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1.
J Med Genet ; 61(8): 759-768, 2024 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-38670634

RESUMO

BACKGROUND: Pontocerebellar hypoplasia (PCH) may present with supratentorial phenotypes and is often accompanied by microcephaly. Damaging mutations in the X-linked gene CASK produce self-limiting microcephaly with PCH in females but are often lethal in males. CASK deficiency leads to early degeneration of cerebellar granule cells but its role in other regions of the brain remains uncertain. METHOD: We generated a conditional Cask knockout mice and deleted Cask ubiquitously after birth at different times. We examined the clinical features in several subjects with damaging mutations clustered in the central part of the CASK protein. We have performed phylogenetic analysis and RT-PCR to assess the splicing pattern within the same protein region and performed in silico structural analysis to examine the effect of splicing on the CASK's structure. RESULT: We demonstrate that deletion of murine Cask after adulthood does not affect survival but leads to cerebellar degeneration and ataxia over time. Intriguingly, damaging hemizygous CASK mutations in boys who display microcephaly and cerebral dysfunction but without PCH are known. These mutations are present in two vertebrate-specific CASK exons. These exons are subject to alternative splicing both in forebrain and hindbrain. Inclusion of these exons differentially affects the molecular structure and hence possibly the function/s of the CASK C-terminus. CONCLUSION: Loss of CASK function disproportionately affects the cerebellum. Clinical data, however, suggest that CASK may have additional vertebrate-specific function/s that play a role in the mammalian forebrain. Thus, CASK has an ancient function shared between invertebrates and vertebrates as well as novel vertebrate-specific function/s.


Assuntos
Guanilato Quinases , Camundongos Knockout , Animais , Guanilato Quinases/genética , Guanilato Quinases/química , Camundongos , Masculino , Humanos , Feminino , Microcefalia/genética , Microcefalia/patologia , Mutação , Éxons/genética , Processamento Alternativo/genética , Filogenia , Cerebelo/metabolismo , Cerebelo/anormalidades , Cerebelo/patologia
2.
J Mol Med (Berl) ; 101(8): 1015-1028, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37462767

RESUMO

Multiple molecular pathways including the receptor for advanced glycation end-products-diaphanous related formin 1 (RAGE-Diaph1) signaling are known to play a role in diabetic peripheral neuropathy (DPN). Evidence suggests that neuropathological alterations in type 1 diabetic spinal cord may occur at the same time as or following peripheral nerve abnormalities. We demonstrated that DPN was associated with perturbations of RAGE-Diaph1 signaling pathway in peripheral nerve accompanied by widespread spinal cord molecular changes. More than 500 differentially expressed genes (DEGs) belonging to multiple functional pathways were identified in diabetic spinal cord and of those the most enriched was RAGE-Diaph1 related PI3K-Akt pathway. Only seven of spinal cord DEGs overlapped with DEGs from type 1 diabetic sciatic nerve and only a single gene cathepsin E (CTSE) was common for both type 1 and type 2 diabetic mice. In silico analysis suggests that molecular changes in spinal cord may act synergistically with RAGE-Diaph1 signaling axis in the peripheral nerve. KEY MESSAGES: Molecular perturbations in spinal cord may be involved in the progression of diabetic peripheral neuropathy. Diabetic peripheral neuropathy was associated with perturbations of RAGE-Diaph1 signaling pathway in peripheral nerve accompanied by widespread spinal cord molecular changes. In silico analysis revealed that PI3K-Akt signaling axis related to RAGE-Diaph1 was the most enriched biological pathway in diabetic spinal cord. Cathepsin E may be the target molecular hub for intervention against diabetic peripheral neuropathy.


Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Neuropatias Diabéticas , Hiperglicemia , Animais , Camundongos , Receptor para Produtos Finais de Glicação Avançada/genética , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Neuropatias Diabéticas/genética , Neuropatias Diabéticas/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/complicações , Catepsina E , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Nervo Isquiático/patologia , Hiperglicemia/genética , Hiperglicemia/patologia
3.
Neurosci Bull ; 38(10): 1248-1262, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35729453

RESUMO

This review reflects upon our own as well as other investigators' studies on the role of receptor for advanced glycation end-products (RAGE), bringing up the latest information on RAGE in physiology and pathology of the nervous system. Over the last ten years, major progress has been made in uncovering many of RAGE-ligand interactions and signaling pathways in nervous tissue; however, the translation of these discoveries into clinical practice has not come to fruition yet. This is likely, in part to be the result of our incomplete understanding of this crucial signaling pathway. Clinical trials examining the therapeutic efficacy of blocking RAGE-external ligand interactions by genetically engineered soluble RAGE or an endogenous RAGE antagonist, has not stood up to its promise; however, other trials with different blocking agents are being considered with hope for therapeutic success in diseases of the nervous system.


Assuntos
Doenças do Sistema Nervoso , Humanos , Ligantes , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Transdução de Sinais/fisiologia
4.
Cells ; 11(7)2022 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-35406695

RESUMO

Most human disease manifests as a result of tissue pathology, due to an underlying disease process (pathogenesis), rather than the acute loss of specific molecular function(s). Successful therapeutic strategies thus may either target the correction of a specific molecular function or halt the disease process. For the vast majority of brain diseases, clear etiologic and pathogenic mechanisms are still elusive, impeding the discovery or design of effective disease-modifying drugs. The development of valid animal models and their proper characterization is thus critical for uncovering the molecular basis of the underlying pathobiological processes of brain disorders. MICPCH (microcephaly and pontocerebellar hypoplasia) is a monogenic condition that results from variants of an X-linked gene, CASK (calcium/calmodulin-dependent serine protein kinase). CASK variants are associated with a wide range of clinical presentations, from lethality and epileptic encephalopathies to intellectual disabilities, microcephaly, and autistic traits. We have examined CASK loss-of-function mutations in model organisms to simultaneously understand the pathogenesis of MICPCH and the molecular function/s of CASK. Our studies point to a highly complex relationship between the potential molecular function/s of CASK and the phenotypes observed in model organisms and humans. Here we discuss the implications of our observations from the pathogenesis of MICPCH as a cautionary narrative against oversimplifying molecular interpretations of data obtained from genetically modified animal models of human diseases.


Assuntos
Deficiência Intelectual Ligada ao Cromossomo X , Microcefalia , Malformações do Sistema Nervoso , Animais , Guanilato Quinases/genética , Deficiência Intelectual Ligada ao Cromossomo X/complicações , Camundongos , Microcefalia/genética , Malformações do Sistema Nervoso/genética
5.
J Med Genet ; 59(11): 1044-1057, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35149592

RESUMO

BACKGROUND: Heterozygous loss of X-linked genes like CASK and MeCP2 (Rett syndrome) causes developmental delay in girls, while in boys, loss of the only allele of these genes leads to epileptic encephalopathy. The mechanism for these disorders remains unknown. CASK-linked cerebellar hypoplasia is presumed to result from defects in Tbr1-reelin-mediated neuronal migration. METHOD: Here we report clinical and histopathological analyses of a deceased 2-month-old boy with a CASK-null mutation. We next generated a mouse line where CASK is completely deleted (hemizygous and homozygous) from postmigratory neurons in the cerebellum. RESULT: The CASK-null human brain was smaller in size but exhibited normal lamination without defective neuronal differentiation, migration or axonal guidance. The hypoplastic cerebellum instead displayed astrogliosis and microgliosis, which are markers for neuronal loss. We therefore hypothesise that CASK loss-induced cerebellar hypoplasia is the result of early neurodegeneration. Data from the murine model confirmed that in CASK loss, a small cerebellum results from postdevelopmental degeneration of cerebellar granule neurons. Furthermore, at least in the cerebellum, functional loss from CASK deletion is secondary to degeneration of granule cells and not due to an acute molecular functional loss of CASK. Intriguingly, female mice with heterozygous deletion of CASK in the cerebellum do not display neurodegeneration. CONCLUSION: We suggest that X-linked neurodevelopmental disorders like CASK mutation and Rett syndrome are pathologically neurodegenerative; random X-chromosome inactivation in heterozygous mutant girls, however, results in 50% of cells expressing the functional gene, resulting in a non-progressive pathology, whereas complete loss of the only allele in boys leads to unconstrained degeneration and encephalopathy.


Assuntos
Doenças Cerebelares , Doenças Neurodegenerativas , Síndrome de Rett , Masculino , Humanos , Animais , Feminino , Camundongos , Lactente , Genes Ligados ao Cromossomo X/genética , Guanilato Quinases/genética , Síndrome de Rett/genética , Doenças Cerebelares/genética , Doenças Neurodegenerativas/genética
6.
Eur J Neurosci ; 54(6): 5982-5999, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34449932

RESUMO

Neuropathy, or dysfunction of peripheral nerve, is one of the most common neurological manifestation in patients with diabetes mellitus (DM). DM is typically associated with a hyperglycaemic milieu, which promotes non-enzymatic glycation of proteins. Proteins with advanced glycation are known to engage a cell-surface receptor called the receptor for advanced glycation end products (RAGE). Thus, it is reasonable to assume that RAGE and its associated molecule-mediated cellular signalling may contribute to DM-induced symmetrical axonal (length-dependent) neuropathy. Of particular interest is diaphanous related formin 1 (DIAPH1), a cytoskeletal organizing molecule, which interacts with the cytosolic domain of RAGE and whose dysfunction may precipitate axonopathy/neuropathy. Indeed, it has been demonstrated that both RAGE and DIAPH1 are expressed in the motor and sensory fibres of nerve harvested from DM animal models. Although the detailed molecular role of RAGE and DIAPH1 in diabetic neurological complications remains unclear, here we will discuss available evidence of their involvement in peripheral diabetic neuropathy. Specifically, we will discuss how a hyperglycaemic environment is not only likely to elevate advanced glycation end products (ligands of RAGE) and induce a pro-inflammatory environment but also alter signalling via RAGE and DIAPH1. Further, hyperglycaemia may regulate epigenetic mechanisms that interacts with RAGE signalling. We suggest the cumulative effect of hyperglycaemia on RAGE-DIAPH1-mediated signalling may be disruptive to axonal cytoskeletal organization and transport and is therefore likely to play a key role in pathogenesis of diabetic symmetrical axonal neuropathy.


Assuntos
Complicações do Diabetes , Diabetes Mellitus , Neuropatias Diabéticas , Animais , Forminas , Humanos , Ligantes , Receptor para Produtos Finais de Glicação Avançada , Transdução de Sinais
7.
Biochem Biophys Res Commun ; 559: 92-98, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-33933994

RESUMO

Synaptic transmission is mediated by neurotransmitters that are stored in synaptic vesicles (SV) and released at the synaptic active zone (AZ). While in recent years major progress has been made in unraveling the molecular machinery responsible for SV docking, fusion and exocytosis, the mechanisms governing AZ protein and SV trafficking through axons still remain unclear. Here, we performed stop-flow nerve ligation to examine axonal trafficking of endogenous AZ and SV proteins. Rat sciatic nerves were collected 1 h, 3 h and 8 h post ligation and processed for immunohistochemistry and electron microscopy. First, we followed the transport of an integral synaptic vesicle protein, SV2A and a SV-associated protein involved in SV trafficking, Rab3a, and observed that while SV2A accumulated on both sides of ligation, Rab3a was only noticeable in the proximal segment of the ligated nerve indicating that only SV trans-membrane protein SV2A displayed a bi-directional axonal transport. We then demonstrate that multiple AZ proteins accumulate rapidly on either side of the ligation with a timescale similar to that of SV2A. Overall, our data uncovers an unexpected robust bi-directional, coordinated -trafficking of SV and AZ proteins in peripheral nerves. This implies that pathological disruption of axonal trafficking will not only impair trafficking of newly synthesized proteins to the synapse but will also affect retrograde transport, leading to neuronal dysfunction and likely neurodegeneration.


Assuntos
Transporte Axonal , Proteínas do Tecido Nervoso/metabolismo , Nervos Periféricos/fisiologia , Transporte Proteico , Vesículas Sinápticas/metabolismo , Animais , Masculino , Ratos Sprague-Dawley , Transmissão Sináptica
8.
Mol Genet Genomic Med ; 8(10): e1426, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32696595

RESUMO

BACKGROUND: CASK is an X-linked gene in mammals and its deletion in males is incompatible with life. CASK heterozygous mutations in female patients associate with intellectual disability, microcephaly, pontocerebellar hypoplasia, and optic nerve hypoplasia, whereas CASK hemizygous mutations in males manifest as early infantile epileptic encephalopathy with a grim prognosis. Here, we report a rare case of survival of a male patient harboring a CASK null mutation to adolescent age. METHODS: Trio whole exome sequencing analysis was performed from blood genomic DNA. Magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS), and electroencephalogram (EEG) analyses were performed to determine anomalies in brain development, metabolite concentrations, and electrical activity, respectively. RESULTS: Trio-WES analysis identified a de novo c.79C>T (p.Arginine27Ter) mutation in CASK causing a premature translation termination at the very N-terminus of the protein. The 17-years, and 11-month-old male patient displayed profound intellectual disability, microcephaly, dysmorphism, ponto-cerebellar hypoplasia, and intractable epilepsy. His systemic symptoms included overall reduced somatic growth, dysautonomia, ventilator and G tube dependence, and severe osteopenia. Brain MRI revealed a severe cerebellar and brain stem hypoplasia with progressive cerebral atrophy. EEG spectral analysis revealed a global functional defect with generalized background slowing and delta waves dominating even in the awake state. CONCLUSION: This case study is the first to report survival of a male patient carrying a CASK loss-of-function mutation to adolescence and highlights that improved palliative care could extend survival. Moreover, the genomic position encoding Arg27 in CASK may possess an increased susceptibility to mutations.


Assuntos
Anormalidades Múltiplas/genética , Epilepsia/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Guanilato Quinases/genética , Deficiência Intelectual/genética , Mutação com Perda de Função , Anormalidades Múltiplas/patologia , Adolescente , Epilepsia/patologia , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Humanos , Deficiência Intelectual/patologia , Masculino
10.
Invest Ophthalmol Vis Sci ; 60(10): 3584-3594, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31425583

RESUMO

Purpose: Heterozygous mutations in the essential X-linked gene CASK associate with optic nerve hypoplasia (ONH) and other retinal disorders in girls. CASK+/- heterozygous knockout mice with mosaic CASK expression exhibit ONH with a loss of retinal ganglion cells (RGCs) but no changes in retinal morphology. It remains unclear if CASK deficiency selectively affects RGCs or also affects other retinal cells. Furthermore, it is not known if CASK expression in RGCs is critical for optic nerve (ON) development and maintenance. Methods: The visual behavior of CASK+/- mice was assessed and electroretinography (ERG) was performed. Using a mouse line with a floxed CASK gene that expresses approximately 40% CASK globally in all cells (hypomorph) under hemizygous and homozygous conditions, we investigated effects of CASK reduction on the retina and ON. CASK then was completely deleted from RGCs to examine its cell-autonomous role. Finally, for the first time to our knowledge, we describe a hemizygous CASK missense mutation in a boy with ONH. Results: CASK+/- heterozygous mutant mice display reduced visual contrast sensitivity, but ERG is indistinguishable from wildtype. CASK hypomorph mice exhibit ONH, but deletion of CASK from RGCs in this background does not exacerbate the condition. The boy with ONH harbors a missense mutation (p.Pro673Leu) that destabilizes CASK and weakens the crucial CASK-neurexin interaction. Conclusions: Our results demonstrate that mosaic or global reduction in CASK expression and/or function disproportionately affects RGCs. CASK expression in RGCs does not appear critical for cell survival, indicating a noncell autonomous role for CASK in the development of ON.


Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Guanilato Quinases/genética , Hipoplasia do Nervo Óptico/genética , Animais , Sobrevivência Celular , Pré-Escolar , Sensibilidades de Contraste/fisiologia , Eletrorretinografia , Feminino , Humanos , Immunoblotting , Imuno-Histoquímica , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação de Sentido Incorreto , Hipoplasia do Nervo Óptico/fisiopatologia , Retina/fisiopatologia , Células Ganglionares da Retina/enzimologia
11.
Sci Rep ; 9(1): 5752, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30962477

RESUMO

Mitochondrial DNA (mtDNA) 3243A > G tRNALeu(UUR) heteroplasmic mutation (m.3243A > G) exhibits clinically heterogeneous phenotypes. While the high mtDNA heteroplasmy exceeding a critical threshold causes mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes (MELAS) syndrome, the low mtDNA heteroplasmy causes maternally inherited diabetes with or without deafness (MIDD) syndrome. How quantitative differences in mtDNA heteroplasmy produces distinct pathological states has remained elusive. Here we show that despite striking similarities in the energy metabolic gene expression signature, the mitochondrial bioenergetics, biogenesis and fuel catabolic functions are distinct in cells harboring low or high levels of the m.3243 A > G mutation compared to wild type cells. We further demonstrate that the low heteroplasmic mutant cells exhibit a coordinate induction of transcriptional regulators of the mitochondrial biogenesis, glucose and fatty acid metabolism pathways that lack in near homoplasmic mutant cells compared to wild type cells. Altogether, these results shed new biological insights on the potential mechanisms by which low mtDNA heteroplasmy may progressively cause diabetes mellitus.


Assuntos
DNA Mitocondrial/genética , Metabolismo Energético , Síndrome MELAS/genética , Mutação , Ácidos Graxos/metabolismo , Glucose/metabolismo , Humanos , Biogênese de Organelas
12.
Am J Med Genet A ; 179(1): 94-103, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30549415

RESUMO

Heterozygous loss-of-function mutations in the X-linked gene CASK are associated with mental retardation and microcephaly with pontine and cerebellar hypoplasia (MICPCH) and ophthalmological disorders including optic nerve atrophy (ONA) and optic nerve hypoplasia (ONH). Recently, we have demonstrated that CASK(+/-) mice display ONH with 100% penetrance but exhibit no change in retinal lamination or structure. It is not clear if CASK loss-of-function predominantly affects retinal ganglion cells, or if other retinal cells like photoreceptors are also involved. Here, we report a heterozygous missense mutation in the N-terminal calcium/calmodulin-dependent kinase (CaMK) domain of the CASK protein in which a highly conserved leucine is mutated to the cyclic amino acid proline. In silico analysis suggests that the mutation may produce destabilizing structural changes. Experimentally, we observe pronounced misfolding and insolubility of the CASKL209P protein. Interestingly, the remaining soluble mutant protein fails to interact with Mint1, which specifically binds to CASK's CaMK domain, suggesting a mechanism for the phenotypes observed with the CASKL209P mutation. In addition to microcephaly, cerebellar hypoplasia and delayed development, the subject with the L209P mutation also presented with bilateral retinal dystrophy and ONA. Electroretinography indicated that rod photoreceptors are the most prominently affected cells. Our data suggest that the CASK interactions mediated by the CaMK domain may play a crucial role in retinal function, and thus, in addition to ONH, individuals with mutations in the CASK gene may exhibit other retinal disorders, depending on the nature of mutation.


Assuntos
Atrofia/genética , Guanilato Quinases/genética , Microcefalia/genética , Distrofias Retinianas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Atrofia/diagnóstico por imagem , Atrofia/fisiopatologia , Criança , Feminino , Guanilato Quinases/química , Células HEK293 , Heterozigoto , Humanos , Mutação com Perda de Função/genética , Microcefalia/diagnóstico por imagem , Microcefalia/fisiopatologia , Simulação de Dinâmica Molecular , Mutação de Sentido Incorreto/genética , Proteínas do Tecido Nervoso/genética , Nervo Óptico/fisiopatologia , Células Fotorreceptoras/patologia , Dobramento de Proteína , Distrofias Retinianas/diagnóstico por imagem , Distrofias Retinianas/fisiopatologia , Células Ganglionares da Retina/patologia , Sequenciamento do Exoma
13.
Hum Genet ; 137(3): 231-246, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29426960

RESUMO

Deletion and truncation mutations in the X-linked gene CASK are associated with severe intellectual disability (ID), microcephaly and pontine and cerebellar hypoplasia in girls (MICPCH). The molecular origin of CASK-linked MICPCH is presumed to be due to disruption of the CASK-Tbr-1 interaction. This hypothesis, however, has not been directly tested. Missense variants in CASK are typically asymptomatic in girls. We report three severely affected girls with heterozygous CASK missense mutations (M519T (2), G659D (1)) who exhibit ID, microcephaly, and hindbrain hypoplasia. The mutation M519T results in the replacement of an evolutionarily invariant methionine located in the PDZ signaling domain known to be critical for the CASK-neurexin interaction. CASKM519T is incapable of binding to neurexin, suggesting a critically important role for the CASK-neurexin interaction. The mutation G659D is in the SH3 (Src homology 3) domain of CASK, replacing a semi-conserved glycine with aspartate. We demonstrate that the CASKG659D mutation affects the CASK protein in two independent ways: (1) it increases the protein's propensity to aggregate; and (2) it disrupts the interface between CASK's PDZ (PSD95, Dlg, ZO-1) and SH3 domains, inhibiting the CASK-neurexin interaction despite residing outside of the domain deemed critical for neurexin interaction. Since heterozygosity of other aggregation-inducing mutations (e.g., CASKW919R) does not produce MICPCH, we suggest that the G659D mutation produces microcephaly by disrupting the CASK-neurexin interaction. Our results suggest that disruption of the CASK-neurexin interaction, not the CASK-Tbr-1 interaction, produces microcephaly and cerebellar hypoplasia. These findings underscore the importance of functional validation for variant classification.


Assuntos
Moléculas de Adesão Celular Neuronais/genética , Cerebelo/anormalidades , Doenças Genéticas Ligadas ao Cromossomo X/genética , Guanilato Quinases/genética , Microcefalia/genética , Proteínas do Tecido Nervoso/genética , Malformações do Sistema Nervoso/genética , Proteínas de Ligação ao Cálcio , Moléculas de Adesão Celular Neuronais/química , Cerebelo/diagnóstico por imagem , Cerebelo/fisiopatologia , Criança , Pré-Escolar , Deficiências do Desenvolvimento/diagnóstico por imagem , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/fisiopatologia , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/fisiopatologia , Guanilato Quinases/química , Humanos , Deficiência Intelectual/diagnóstico por imagem , Deficiência Intelectual/genética , Deficiência Intelectual/fisiopatologia , Microcefalia/diagnóstico por imagem , Microcefalia/fisiopatologia , Mutação de Sentido Incorreto/genética , Proteínas do Tecido Nervoso/química , Malformações do Sistema Nervoso/diagnóstico por imagem , Malformações do Sistema Nervoso/fisiopatologia , Moléculas de Adesão de Célula Nervosa , Domínios PDZ/genética , Fenótipo , Agregados Proteicos/genética , Ligação Proteica , Mapas de Interação de Proteínas/genética , Proteínas com Domínio T/genética , Domínios de Homologia de src/genética
14.
BMC Res Notes ; 10(1): 743, 2017 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-29258560

RESUMO

OBJECTIVES: Children with microcephaly face lifelong psychomotor, cognitive, and communications skills disabilities. Etiology of microcephaly is heterogeneous but presentation often includes seizures, hypotonia, ataxia, stereotypic movements, attention deficits, excitability, cognitive delays, and poor communication skills. Molecular diagnostics have outpaced available interventions and most children receive generic physical, speech, and occupational therapies with little attention to the efficacy of such treatments. Mutations in the X-linked intellectual disability gene (XLID) CASK is one etiology associated with microcephaly which produces mental retardation and microcephaly with pontine and cerebellar hypoplasia (MICPCH; OMIM# 300749). We pilot-tested an intensive therapy in three girls with heterozygous mutation in the gene CASK and MICPCH. Child A = 54 months; Child B = 89 months; and Child C = 24 months received a targeted treatment to improve gross/fine motor skills, visual-motor coordination, social interaction, and communication. Treatment was 4 h each weekday for 10 treatment days. Operant training promoted/refined goal-directed activities. The Peabody Developmental Motor Scales 2 was administered pre- and post-treatment. RESULTS: Child A gained 14 developmental months; Child B gained 20 developmental months; and Child C gained 39 developmental months. This case series suggests that children with MICPCH are responsive to intensive therapy aimed at increasing functional skills/independence. Trial Registration ClinicalTrials.gov Registration Number: NCT03325946; Release Date: October 30, 2017.


Assuntos
Cognição/fisiologia , Guanilato Quinases/genética , Destreza Motora/fisiologia , Mutação , Reabilitação Neurológica/métodos , Criança , Pré-Escolar , Feminino , Humanos , Deficiência Intelectual Ligada ao Cromossomo X/genética , Deficiência Intelectual Ligada ao Cromossomo X/fisiopatologia , Deficiência Intelectual Ligada ao Cromossomo X/terapia , Microcefalia/genética , Microcefalia/fisiopatologia , Microcefalia/terapia , Projetos Piloto , Resultado do Tratamento
15.
Invest Ophthalmol Vis Sci ; 58(12): 5485-5496, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-29067402

RESUMO

Purpose: Optic nerve hypoplasia (ONH) is the most common cause of childhood congenital blindness in developed nations, yet the fundamental pathobiology of ONH remains unknown. The objective of this study was to employ a 'face validated' murine model to determine the timing of onset and the pathologic characteristics of ONH. Methods: Based on the robust linkage between X-linked CASK haploinsufficiency and clinically diagnosed ONH, we hypothesized that heterozygous deletion of CASK (CASK(+/-)) in rodents will produce an optic nerve pathology closely recapitulating ONH. We quantitatively analyzed the entire subcortical visual system in female CASK(+/-) mice using immunohistochemistry, anterograde axonal tracing, toluidine blue staining, transmission electron microscopy, and serial block-face scanning electron microscopy. Results: CASK haploinsuffiency in mice phenocopies human ONH with complete penetrance, thus satisfying the 'face validity'. We demonstrate that the optic nerve in CASK(+/-) mice is not only thin, but is comprised of atrophic retinal axons and displays reactive astrogliosis. Myelination of the optic nerve axons remains unchanged. Moreover, we demonstrate a significant decrease in retinal ganglion cell (RGC) numbers and perturbation in retinothalamic connectivity. Finally, we used this mouse model to define the onset and progression of ONH pathology, demonstrating for the first time that optic nerve defects arise at neonatally in CASK(+/-)mice. Conclusions: Optic nerve hypoplasia is a complex neuropathology of the subcortical visual system involving RGC loss, axonopathy, and synaptopathy and originates at a developmental stage in mice that corresponds to the late third trimester development in humans.


Assuntos
Doenças do Nervo Óptico/congênito , Nervo Óptico/patologia , Animais , Axônios/patologia , Modelos Animais de Doenças , Progressão da Doença , Guanilato Quinases/genética , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Doenças do Nervo Óptico/genética , Doenças do Nervo Óptico/patologia , Células Ganglionares da Retina/patologia
16.
Methods Mol Biol ; 1647: 171-183, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28809002

RESUMO

Eukaryotic protein kinases are an intensely investigated class of enzymes which have garnered attention due to their usefulness as drug targets. Determining the regulation of ATP binding to a protein kinase is not only critical for understanding function in a cellular context but also for designing kinase-specific molecular inhibitors. Here, we provide a general procedure for characterizing ATP binding to eukaryotic protein kinases. The protocol can be adapted to identify the conditions under which a particular kinase is activated. The approach is simple, requiring only a fluorescent ATP analog such as TNP-ATP or MANT-ATP and an instrument to monitor changes in fluorescence. Although the interaction kinetics between a kinase and a given ATP analog may differ from that of native ATP, this disadvantage is offset by the ease of performing and interpreting this assay. Importantly, it can be optimized to probe a large variety of conditions under which the kinase-nucleotide binding might be affected.


Assuntos
Trifosfato de Adenosina/análogos & derivados , Corantes Fluorescentes/química , Proteínas Quinases/química , ortoaminobenzoatos/química , Trifosfato de Adenosina/química , Eucariotos/enzimologia , Guanilato Quinases/química , Guanilato Quinases/metabolismo , Cinética , Proteínas Quinases/metabolismo , Espectrometria de Fluorescência
17.
PLoS One ; 12(7): e0181140, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28700683

RESUMO

[This corrects the article DOI: 10.1371/journal.pone.0125185.].

19.
Matters (Zur) ; 20172017 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-30148136

RESUMO

Scavenger receptors (SRs) are a family of receptors displaying affinity for a wide variety of ligands including modified lipoproteins. SRs may play a range of physiological functions including intracellular transport, lipid transport and pathogen clearance. The role of SRs has been documented in pathologies such as atherosclerosis and Alzheimer's disease. Although most studies on SRs have focused on macrophages, they are also present in other cells like endothelium, smooth muscles and brain tissue. Within brain, due to its functional similarity, SRs have been studied mostly in microglia. However, in situ images from Allen's brain atlas suggest SRs are abundant in neurons. In this study we have used two fluorophore labeled well characterized SR ligand, maleylated-BSA (MBSA) and polyguanylic acid (poloyG) to probe acute cortical slices. Our data indicate that within cortex, neurons avidly endocytose both ligands. Thus in cerebral cortex neurons may have higher number of functional SRs on the surface than other cell-types.

20.
J Vis Exp ; (113)2016 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-27501303

RESUMO

Human brain is a high energy consuming organ that mainly relies on glucose as a fuel source. Glucose is catabolized by brain mitochondria via glycolysis, tri-carboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) pathways to produce cellular energy in the form of adenosine triphosphate (ATP). Impairment of mitochondrial ATP production causes mitochondrial disorders, which present clinically with prominent neurological and myopathic symptoms. Mitochondrial defects are also present in neurodevelopmental disorders (e.g. autism spectrum disorder) and neurodegenerative disorders (e.g. amyotrophic lateral sclerosis, Alzheimer's and Parkinson's diseases). Thus, there is an increased interest in the field for performing 3D analysis of mitochondrial morphology, structure and distribution under both healthy and disease states. The brain mitochondrial morphology is extremely diverse, with some mitochondria especially those in the synaptic region being in the range of <200 nm diameter, which is below the resolution limit of traditional light microscopy. Expressing a mitochondrially-targeted green fluorescent protein (GFP) in the brain significantly enhances the organellar detection by confocal microscopy. However, it does not overcome the constraints on the sensitivity of detection of relatively small sized mitochondria without oversaturating the images of large sized mitochondria. While serial transmission electron microscopy has been successfully used to characterize mitochondria at the neuronal synapse, this technique is extremely time-consuming especially when comparing multiple samples. The serial block-face scanning electron microscopy (SBFSEM) technique involves an automated process of sectioning, imaging blocks of tissue and data acquisition. Here, we provide a protocol to perform SBFSEM of a defined region from rodent brain to rapidly reconstruct and visualize mitochondrial morphology. This technique could also be used to provide accurate information on mitochondrial number, volume, size and distribution in a defined brain region. Since the obtained image resolution is high (typically under 10 nm) any gross mitochondrial morphological defects may also be detected.


Assuntos
Encéfalo , Mitocôndrias , Transtorno do Espectro Autista , Humanos , Microscopia Eletrônica de Varredura , Sinapses
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