The Joubert Syndrome Gene arl13b is Critical for Early Cerebellar Development in Zebrafish / 神经科学通报·英文版

Joubert syndrome is characterized by unique malformation of the cerebellar vermis. More than thirty Joubert syndrome genes have been identified, including ARL13B. However, its role in cerebellar development remains unexplored. We found that knockdown or knockout of arl13b impaired balance and locomotion in zebrafish larvae. Granule cells were selectively reduced in the corpus cerebelli, a structure homologous to the mammalian vermis. Purkinje cell progenitors were also selectively disturbed dorsomedially. The expression of atoh1 and ptf1, proneural genes of granule and Purkinje cells, respectively, were selectively down-regulated along the dorsal midline of the cerebellum. Moreover, wnt1, which is transiently expressed early in cerebellar development, was selectively reduced. Intriguingly, activating Wnt signaling partially rescued the granule cell defects in arl13b mutants. These findings suggested that Arl13b is necessary for the early development of cerebellar granule and Purkinje cells. The arl13b-deficient zebrafish can serve as a model organism for studying Joubert syndrome.

Neuronal Activity in the Cerebellum During the Sleep-Wakefulness Transition in Mice / 神经科学通报·英文版

Cerebellar malfunction can lead to sleep disturbance such as excessive daytime sleepiness, suggesting that the cerebellum may be involved in regulating sleep and/or wakefulness. However, understanding the features of cerebellar regulation in sleep and wakefulness states requires a detailed characterization of neuronal activity within this area. By performing multiple-unit recordings in mice, we showed that Purkinje cells (PCs) in the cerebellar cortex exhibited increased firing activity prior to the transition from sleep to wakefulness. Notably, the increased PC activity resulted from the inputs of low-frequency non-PC units in the cerebellar cortex. Moreover, the increased PC activity was accompanied by decreased activity in neurons of the deep cerebellar nuclei at the non-rapid eye-movement sleep-wakefulness transition. Our results provide in vivo electrophysiological evidence that the cerebellum has the potential to actively regulate the sleep-wakefulness transition.

The Joubert Syndrome Gene arl13b is Critical for Early Cerebellar Development in Zebrafish / 神经科学通报·英文版

Joubert syndrome is characterized by unique malformation of the cerebellar vermis. More than thirty Joubert syndrome genes have been identified, including ARL13B. However, its role in cerebellar development remains unexplored. We found that knockdown or knockout of arl13b impaired balance and locomotion in zebrafish larvae. Granule cells were selectively reduced in the corpus cerebelli, a structure homologous to the mammalian vermis. Purkinje cell progenitors were also selectively disturbed dorsomedially. The expression of atoh1 and ptf1, proneural genes of granule and Purkinje cells, respectively, were selectively down-regulated along the dorsal midline of the cerebellum. Moreover, wnt1, which is transiently expressed early in cerebellar development, was selectively reduced. Intriguingly, activating Wnt signaling partially rescued the granule cell defects in arl13b mutants. These findings suggested that Arl13b is necessary for the early development of cerebellar granule and Purkinje cells. The arl13b-deficient zebrafish can serve as a model organism for studying Joubert syndrome.

Screening and Verification of RNA-seq-based PBCRS for Treating Key Genes of Breast Cancer / 中国实验方剂学杂志

Objective: To study the inhibitory effect of the medicine group of promoting blood circulation and removing stasis (PBCRS) on breast cancer induced by 7, 12-dimethylbenz(a)anthracene (DMBA) in rats, and screen out and verify key genes based on RNA Sequencing (RNA-seq) technology and Ingenuity Pathway Analysis (IPA). Method: Totally 96 Sprague-Dawley (SD) rats were randomly divided into blank group, DMBA model control group, tamoxifen (TAM) group (1.9 mg·kg-1·d-1), high-dose, middle-dose and low-dose PBCRS groups (12.96, 6.48, 3.24 g·kg-1·d-1). One week after drug intervention, except for the blank group, the DMBA was used to induce the rat model of breast cancer (with an interval of a week, irrigation for two times at the dose of 100 mg·kg-1). After 10 weeks, the changes in tumor weight and tumor volume were observed. The total RNA was extracted by total RNA extraction kit, and three RNA samples were collected from the DMBA model control group and the middle-dose PBCRS group for genetic testing. Based on RNA-seq, key differential genes were screened out and verified by Real-time PCR. Result: Comparing with the DMBA model control group, the tumor volume and tumor weight in middle-dose PBCRS group were decreased significantly (PPConclusion: PBCRS may inhibit the occurrence of breast cancer by interfering with the expression of FBP1 in breast cancer tissue.

Neuroprotective effect of melatonin in cerebellums in Alzheimer's disease animal models via MAPK/ERK signaling pathway / 中国药理学通报

Aim: To investigate the effect of melatonin on neuroprotection in cerebellums of rats with Alzheimer' s disease via MAPK/ERK signaling pathway. Methods: Thirty-two male Sprague-Dawley rats were randomly divided into four groups: control group, Aβ1-42 lateral ventricle injection group (AD), and melatonin intraperitoneal injection group (MT), and Aβ1-42 lateral ventricle injection combined with melatonin intraperitoneal injection group (AD + MT). The pathological changes of rat cerebellar cortex were detected by HE staining; the expression of NeuN (neuronal marker), Calbindin(Purkinje cell marker) and p-ERK protein in each group was detected by immunofluorescence; the expression of ERK and p-ERK in each group was determined by Western blot. Results: The HE staining showed that the expression of neurons decreased, followed with the disordered arrangement and morphological alteration of cells in AD. Melatonin could significantly alleviate the pathological damage in cerebellum. Immunofluorescence results showed that compared with AD group, the expression of NeuN (neuronal marker) increased, the number of Purkinje cells marked by Calbindin significantly was up-regulated(P < 0. 01), and the expression of p-ERK was down-regulated in AD + MT group. Western blot showed that the expression of p-ERK was down-regulated by melatonin. Conclusions: Melatonin may exert the neuroprotective effect and relieve the pathological changes by inhibiting the activation of MAPK/ERK signaling pathway.

Effect of intracerebellar microinjection of cdc2-siRNA on the coordination of mice with Niemann-Pick disease type C / 中华行为医学与脑科学杂志

Objective To observe the effect of inhibiting the abnormal activation of cdc2 gene on the coordination of mice with Niemann-Pick disease type C(NPC).Methods Recombinant adeno-associated virus(rAAV) encoding cdc2-siRNA was packaged,and then was injected into the cerebellum of 2 weeks old npc-/-mice.Footprint test and vertical screen test were performed to assess the coordination of mice at the age of 8 weeks.Purkinje cells visualized by HE staining in cerebellum were counted,and the phosphorylation of microtubule-associated protein Tau recognized by PHF-1 antibody was detected by immunoblotting technology.Results (1) Footprint test showed that the stride length in cdc2-siRNA npc-/-group((4.92±0.31)cm) was markedly longer than that in empty vector npc-/-group((4.05 ± 0.19) cm) (P＜ 0.05).(2) Vertical screen test showed that the latency to turn head upwards or reach the upper edge of the screen in cdc2-siRNA npc-/-group((26.01± 1.82) s,(50.93±1.98) s) was significantly shorter than that in the empty vector npc-/-group ((31.96± 3.47) s,(56.89 ± 2.97) s),respectively (P＜ 0.05 for all comparisons).(3) The number of Purkinje cells in cerebellum was dramatically increased in cdc2-siRNA npc-/-group(11.0±2.5) compared with the empty vector npc-/-group (5.1 ± 2.2) (P＜0.05).(4)The relative optical densities of cdc2 and phosphorylated Tau immunoreactive bands in cdc2-siRNA npc-/-group(1.42±0.22,0.95±0.31)were significantly lower than those in the empty vector npc-/-group(2.11±0.29,2.61±0.62),respectively (P＜0.05 for all comparisons).Conclusion Inhibiting the abnormal activation of cdc2 gene can improve the coordination of npc-/-mice by ameliorating Purkinje cell's loss and reducing the hyperphosphorylation of Tau in cerebellum.

Effect of gestational diabetes on neuronal cells in rat cerebellum in early postnatal life / Efecto de la diabetes gestacional sobre las células neuronales del cerebelo de ratas en el período postnatal

Previous study has shown the adverse effects of gestational diabetes on hippocampal neuronal density in animal model. This study was conducted to determine the effect of gestational diabetes on rat cerebellum in early postnatal life. In this experimental study, 10 dams randomly allocated into control and diabetic groups on day 1 of gestation. Five dams in diabetic group were administered 40 mg/kg/BW (intraperitoneally) of streptozotocin and control animals received normal saline. Six offspring of each gestational diabetes mellitus and controls were randomly selected at day 7 of postnatal life. Offspring were sacrificed and coronal sections were taken from the cerebellum and stained with cresyl violet. The number of Purkinje and granular cells and thickness of layers of cerebellum were evaluated by quantitative computer-assisted morphometric method. The Purkinje cells density at apex and depth of cerebellar lobules in the experimental group (14.40±0.7, 14.86±0.6) significantly reduced in comparison with the control group (16.72±0.3, 17.85±0.7) (P<0.05). The granular cell density at apex and depth of cerebellar lobules in the experimental group (23.94±0.6, 22.81±0.5) significantly reduced in comparison with the control group (29.20±0.8, 28.1±0.8) (P<0.05). The thickness of the Purkinje and internal granular and molecular layers at apex and depth of cerebellar cortex significantly reduced in diabetics group compared to controls (P<0.05). This study revealed that gestational diabetes induces loss of number and size of the Purkinje cells and the granular cells and reduction of thickness of the Purkinje and internal granular layer of the cerebellar cortex in neonatal mice.

Estudios previos han demostrado los efectos adversos de la diabetes gestacional sobre la densidad neuronal del hipocampo en modelos animales. Este estudio se realizó para determinar el efecto de la diabetes gestacional en el cerebelo de ratas durante la edad temprana postnatal. Fueron asignadas 10 ratas hembras al azar en grupos control y diabético en el primer día de gestación. Cinco en el grupo diabético recibieron una dosis de 40 mg/kg/Peso corporal de estreptozotocina (intraperitoneal) y los control una solución salina normal. Seis crías de cada una de las hembras del grupo diabetes mellitus gestacional y del grupo controles fueron seleccionados al azar el día 7 de vida postnatal. Fueron sacrificadas y se obtuvieron secciones coronales desde el cerebelo que fueron teñidas con violeta de cresilo. El número de células granulares de Purkinje y espesor de las capas de cerebelo fueron evaluadas por método morfométrico y ordenador cuantitativo. La densidad de células de Purkinje en el ápice y profundidad de los lóbulos del cerebelo en el grupo experimental (14,40±0,7 y 14,86±0,6) se redujeron significativamente en comparación con el grupo control (16,72±0,3 y 17,85±0,7) (P<0,05). La densidad de células granulares en el ápice y profundidad de los lóbulos del cerebelo en el grupo experimental (23,94±0,6 y 22,81±0,5) se redujo significativamente en comparación con el grupo control (29,20±0,8 y 28,1±0,8) (P<0,05). En el espesor de células Purkinje, las capas moleculares y granulares internas en el ápice y profundidad de la corteza del cerebelo, se observó una reducción significativa en el grupo diabéticos en comparación con los controles (P<0,05). Se observó que la diabetes gestacional induce la pérdida del número y tamaño de las células Purkinje y de células granulares, así como la reducción del espesor de las capas de Purkinje y granular interna de la corteza del cerebelo en ratones neonatos.

Characterization of Membrane Excitability of Purkinje Cells in Vestibulocerebellum Using Patch Clamp Techniques / 대한이비인후과학회지

Cerebellum is known as a center for sensory/motor coordination and memory storage in motor learning. The vestibular nuclei have extensive afferent and efferent connections with posterior cerebellum which can be referred to as vestibulocerebellum. While secondary vestibular afferents are distributed bilaterally in the vestibulocerebellum, primary afferents may directly project to ipsilateral vestibulocerebellum. The Purkinje cells which are the only output neurons from the cerebellar cortex receive vestibular information via parallel and climbing fibers. That information is integrated and encoded in the Purkinje cells and then conveyed into the vestibular nucleus or deep cerebellar nucleus, which permits adaptive guidance of vestibular function by the vestibulocerebellum.

Diferenciação de células-tronco embrionárias murinas (mESCs) em células produtoras de insulina (IPCs) e caracterização funcional do gene Purkinje cell protein 4 (Pcp4) neste processo / Differentiation of murine embryonic stem cells (mESCs) into insulin-producing cells (IPCs) and functional characterization of the Purkinje Cell Protein 4 (Pcp4) gene in this process

Fontes alternativas de células ß têm sido estudadas para o tratamento de Diabetes mellitus tipo 1, dentre as quais a mais promissora consiste das células-tronco diferenciadas em células produtoras de insulina (IPCs). Alguns trabalhos demonstram a capacidade de células-tronco embrionárias murinas (mESCs) de formarem estruturas semelhantes a ilhotas pancreáticas, porém, os níveis de produção de insulina são insuficientes para a reversão do diabetes em camundongos diabetizados. Este trabalho visa desenvolver um protocolo adequado para geração de IPCs e contribuir para a identificação e caracterização funcional de novos genes associados à organogênese pancreática. Logo no início da diferenciação das mESCs em IPCs, foi possível verificar o surgimento de células progenitoras, evidenciado pela expressão de marcadores importantes da diferenciação beta-pancreática. Ao final do processo de diferenciação in vitro, ocorreu a formação de agrupamentos (clusters) semelhantes a ilhotas, corando positivamente por ditizona, que é específica para células ß-pancreáticas. Para avaliar seu potencial in vivo, estes clusters foram microencapsulados em Biodritina® e transplantados em camundongos diabetizados. Apesar dos níveis de insulina produzidos não serem suficientes para estabelecer a normoglicemia, os animais tratados com IPCs apresentaram melhores condições, quando comparados ao grupo controle, tendo melhor controle glicêmico, ganho de massa corpórea e melhor aparência da pelagem, na ausência de apatia. Além disso, análise dos clusters transplantados nestes animais indicou aumento da expressão de genes relacionados à maturação das células ß. Porém, quando estes clusters foram microencapsuladas em Bioprotect® e submetidos à maturação in vivo em animais normais, ocorreu um aumento drástico na expressão de todos os genes analisados, indicando sua maturação completa em células beta. O transplante destas células completamente maturadas em animais diabetizados, tornou-os normoglicêmicos e capazes de responder ao teste de tolerância à glicose (OGTT) de forma semelhante aos animais normais. A segunda parte do trabalho visou analisar genes diferencialmente expressos identificados em estudo anterior do nosso grupo, comparando, através de DNA microarray, mESCs indiferenciadas e diferenciadas em IPCs. Um dos genes diferencialmente expressos é aquele que codifica para a Purkinge cell protein 4 (Pcp4), sendo 3.700 vezes mais expresso em IPCs. Para investigar o possível papel do gene Pcp4 em células ß e no processo de diferenciação ß-pancreática, adotou-se o enfoque de genômica funcional, superexpressando e inibindo sua expressão em células MIN-6 e mESCs. Apesar da alteração na expressão de Pcp4 em células MIN-6 não ter interferido de forma expressiva na expressão dos genes analisados, quando inibido, modificou o perfil da curva de crescimento celular, aumentando seu tempo de dobramento de forma significativa e diminuindo da viabilidade celular em ensaios de indução de apoptose. Já na diferenciação de mESCs em IPCs, a superexpressão de Pcp4 interferiu de forma positiva apresentando uma tendência a aumentar a expressão dos genes relacionado à diferenciaçãoß-pancreática. Concluindo, desenvolvemos um novo protocolo de diferenciação de mESCs em IPCs as quais foram capazes de reverter o diabetes em camundongos diabetizados e descrevemos, pela primeira vez, o gene Pcp4 como sendo expresso em células ß-pancreáticas, podendo estar relacionado à manutenção da viabilidade celular e maturação destas células

New cellular sources for type 1 Diabetes mellitus treatment have been previously investigated, the most promising of which seems to be the insulin producing cells (IPCs), obtained by stem cells differentiation. Some reports show that murine embryonic stem cells (mESCs) are able to form islet-like structures, however, their insulin production is insufficient to render diabetic mice normoglycemic. This work aims at developing an adequate protocol for generation of IPCs and searching for new genes which could be involved in the pancreatic organogenesis process. Early on during mESCs differentiation into IPCs, we observed the presence of progenitor cells, which were able to express pancreatic ß-cell markers. At the end of the differentiation process, the islet-like clusters positively stained for the insulin-specific dithizone. These clusters were microencapsulated in Biodritin® microcapsules, and then transplanted into diabetized mice. Although the levels of insulin production were insufficient for the animals to achieve normoglycemia, those which received IPCs displayed improved conditions, when compared to the control group, as judged by a better glycemic control, body weight gain and healthy fur appearance, in the absence of apathy. In addition, when these transplantated clusters were retrieved, high levels of expression of the genes related to ß-cell maturation were detected. IPCs were also microencapsulated in Bioprotect® and subjected to in vivo maturation in normal animals. A dramatic increase of the analyzed genes expression was observed, indicating complete maturation of the differentiated cells. When these cells were transplanted into diabetized mice, these animals achieved normoglycemia and were able to display glucose tolerance test (OGTT) response very similar to that of normal mice. In the second part of this work, we analyzed upregulated genes described in previous work from our group, comparing undifferentiated mESCs to IPCs using a microarray platform. One of these genes is that coding for the Purkinje cell protein 4 (Pcp4), which is 3,700 more expressed than in undifferentiated mESC cells. We adopted a functional genomics approach to investigate the role played by the Pcp4 gene in ß-cells and in ß-cell differentiation, by inducing overexpression and knocking down this gene in MIN-6 and mESC cells. Although the differential expression of Pcp4 in MIN-6 was not able to interfere with the expression of the genes analyzed, we observed different cell growth rates, with increased doubling time and decreased cell viability when its expression was knocked down. In addition, overexpression of Pcp4 in mESCs subjected to differentiation into IPCs apparently increases the expression of genes related to ß-cell differentiation. In conclusion, we developed a new protocol for ESCs differentiation into IPCs, which is able to revert diabetes in diabetized mice, and we also describe here, for the first time, the Pcp4 gene as being expressed in pancreatic ß-cells and possibly being related to maintenance of cell viability and ß-cell maturation

TRPC-Mediated Current Is Not Involved in Endocannabinoid-Induced Short-Term Depression in Cerebellum

It has been reported that activation of metabotropic glutamate receptor 1 (mGluR1) can mediate endocannabinoid-induced short-term depression of synaptic transmission in cerebellar parallel fiber (PF)-Purkinje cell (PC) synapse. mGluR1 has signaling pathways involved in intracellular calcium increase which may contribute to endocannabinoid release. Two major mGluR1-evoked calcium signaling pathways are known: (1) slow-kinetic inward current carried by transient receptor potential canonical (TRPC) channel which is permeable to Ca2+; (2) IP3-induced calcium release from intracellular calcium store. However, it is unclear how much each calcium source contributes to endocannabinoid signaling. Here, we investigated whether calcium influx through mGluR1-evoked TRPC channel contributes to endocannabinoid signaling in cerebellar Purkinje cells. At first, we applied SKF96365 to inhibit TRPC, which blocked endocannabinoid-induced short-term depression completely. However, an alternative TRP channel inhibitor, BTP2 did not affect endocannabinoid-induced short-term depression although it blocked mGluR1-evoked TRPC currents. Endocannabinoid signaling occurred normally even though the TRPC current was mostly blocked by BTP2. Our data imply that TRPC current does not play an important role in endocannabinoid signaling. We also suggest precaution in applying SKF96365 to inhibit TRP channels and propose BTP2 as an alternative TRPC inhibitor.

Numbers of Purkinje Cell with Increasing Age-A Post Mortem Study.

The change in the number of Purkinje cells with increasing age is evident especially in disorders of fine movement, equilibrium, hypotonia, postural changes, and disturbances of voluntary movement. The present study was done to see the changes in the number of Purkinje cells per square mm in different age groups of Bangladeshi people. This cross sectional descriptive type of study was designed and done in the Department of Anatomy, Dhaka Medical College, Dhaka, from January to December 2010, which was performed on the cerebellum of 28 Bangladeshi people, collected during autopsy examination of unclaimed dead bodies from Department of Forensic Medicine. Paraffin blocks of cerebellum were cut at 5mm thickness and stained with routine Harris' Haematoxylin and Eosin (H & E) stain. Estimation of number of Purkinje cell was done by using the counting circle and examined under the light microscope. The mean ± SD of number of Purkinje cell was 160.71 ± 24.47 in group A (Age 20-29 years) and 152.20 ± 6.49 in group D (age> 50 years), the mean reduction was 2.5% per decade. Histological studies revealed the number of Purkinje cell per square mm decreased with age which was statistically significant and further cytological study of Purkinje cell with larger sample size is recommended.

Effects of Fragile Mental Retardation Protein on Cerebellar Neuron Development and Migration in Mouse Model / 中国医科大学学报

Objective To investigate the effects of fragile X mental retardation protein（FMRP）on the development and migration of cerebellar neurons in mouse model.Methods Plasmids containing FMRPmutant-EGFP or EGFP were established and transfected into the lateral ventricle of the embryo mouse.Fragile X syndrome（FXS）genotype of the mouse model was identified.Nissl staining and immunofluorescence staining were conducted to assess the changes in neuron development and migration.Results In the experimental group,Nissl staining showed that the deep cerebellar neuclei contracted and divided by white matter,and the non-polarized Purkinje cells retained in internal granular layer;while immunofluorescence staining showed that Tbr2-positive unipolar brush cells changed the migration pathway and accumulated in the ventricular zone.Conclusion Cerebellar neurons showed abnormal formation and migration with the absence of FMRP.

Differential regulation of Purkinje cell dendritic spines in rolling mouse Nagoya (tg(rol)/tg(rol)), P/Q type calcium channel (alpha1(A)/Ca(v)2.1) mutant / 대한해부학회지

Voltage dependent calcium channels (VDCC) participate in regulation of neuronal Ca2+. The Rolling mouse Nagoya (Cacna1a(tg-rol) ) is a spontaneous P/Q type VDCC mutant, which has been suggested as an animal model for some human neurological diseases such as autosomal dominant cerebellar ataxia (SCA6), familial hemiplegic migraine and episodic ataxia type-2. Morphology of Purkinje cell (PC) dendritic spine is suggested to be regulated by signal molecules such as Ca2+ and by interactions with afferent inputs. The amplitude of excitatory postsynaptic current was decreased in parallel fiber (PF) to PC synapses, whereas apparently increased in climbing fiber (CF) to PC synapses in rolling mice Nagoya. We have studied synaptic morphology changes in cerebella of this mutant strain. We previously found altered synapses between PF varicosity and PC dendritic spines. To study dendritic spine plasticity of PC in the condition of insufficient P/Q type VDCC function, we used high voltage electron microscopy (HVEM). We measured the density and length of PC dendritic spines at tertiary braches. We observed statistically a significant decrease in spine density as well as shorter spine length in rolling mice compared to wild type mice at tertiary dendritic braches. In proximal PC dendrites, however, there were more numerous dendritic spines in rolling mice Nagoya. The differential regulation of rolling PC spines at tertiary and proximal dendrites in rolling mice Nagoya suggests that two major excitatory afferent systems may be regulated reciprocally in the cerebellum of rolling mouse Nagoya.

Optimized Immunohistochemical Analysis of Cerebellar Purkinje Cells Using a Specific Biomarker, Calbindin D28k

Cerebellar Purkinje cells (PCs) play a crucial role in motor functions and their progressive degeneration is closely associated with spinocerebellar ataxias. Although immunohistochemical (IHC) analysis can provide a valuable tool for understanding the pathophysiology of PC disorders, the method validation of IHC analysis with cerebellar tissue specimens is unclear. Here we present an optimized and validated IHC method using antibodies to calbindin D28k, a specific PC marker in the cerebellum. To achieve the desired sensitivity, specificity, and reproducibility, we modified IHC analysis procedures for cerebellar tissues. We found that the sensitivity of staining varies depending on the commercial source of primary antibody. In addition, we showed that a biotin-free signal amplification method using a horseradish peroxidase polymer-conjugated secondary antibody increases both the sensitivity and specificity of ICH analysis. Furthermore, we demonstrated that dye filtration using a 0.22 micrometer filter eliminates or minimizes nonspecific staining while preserving the analytical sensitivity. These results suggest that our protocol can be adapted for future investigations aiming to understand the pathophysiology of cerebellar PC disorders and to evaluate the efficacy of therapeutic strategies for treating these diseases.

Expression of HSP27 and alphaB-crystallin in Avian Cerebellum during Development / 대한해부학회지

It is well known that small heat shock proteins play a role as molecular chaperone. However, during normal development of the cerebellum, expression and distribution of HSP27 and alphaB-crystallin (alphaBC) which are small heat shock proteins have not been reported. To verify the protective role of HSP27 and alphaBC in neurons and glial cells, we examined the expression and distribution of HSP27 and alphaBC in the developing chick cerebellum using immunoblot, immunohistochemical and double immunofluorescence staining. Expression of both HSP27 and alphaBC was first identified in the cerebellum of the embryonic day 14 (E14) embryo, and was increased at E18. Double immunofluorescence analysis with myelin-basic protein (MBP) demonstrated that alphaBC positive (+) cells were mature myelinating oligodendrocytes. alphaBC+ cells were observed in the white matter of the E14 cerebellum. At E18, there were a number of alphaBC+ cells in the white matter and a few cells in the granular layer of the gray matter. On the other hand, HSP27+ cells were observed in the white matter and the Purkinje cell layer at E14. At E18, HSP27+ signals were observed in Purkinje cells and neurons of cerebellar nucleus as well as oligodendrocytes in the white matter and the granular layer. The results that HSP27 and alphaBC were expressed in specific neurons and glial cells in the developing cerebellum suggest that HSP27 and alphaBC may be involved in the protective mechanism for the apoptosis of neurons and the physiological stress occurred in oligodendrocyts during cell maturation.

Neurodegeneration-related Aminoacyl-tRNA Synthetases / 生物化学与生物物理进展

Aminoacyl-tRNA synthetases (AARSs) catalyze aminoacylation of their tRNAs for protein biosynthesis. As belong to one of the most ancient and conserved enzyme family their additional functions in mammalian cells were focused recently. Mutations in tyrosyl-tRNA synthetase, glycyl-tRNA synthetase and alanyl-tRNA synthetase from patients and mice models were identified to cause two subtypes of Charcot-Marie-Tooth disease and cerebellar Purkinje cell loss, respectively. These mutations affect different functions of the three enzymes including aminoacylation, editing and unknown functions. These results combined AARSs with neurodegeneration and gave new sights into neuropathy.

Postnatal Developmental Expression of TEM7 mRNA and its Putative Ligand in the Rat Cerebellum / 대한해부학회지

Tumor endothelial marker 7 (TEM7) is a putative transmembrane protein that is highly expressed in the tumor endothelium and cerebellar neurons. In the present study, the expression profile of TEM7 mRNA and its putative ligand in the developing cerebellum of the rat was investigated using in situ hybridization and ligand binding assay. The secreted recombinant ectodomain of TEM7 was employed to label the expression of putative ligand of TEM7 in the cerebellum. The expression of a putative ligand of TEM7 demonstrated by using TEM7 ectodomain was found in the molecular layer of the cerebellum, where the dendritic trees of Purkinje cells are present. A developmental study has shown that TEM7 mRNA expression in the Purkinje neurons was increased with age during postnatal development, whereas the putative ligand labeling in the molecular layer was observed throughout the developmental period. These findings indicate that TEM7-ligand interaction plays a role in the differentiation of Purkinje cells during postnatal development.

A Case of Paraneoplastic Limbic Encephalitis presenting Anti-Purkinje Cell Antibody

No abstract available.

Effect of Maternal Thyroxine Treatment on the Postnatal Development of Brain-derived Neurotrophic Factor-containing Purkinje Cell in Fetal Alcohol Effects in the Rat Cerebellum / 체질인류학회지

Maternal alcohol abuse is thought to be the common cause of mental retardation. Even moderate maternal alcohol consumption may produce fetal alcohol effects with behavioral and learning difficulties, if the drinking is associated with malnutrition. Especially, continuous alcohol consumption during critical period of brain development is very likely to produce fetal alcohol effects. The aims of this study are to investigate whether exogenous thyroxine treatment to alcohol -fed dams may ameliorate the detrimental effects of alcohol on the postnatal development of BDNF -containing Purkinje cell of the cerebellar cortex of the offspring. The morphological features of the growth and maturation were observed at 0, 7, 14, 21, 28 postnatal days via immunohistochemistry. In addition, electron microscopic finding of BDNF -containing Purkinje cell at P14 was also examined. Time -pregnant rats were divided into three groups. Alcohol -fed group received 35 calories of liquid alcohol diet daily from gestation day 6; control pair -fed group was fed a liquid diet in which dextrin replaced alcohol isocalorically; alcohol +/-T4 group received 35 calories liquid alcohol diet and exogenous thyroxine subcutaneously. As a result, a similar developmental pattern of BDNF -immunoreactive Purkinje cells was observed in control pair - fed and alcohol+/-T4 group on and after P14. These cells of alcohol -fed group showed immature features. Single -layer arrangement of these cells in alcohol -fed group was not completely achieved throughout postnatal life. Electron microscopic observations of BDNF -immunoreactive Purkinje cells at P14 revealed large nucleus, small cytoplasm, small amount of ribosomal collection and rudimentary cytoplasmic organelles in alcohol -fed group. The morphology of BDNF -immunoreactive Purkinje cell in alcohol +/-T4 group was similar to that in control pair -fed group. It was characterized by numerous short segments of rough endoplasmic reticulum, many of which showed a tendency of parallel alignment that suggested an attempt at Nissl body configuration. The cytology of Golgi complexes was also found within the cytoplasm in perinuclear location. Those observed differences of postnatal maturation patterns between alcohol -fed and alcohol +/-T4 group may indicate the beneficial effects on the postnatal development of BDNF -containing Purkinje cells in cerebellar cortex in the pups of thyroxine -treated alcohol -exposed dams. These results suggest that the increase of BDNF synthesis during early postnatal life caused by maternal administration of exogenous thyroxine may ameliorate fetal alcohol effects as a result of the dysthyroid state following maternal alcohol abuse.

Abnormal Expression of Neuropeptide Y in the Cerebellar Purkinje Cells of the Ataxic Mutant Mice, Pogo / 체질인류학회지

The pogo mouse is an autosomal recessive ataxic mutant that arose spontaneously in the inbred KJR/MsKist strain derived originally from Korean wild mice. The ataxic phenotype is characterized by difficulty in maintaining posture and the consequent inability to walk straight. In our previous study about pogo mice cerebellum, we reported the Purkinje cell abnormalities and ectopic expression of tyrosine hydroxylase (TH) in Purkinje cell. In this study, we have provided an abnormal expression of NPY in ataxic mutant pogo mice for the first time. There was increased immunoreactivity for NPY in Purkinje cell of ataxic pogo (pogo/pogo) mice compared to those of heterozygote non-ataxic pogo mice (pogo/+, control group). In our previous study, TH is also expressed abnormally in Purkinje cells of ataxic mutant pogo (pogo/pogo) mouse cerebellum. To compare the expression patterns of TH and NPY within some Purkinje cell using double immunofluorescence, most of NPY-immunoreactive Purkinje cells in the ataxic pogo mice are TH-immunoreactive Purkinje cells. However, all of TH-immunoreactive Purkinje cells are not express the NPY. These data reveal that abnormal NPY-immunoreactivity in the ataxic pogo (pogo/pogo) cerebellum is restricted to a subset of cells within the ectopic TH-immunoreactive Purkinje cell subset. These results further suggest that Purkinje cell abnormalities contribute to motor ataxia in the ataxic pogo mouse.