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1.
Neurobiol Dis ; 152: 105291, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33556542

RESUMO

Abnormal aggregation of the α-synuclein protein is a key molecular feature of Parkinson's disease and other neurodegenerative diseases. The precise mechanisms that trigger α-synuclein aggregation are unclear, and it is not known what role aggregation plays in disease pathogenesis. Here we use an in vivo zebrafish model to express several different forms of human α-synuclein and measure its aggregation in presynaptic terminals. We show that human α-synuclein tagged with GFP can be expressed in zebrafish neurons, localizing normally to presynaptic terminals and undergoing phosphorylation at serine-129, as in mammalian neurons. The visual advantages of the zebrafish system allow for dynamic in vivo imaging to study α-synuclein, including the use of fluorescence recovery after photobleaching (FRAP) techniques to probe protein mobility. These experiments reveal three distinct terminal pools of α-synuclein with varying mobility, likely representing different subpopulations of aggregated and non-aggregated protein. Human α-synuclein is phosphorylated by an endogenous zebrafish Polo-like kinase activity, and there is a heterogeneous population of neurons containing either very little or extensive phosphorylation throughout the axonal arbor. Both pharmacological and genetic manipulations of serine-129 show that phosphorylation of α-synuclein at this site does not significantly affect its mobility. This suggests that serine-129 phosphorylation alone does not promote α-synuclein aggregation. Together our results show that human α-synuclein can be expressed and measured quantitatively in zebrafish, and that disease-relevant post-translational modifications occur within neurons. The zebrafish model provides a powerful in vivo system for measuring and manipulating α-synuclein function and aggregation, and for developing new treatments for neurodegenerative disease.


Assuntos
Modelos Animais de Doenças , Doença de Parkinson , Terminações Pré-Sinápticas/patologia , Agregação Patológica de Proteínas/metabolismo , alfa-Sinucleína/metabolismo , Animais , Animais Geneticamente Modificados , Humanos , Fosforilação , Serina/metabolismo , Peixe-Zebra
2.
J Undergrad Neurosci Educ ; 13(3): A215-24, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26240532

RESUMO

The lateral line sensory system in fish detects movements in the water and allows fish to respond to predators, prey, and other stimuli. As the lateral line forms in the first two days of zebrafish development, axons extend caudally along the lateral surface of the fish, eventually forming synapses with hair cells of neuromasts. Growing lateral line axons are located superficially under the skin and can be labeled in living zebrafish using fluorescent protein expression. This system provides a relatively straightforward approach for in vivo time-lapse imaging of neuronal development in an undergraduate setting. Here we describe an upper-level neurobiology laboratory module in which students investigate aspects of axonal development in the zebrafish lateral line system. Students learn to handle and image living fish, collect time-lapse videos of moving mitochondria, and quantitatively measure mitochondrial dynamics by generating and analyzing kymographs of their movements. Energy demands may differ between axons with extending growth cones versus axons that have already reached their targets and are forming synapses. Since relatively little is known about this process in developing lateral line axons, students generate and test their own hypotheses regarding how mitochondrial dynamics may differ at two different time points in axonal development. Students also learn to incorporate into their analysis a powerful yet accessible quantitative tool, the kymograph, which is used to graph movement over time. After students measure and quantify dynamics in living fish at 1 and 2 days post fertilization, this module extends into independent projects, in which students can expand their studies in a number of different, inquiry-driven directions. The project can also be pared down for courses that wish to focus solely on the quantitative analysis (without fish handling), or vice versa. This research module provides a useful approach for the design of open-ended laboratory research projects that integrate the scientific process into undergraduate Biology courses, as encouraged by the AAAS and NSF Vision and Change Initiative.

3.
Mol Vis ; 18: 2309-22, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22977299

RESUMO

PURPOSE: To characterize the expression pattern of cadherin 23 (cdh23) in the zebrafish visual system, and to determine whether zebrafish cdh23 mutants have retinal defects similar to those present in the human disease Usher syndrome 1D. METHODS: In situ hybridization and immunohistochemistry were used to characterize cdh23 expression in the zebrafish, and to evaluate cdh23 mutants for retinal degeneration. Visual function was assessed by measurement of the optokinetic response in cdh23 siblings and mutants. RESULTS: We detected cdh23 mRNA expression in multiple nuclei of both the developing and adult central nervous system. In the retina, cdh23 mRNA was expressed in a small subset of amacrine cells, beginning at 70 h postfertilization and continuing through adulthood. No expression was detected in photoreceptors. The cdh23-positive population of amacrine cells was GABAergic. Examination of homozygous larvae expressing two different mutant alleles of cdh23-cdh23(tc317e) or cdh23(tj264a)-revealed no detectable morphological retinal defects or degeneration. In addition, the optokinetic response to moving gratings of varied contrast or spatial frequency was normal in both mutants. CONCLUSIONS: Unlike in other vertebrates, cdh23 is not detectable in zebrafish photoreceptors. Instead, cdh23 is expressed by a small subset of GABAergic amacrine cells. Moreover, larvae with mutations in cdh23 do not exhibit any signs of gross retinal degeneration or dysfunction. The role played by cdh23 in human retinal function is likely performed by either a different gene or an unidentified cdh23 splice variant in the retina that is not affected by the above mutations.


Assuntos
Células Amácrinas/metabolismo , Caderinas/genética , Regulação da Expressão Gênica no Desenvolvimento , Larva/genética , Mutação , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Alelos , Processamento Alternativo , Células Amácrinas/citologia , Animais , Proteínas Relacionadas a Caderinas , Movimentos Oculares , Homozigoto , Humanos , Imuno-Histoquímica , Hibridização In Situ , Células Fotorreceptoras/citologia , Células Fotorreceptoras/metabolismo , RNA Mensageiro/biossíntese , Receptores de GABA/genética , Degeneração Retiniana/genética , Síndromes de Usher/genética
4.
J Cell Biol ; 180(4): 803-12, 2008 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-18299350

RESUMO

When a growing cell expands, lipids and proteins must be delivered to its periphery. Although this phenomenon has been observed for decades, it remains unknown how the secretory pathway responds to growth signaling. We demonstrate that control of Golgi phosphatidylinositol-4-phosphate (PI(4)P) is required for growth-dependent secretion. The phosphoinositide phosphatase SAC1 accumulates at the Golgi in quiescent cells and down-regulates anterograde trafficking by depleting Golgi PI(4)P. Golgi localization requires oligomerization of SAC1 and recruitment of the coat protein (COP) II complex. When quiescent cells are stimulated by mitogens, SAC1 rapidly shuttles back to the endoplasmic reticulum (ER), thus releasing the brake on Golgi secretion. The p38 mitogen-activated kinase (MAPK) pathway induces dissociation of SAC1 oligomers after mitogen stimulation, which triggers COP-I-mediated retrieval of SAC1 to the ER. Inhibition of p38 MAPK abolishes growth factor-induced Golgi-to-ER shuttling of SAC1 and slows secretion. These results suggest direct roles for p38 MAPK and SAC1 in transmitting growth signals to the secretory machinery.


Assuntos
Retículo Endoplasmático/enzimologia , Complexo de Golgi/enzimologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Animais , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/ultraestrutura , Células COS , Chlorocebus aethiops , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Inibidores Enzimáticos/farmacologia , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Camundongos , Mitógenos/farmacologia , Células NIH 3T3 , Transporte Proteico/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
5.
J Neurosci ; 22(24): 10643-52, 2002 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-12486157

RESUMO

The glutamate transporter excitatory amino acid transporter 3 (EAAT3) is polarized to the apical surface in epithelial cells and localized to the dendritic compartment in hippocampal neurons, where it is clustered adjacent to postsynaptic sites. In this study, we analyzed the sequences in EAAT3 that are responsible for its polarized localization in Madin-Darby canine kidney (MDCK) cells and neurons. Confocal microscopy and cell surface biotinylation assays demonstrated that deletion of the EAAT3 C terminus or replacement of the C terminus of EAAT3 with the analogous region in EAAT1 eliminated apical localization in MDCK cells. The C terminus of EAAT3 was sufficient to redirect the basolateral-preferring EAAT1 and the nonpolarized EAAT2 to the apical surface. Using alanine substitution mutants, we identified a short peptide motif in the cytoplasmic C-terminal region of EAAT3 that directs its apical localization in MDCK cells. Mutation of this sequence also impairs dendritic targeting of EAAT3 in hippocampal neurons but does not interfere with the clustering of EAAT3 on dendritic spines and filopodia. These data provide the first evidence that an identical cytoplasmic motif can direct apical targeting in epithelia and somatodendritic targeting in neurons. Moreover, our results demonstrate that the two fundamental features of the localization of EAAT3 in neurons, its restriction to the somatodendritic domain and its clustering near postsynaptic sites, are mediated by distinct molecular mechanisms.


Assuntos
Sistema X-AG de Transporte de Aminoácidos/análise , Sistema X-AG de Transporte de Aminoácidos/química , Hipocampo/química , Neurônios/química , Simportadores/análise , Simportadores/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Sistema X-AG de Transporte de Aminoácidos/genética , Sistema X-AG de Transporte de Aminoácidos/metabolismo , Animais , Linhagem Celular , Polaridade Celular , Células Cultivadas , Dendritos/química , Cães , Transportador 1 de Aminoácido Excitatório , Transportador 2 de Aminoácido Excitatório , Transportador 3 de Aminoácido Excitatório , Proteínas de Transporte de Glutamato da Membrana Plasmática , Hipocampo/citologia , Hipocampo/metabolismo , Dados de Sequência Molecular , Mutação , Neurônios/metabolismo , Sinais Direcionadores de Proteínas , Transporte Proteico , Ratos , Simportadores/genética , Simportadores/metabolismo
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