaly, alopecia, cutis laxa, and scoliosis: MACS syndrome.

Inherited disorders of elastic tissue represent a complex and heterogeneous group of diseases, characterized often by sagging skin and occasionally by life-threatening visceral complications. In the present study, we report on an autosomal-recessive disorder that we have termed MACS syndrome (macrocephaly, alopecia, cutis laxa, and scoliosis). The disorder was mapped to chromosome 20p11.21-p11.23, and a homozygous frameshift mutation in RIN2 was found to segregate with the disease phenotype in a large consanguineous kindred. The mutation identified results in decreased expression of RIN2, a ubiquitously expressed protein that interacts with Rab5 and is involved in the regulation of endocytic trafficking. RIN2 deficiency was found to be associated with paucity of dermal microfibrils and deficiency of fibulin-5, which may underlie the abnormal skin phenotype displayed by the patients.

A mutation in SNAP29, coding for a SNARE protein involved in intracellular trafficking, causes a novel neurocutaneous syndrome characterized by cerebral dysgenesis, neuropathy, ichthyosis, and palmoplantar keratoderma.

Neurocutaneous syndromes represent a vast, largely heterogeneous group of disorders characterized by neurological and dermatological manifestations, reflecting the common embryonic origin of epidermal and neural tissues. In the present report, we describe a novel neurocutaneous syndrome characterized by cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma (CEDNIK syndrome). Using homozygosity mapping in two large families, we localized the disease gene to 22q11.2 and identified, in all patients, a 1-bp deletion in SNAP29, which codes for a SNARE protein involved in vesicle fusion. SNAP29 expression was decreased in the skin of the patients, resulting in abnormal maturation of lamellar granules and, as a consequence, in mislocation of epidermal lipids and proteases. These data underscore the importance of vesicle trafficking regulatory mechanisms for proper neuroectodermal differentiation.

Frequency and pattern of DNA deletions in Duchenne (DMD) and Becker (BMD) patients: comparison of different racial background and isolated versus inherited cases

Foram estudadas 164 pacientes afetados por Distrofia Muscular tipo Duchenne (DMD) e 21 pacientes afetados por Distrofia Muscular tipo Becker (DMB), todos pertencentes a genealogias diferentes e a grupos raciais variados. A triagem de deleçöes de DNA foi feita na regiäo central do gene (sondas Cf23a e Cf56a) da distrofina. Os pacientes foram classificados em dois grupos raciais (caucasóides e negróides) e a freqüência de deleçöes entre os dois grupos foi comparada. Dentre as genealogias com pacientes afetados por DMD, 51 tinham dois ou mais afetados, em 107 o probando era caso isolado e 6 eram adotivos. A freqüência de deleçöes encontrada foi de 42,7% (70/164), sem diferença significativa entre caucasóides e negróides. Porém, uma diferença significante (P<0,02) foi encontrada na freqüência de deleçöes entre os casos isolados (51,4%) e os famíliais (29,4%). Nos afetados por DMB a freqüência de deleçöes foi de n38%, todas elas com início na regiäo correspondente a sonda Cf23a

Estudo imuno-histoquímico da distrofina em musculo de afetados por distrofias musculares progressivas

A deficiência da proteína Distrofina (locvalizada na membrana das fibras musculares) foi descrita recentemente como provável causa da distrofia muscular tipo Duchenne (DMD). No presente trabalho, foi feita uma reaçäo imuno-histoquímica para a detecçäo da distrofina em cortes histológicos de congelaçäo, obtidos a partir de biópsias musculares. Foram estudadas 56 indivíduos: Em quatro biópsias de musculo normal, ocorreu uma marcaçäo nítida e homogênea em todo o sarcolema das fibras musculares. No musculo de 20 pacientes com DMD, näo se observou marcaçäo na membrana da maioria das fibras, embora em muitos casos tenha ocorrido uma marcaçäo parcial de algumas delas (entre 4% e 30%). Em 10 afetados pela distrofia tipo Becker (DMB), 8 apresentaram marcaçäo normal em todo o sarcolema das fibras e 2 mostraram uma reaçäo aparentemente mais fraca. Nos quatro afetados por distrofia tipo Cinturas (DMC), apesar das alteraçöes histopatológicos típicas de afecçäo muscular primária, a membrana de todas as fibras mostrou-se com forte padräo imuno-reativo. Foram estudadas também 11 heterozigotas certas quanto ao gene da DMD (8 com atividade de enzimas séricas elevadas e 3 com enzimas normais), 4 filhas de heterozigotas certas com enzimas muito elevadas e 3 heterozigotas para o gene da DMB (todas com enzimas normais). Em todas elas observou-se marcaçäo total e homogênea em todas as fibras musculares. estes resultados säo muito importantes para a padronizaçäo de uma metodologia que possibilite um diagnóstico diferencial entre alguns tipos de distrofia, bem como tentar aprimorar a taxa de detecçäo de portadores, para fins de Aconselhamento Genético

Genetic risk estimates for Duchenne dystrophy (DMD) in the absence of DNA deletions in the central region of the dystrophin gene

Em estudo de triagem de deleçöes em 164 pacientes afetados por distrofias de Duchenne (DMD) näo aparentados, com as sondas de DNAc cf23a e cf56a e cf56a (que detectam cerca de 70% das deleçöes de DNA), observou-se que a frequência de deleçöes era significantemente maior nos esporádicos (0.56) do que naqueles herdados (0.34). Estes achados têm implicaçöes importantes para o Aconselhamento Genético pois mostram qua a ausência de deleçöes de DNA no probando levam a uma deleçäo molecular. Sugere-se também que seja estudada a distrofia muscular no afetado para exclusäo de um possível diagnóstico de uma forma autossômica recessiva de Duchenne, sempre que näo for encontrada deleçäo molecular no propósito

Serum creatine-kinase and pyruvate-kinase levels in females of different racial background at risk for Duchenne muscular dystrophy

As atividades séricas das enzimas creatino-cinase (CK) e piruvato-cinase (PK) foram determinadas em 146 mulheres beterozigotas certas quanto ao gene da distrofia muscular de Duchenne (DMD) e 187 mäes de casos isolados, pertencentes a dois grupos raciais: caucasóide e negroide. A atividade da CK foi medida em 206 mulheres caucasoides e 127 mulheres negroides e a da PK em 148 caucasoides e 92 negroides. Os resultados desta pesquisa mostraram um aumento da média de atividade enzimática no grupo de mulheres heterozigotes e mäes de casos isolados negroides em comparaçäo ao grupo de mulheres caucasoides. Entretanto, as diferenças foram estatísticamente significantes apenas para a CK sérica. Sugere-se portanto, que os resultados da atividade sérica da CK de mulheres em risco de serem portadoras do gene da DMD sejam comparados com os de mulheres normais de mesmo grupo racial

Becker-type muscular dystrophy in a mother of three boys affected by Duchenne muscular dystrophy: a follow-up study

Uma família com três afetados por distrofia tipo Duchenne (DMD) na qual a mäe e uma de suas filhas tinham sinais clínicos de DMD, foi seguida durante 14 anos. A mäe afetada tinha um quadro clínico semelhante a distrofia muscular tipo Becker ou tipo cinturas. Aos 50 anos de idade ela näo conseguia subir escadas e ela parou de andar aos 52 anos. Uma de suas filhas, com idade atual de 32 anos, tem hipertrofia das panturrilhas e altos níveis de creatino-cinase (CK) e piruvato-cinase (PK) desde os 16 anos de idade. Várias hipóteses säo discutidas para explicar a ocorrência de sinais clínicos em duas ou mais portadoras pertencentes à mesma genealogia