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1.
Neurosci Lett ; 810: 137317, 2023 07 27.
Artículo en Inglés | MEDLINE | ID: mdl-37286070

RESUMEN

Fragile X syndrome (FXS) is a developmental disorder characterized by intellectual disability and autistic-like behaviors. These symptoms are supposed to result from dysregulated translation in pre- and postsynapses, resulting in aberrant synaptic plasticity. Although most drug development research on FXS has focused on aberrant postsynaptic functions by excess translation in postsynapses, the effect of drug candidates on FXS in presynaptic release is largely unclear. In this report, we developed a novel assay system using neuron ball culture with beads to induce presynapse formation, allowing for the analysis of presynaptic phenotypes, including presynaptic release. Metformin, which is shown to rescue core phenotypes in FXS mouse model by normalizing dysregulated translation, ameliorated the exaggerated presynaptic release of neurons of FXS model mouse using this assay system. Furthermore, metformin suppressed the excess accumulation of the active zone protein Munc18-1, which is supposed to be locally translated in presynapses. These results suggest that metformin rescues both postsynaptic and presynaptic phenotypes by inhibiting excess translation in FXS neurons.


Asunto(s)
Síndrome del Cromosoma X Frágil , Animales , Ratones , Modelos Animales de Enfermedad , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil/tratamiento farmacológico , Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/metabolismo , Ratones Noqueados , Neuronas/metabolismo , Sinapsis/fisiología
2.
J Vis Exp ; (150)2019 08 02.
Artículo en Inglés | MEDLINE | ID: mdl-31424445

RESUMEN

During neuronal development, synapse formation is an important step to establish neural circuits. To form synapses, synaptic proteins must be supplied in appropriate order by transport from cell bodies and/or local translation in immature synapses. However, it is not fully understood how synaptic proteins accumulate in synapses in proper order. Here, we present a novel method to analyze presynaptic formation by using the combination of neuron ball culture with beads to induce presynapse formation. Neuron balls that is neuronal cell aggregates provide axonal sheets far from cell bodies and dendrites, so that weak fluorescent signals of presynapses can be detected by avoiding overwhelming signals of cell bodies. As beads to trigger presynapse formation, we use beads conjugated with leucine-rich repeat transmembrane neuronal 2 (LRRTM2), an excitatory presynaptic organizer. Using this method, we demonstrated that vesicular glutamate transporter 1 (vGlut1), a synaptic vesicle protein, accumulated in presynapses faster than Munc18-1, an active zone protein. Munc18-1 accumulated translation-dependently in presynapse even after removing cell bodies. This finding indicates the Munc18-1 accumulation by local translation in axons, not transport from cell bodies. In conclusion, this method is suitable to analyze accumulation of synaptic proteins in presynapses and source of synaptic proteins. As neuron ball culture is simple and it is not necessary to use special apparatus, this method could be applicable to other experimental platforms.


Asunto(s)
Neuronas/fisiología , Sinapsis/fisiología , Animales , Axones/metabolismo , Células Cultivadas , Ratones , Proteínas Munc18/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo
3.
Neurosci Res ; 146: 36-47, 2019 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-30240639

RESUMEN

Fragile X mental retardation protein (FMRP), a causative gene (FMR1) product of Fragile X syndrome (FXS), is an RNA-binding protein to regulate local protein synthesis in dendrites for postsynaptic functions. However, involvement of FMRP in local protein synthesis in axons for presynaptic functions remains unclear. Here we investigated role of FMRP in local translation of the active zone protein Munc18-1 during presynapse formation. We found that leucine-rich repeat transmembrane neuronal 2 (LRRTM2)-conjugated beads, which promotes synchronized presynapse formation, induced simultaneous accumulation of FMRP and Munc18-1 in presynapses of axons of mouse cortical neurons in neuronal cell aggregate culture. The LRRTM2-induced accumulation of Munc18-1 in presynapses was observed in axons protein-synthesis-dependently, even physically separated from cell bodies. The accumulation of Munc18-1 was enhanced in Fmr1-knockout (KO) axons as compared to wild type (WT), suggesting FMRP-regulated suppression for local translation of Munc18-1 in axons during presynapse formation. Using naturally formed synapses of dissociated culture, structured illumination microscope revealed that accumulation of Munc18-1 puncta in Fmr1-KO neurons increased significantly at 19 days in vitro, as compared to WT. Our findings lead the possibility that excessive accumulation of Munc18-1 in presynapses at early stage of synaptic development in Fmr1-KO neurons may have a critical role in impaired presynaptic functions in FXS.


Asunto(s)
Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/fisiología , Proteínas de la Membrana/metabolismo , Proteínas Munc18/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neurogénesis/fisiología , Sinapsis/metabolismo , Animales , Axones/metabolismo , Corteza Cerebral , Dendritas/metabolismo , Síndrome del Cromosoma X Frágil , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados
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