Mutant Huntingtin Causes a Selective Decrease in the Expression of Synaptic Vesicle Protein 2C / 神经科学通报·英文版

Huntington's disease (HD) is a neurodegenerative disease caused by a polyglutamine expansion in the huntingtin (Htt) protein. Mutant Htt causes synaptic transmission dysfunctions by interfering in the expression of synaptic proteins, leading to early HD symptoms. Synaptic vesicle proteins 2 (SV2s), a family of synaptic vesicle proteins including 3 members, SV2A, SV2B, and SV2C, plays important roles in synaptic physiology. Here, we investigated whether the expression of SV2s is affected by mutant Htt in the brains of HD transgenic (TG) mice and Neuro2a mouse neuroblastoma cells (N2a cells) expressing mutant Htt. Western blot analysis showed that the protein levels of SV2A and SV2B were not significantly changed in the brains of HD TG mice expressing mutant Htt with 82 glutamine repeats. However, in the TG mouse brain there was a dramatic decrease in the protein level of SV2C, which has a restricted distribution pattern in regions particularly vulnerable in HD. Immunostaining revealed that the immunoreactivity of SV2C was progressively weakened in the basal ganglia and hippocampus of TG mice. RT-PCR demonstrated that the mRNA level of SV2C progressively declined in the TG mouse brain without detectable changes in the mRNA levels of SV2A and SV2B, indicating that mutant Htt selectively inhibits the transcriptional expression of SV2C. Furthermore, we found that only SV2C expression was progressively inhibited in N2a cells expressing a mutant Htt containing 120 glutamine repeats. These findings suggest that the synaptic dysfunction in HD results from the mutant Htt-mediated inhibition of SV2C transcriptional expression. These data also imply that the restricted distribution and decreased expression of SV2C contribute to the brain region-selective pathology of HD.

Synaptic vesicle protein2A decreases in amygdaloid-kindling pharmcoresistant epileptic rats / 华中科技大学学报（医学）（英德文版）

Synaptic vesicle protein 2A (SV2A) involvement has been reported in the animal models of epilepsy and in human intractable epilepsy. The difference between pharmacosensitive epilepsy and pharmacoresistant epilepsy remains poorly understood. The present study aimed to observe the hippocampus SV2A protein expression in amygdale-kindling pharmacoresistant epileptic rats. The pharmacosensitive epileptic rats served as control. Amygdaloid-kindling model of epilepsy was established in 100 healthy adult male Sprague-Dawley rats. The kindled rat model of epilepsy was used to select pharmacoresistance by testing their seizure response to phenytoin and phenobarbital. The selected pharmacoresistant rats were assigned to a pharmacoresistant epileptic group (PRE group). Another 12 pharmacosensitive epileptic rats (PSE group) served as control. Immunohistochemistry, real-time PCR and Western blotting were used to determine SV2A expression in the hippocampus tissue samples from both the PRE and the PSE rats. Immunohistochemistry staining showed that SV2A was mainly accumulated in the cytoplasm of the neurons, as well as along their dendrites throughout all subfields of the hippocampus. Immunoreactive staining level of SV2A-positive cells was 0.483 ± 0.304 in the PRE group and 0.866 ± 0.090 in the PSE group (P < 0.05). Real-time PCR analysis demonstrated that 2(-ΔΔCt) value of SV2A mRNA was 0.30 ± 0.43 in the PRE group and 0.76 ± 0.18 in the PSE group (P < 0.05). Western blotting analysis obtained the similar findings (0.27 ± 0.21 versus 1.12 ± 0.21, P < 0.05). PRE rats displayed a significant decrease of SV2A in the brain. SV2A may be associated with the pathogenesis of intractable epilepsy of the amygdaloid-kindling rats.

Synaptic vesicle cycling is not impaired in a glutamatergic and a cholinergic synapse that exhibit deficits in acidification and filling

The purpose of the present work was to investigate synaptic vesicle trafficking when vesicles exhibit alterations in filling and acidification in two different synapses: a cholinergic frog neuromuscular junction and a glutamatergic ribbon-type nerve terminal in the retina. These synapses display remarkable structural and functional differences, and the mechanisms regulating synaptic vesicle cycling might also differ between them. The lipophilic styryl dye FM1-43 was used to monitor vesicle trafficking. Both preparations were exposed to pharmacological agents that collapse ΔpH (NH4Cl and methylamine) or the whole ΔµH+ (bafilomycin), a necessary situation to provide the driving force for neurotransmitter accumulation into synaptic vesicles. The results showed that FM1-43 loading and unloading in neuromuscular junctions did not differ statistically between control and experimental conditions (P > 0.05). Also, FM1-43 labeling in bipolar cell terminals proved highly similar under all conditions tested. Despite remarkable differences in both experimental models, the present findings show that acidification and filling are not required for normal vesicle trafficking in either synapse.

O objetivo do presente trabalho foi investigar o tráfego de vesículas sinápticas quando estas apresentam alterações no armazenamento de neurotransmissores e acidificação em duas distintas sinapses: a junção neuromuscular colinérgica de rãs versus o terminal nervoso glutamatérgico do tipo ribbon em céulas bipolares da retina. Essas sinapses exibem notáveis diferenças estruturais e funcionais e os mecanismos de regulação de ciclo das vesículas sinápticas podem ser diferentes entre eles. Para monitorar o tráfego de vesícula, foi utilizado o marcador lipofílico FM1-43. Ambas as preparações foram expostas a agentes farmacológicos que provocam o colapso de ΔpH (NH4Cl e metilamina) ou de todo ΔµH+ (bafilomicina), gradientes necessários para o acúmulo de neurotransmissores em vesículas sinápticas. Nossos resultados demonstram que a marcação e desmarcação de FM1-43 nas junções neuromusculares não foi estatisticamente diferente entre as diversas condições experimentais (P > 0,05). Além disso, a marcação de FM1-43 em terminais sinápticos de células bipolares foram bastante semelhantes em todas as condições testadas. Apesar das diferenças marcantes em ambos os modelos experimentais, nossos achados demonstram que a acidificação e o preenchimento de vesículas sinápticas não são necessários para o tráfico normal da vesícula nas sinapses estudadas.

Regulating Proteins Participating in Neurotransmitter Release of Synaptic Vesicles at Nerve Terminals / 上海第二医科大学学报

Neurotransmitter release is controlled by groups of proteins associated with the membranes of synaptic vesicles and the presynaptic membranes.It is a highly dynamic process which is spatially and temporally regulated via a cascade of protein-protein interactions.These proteins participate in each step of the synaptic vesicle circulation at nerve terminals including the formation of soluble N-ethylmaleimide-sensitive factor-attachment protein receptors complex,the targeted trafficking of synaptic vesicles,the vesicle docking,the neurotransmitter release and finally the reuse of the proteins.This article focuses on the physiological function and the interactions of these regulating proteins.

The proteins of synaptic vesicle membranes are affected during ageing of rat brain

Low molecular weight GTP-binding proteins are molecular switches that are believed to play pivotal roles in cell growth, differentiation, cytoskeletal organization, and vesicular trafficking. Rab proteins are key players in the regulation of vesicular transport, while Rho family members control actin-dependent cell functions, i.e. the regulation of cytoskeletal organization in response to extracelluar growth factors and in dendritic neuron development. In this study, we have examined the regulation of small GTP-binding proteins that are implicated in neurosecretion and differentiation of neuron during ageing processes. Comparison of small GTP-binding proteins from the synaptosome and crude synaptic vesicles (LP2 membranes) of 2 months and 20 months old rat brain respectively showed no difference in the level of Rab family proteins (Rab3A and Rab5A). However, Rho family proteins such as RhoA and Cdc42 were elevated in LP2 membranes of the aged brain. The dissociation of Rab3A by Ca2+/calmodulin (CaM) from SV membranes was not changed during aging. Ca2+/CaM stimulated phosphorylation of the 22 and 55-kDa proteins in SV membranes from the aged rat brain, and inhibited phosporylation of 30-kDa proteins. GTPgammaS inhibited phosphorylation of the 100-kDa proteins and stimulated phosphorylation of the 70 kDa in LP2 membranes from both the young and aged rat brains, whereas GDPbetaS caused just the opposite reaction. These results suggest that protein phosphorylation and regulation of Rho family GTPases in rat brain appears to be altered during ageing processes.

The proteins of synaptic vesicle membranes are affected during ageing of rat brain

Low molecular weight GTP-binding proteins are molecular switches that are believed to play pivotal roles in cell growth, differentiation, cytoskeletal organization, and vesicular trafficking. Rab proteins are key players in the regulation of vesicular transport, while Rho family members control actin-dependent cell functions, i.e. the regulation of cytoskeletal organization in response to extracelluar growth factors and in dendritic neuron development. In this study, we have examined the regulation of small GTP-binding proteins that are implicated in neurosecretion and differentiation of neuron during ageing processes. Comparison of small GTP-binding proteins from the synaptosome and crude synaptic vesicles (LP2 membranes) of 2 months and 20 months old rat brain respectively showed no difference in the level of Rab family proteins (Rab3A and Rab5A). However, Rho family proteins such as RhoA and Cdc42 were elevated in LP2 membranes of the aged brain. The dissociation of Rab3A by Ca2+/calmodulin (CaM) from SV membranes was not changed during aging. Ca2+/CaM stimulated phosphorylation of the 22 and 55-kDa proteins in SV membranes from the aged rat brain, and inhibited phosporylation of 30-kDa proteins. GTPgammaS inhibited phosphorylation of the 100-kDa proteins and stimulated phosphorylation of the 70 kDa in LP2 membranes from both the young and aged rat brains, whereas GDPbetaS caused just the opposite reaction. These results suggest that protein phosphorylation and regulation of Rho family GTPases in rat brain appears to be altered during ageing processes.

Radiation Effects on the Ultrastructure of Rat Cerebellar Cortex / 대한해부학회지

Severe irradiation on head may result functional alterations of central nervous system. In this study, the irradiation effect on the cerebellar cortex following heavy X-irradiation on head was studied ultrastructurally. Radiation was produced with the linear accelerator ML-4MV[Mitshubishi Co.], and rats weighing about 200gm each were exposed their heads within the radiation areas of 30cm x 30cm, under the radiation distance of 80cm, and with the radiation depth of 1.2 cm. Radiation doses were 3,000rads or 6,000rads, respectively. Animals were sacrificed on 6 hours, 2 days or 6 days following the radiation. Under anesthesia, animals were perfused with 1% glutaraldehyde-1% paraformaldehyde solution. Two hours after the perfusion, brain were taken out and refixed over night in the perfusion fixative. Small blocks of cerebellar hemispheric cortices were refixed 2 hours in 2% osmium tetroxide solution. Fixed tissues were dehydrated in alcohol, embedded in araldite mixture, and cut with ultratome. Ultrathin sections were contrasted with uranyl acetate and lead citrate solutions, and observed with electron microscope. The results obstained were as follow : 1. On 6th hour following X-irradiations, many cerebellar cortical neurons showed increased electron densities, more complicated nuclear infoldings, depletion of synaptic vesicles, expansion of astroglial territories, etc. 2. On 2nd day following X-irradiations, many organelle-rich cells such as Purkinje cells and Golgi cells were darkly degenerated. Numerous myelin figures formed by the cisternal fusions of Golgi apparatus or granular endoplasmic reticula were observed. Cytoplasmic processes of activated astroglial cells were expanded around capillaries and between granule cells. 3. On 6th day following X-irradiations, morphology of neuropil and neurones in the cerebellar cortex was generally restored, except the expanded territories of astroglial cells. From the above results, it was concluded that the release ofneurotransmitters and transcapillary leakage of blood substance were occurred on 6 hours after heavy X-irradiations. And severe alterations were produced on 2 day after X-irradiation, but the condition was generally restored on 6th day following X-irradiation.