Molecular mechanisms underlying the promotion of wound repair by coenzyme Q10: PI3K/Akt signal activation via alterations to cell membrane domains.

Coenzyme Q10 (CoQ10) promotes wound healing in vitro and in vivo. However, the molecular mechanisms underlying the promoting effects of CoQ10 on wound repair remain unknown. In the present study, we investigated the molecular mechanisms through which CoQ10 induces wound repair using a cellular wound-healing model. CoQ10 promoted wound closure in a dose-dependent manner and wound-mediated cell polarization after wounding in HaCaT cells. A comparison with other CoQ homologs, benzoquinone derivatives, and polyisoprenyl compounds suggested that the whole structure of CoQ10 is required for potent wound repair. The phosphorylation of Akt after wounding and the plasma membrane translocation of Akt were elevated in CoQ10-treated cells. The promoting effect of CoQ10 on wound repair was abrogated by co-treatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor. Immuno-histochemical and biochemical analyses showed that CoQ10 increased the localization of caveolin-1 (Cav-1) to the apical membrane domains of the cells and the Cav-1 content in the membrane-rich fractions. Depletion of Cav-1 suppressed CoQ10-mediated wound repair and PI3K/Akt signaling activation in HaCaT cells. These results indicated that CoQ10 increases the translocation of Cav-1 to the plasma membranes, activating the downstream PI3K/Akt signaling pathway, and resulting in wound closure in HaCaT cells.

Allele-specific regulation of mutant Huntingtin by Wig1, a downstream target of p53.

p53 has been implicated in the pathophysiology of Huntington's disease (HD). Nonetheless, the molecular mechanism of how p53 may play a unique role in the pathology remains elusive. To address this question at the molecular and cellular biology levels, we initially screened differentially expressed molecules specifically dependent on p53 in a HD animal model. Among the candidate molecules, wild-type p53-induced gene 1 (Wig1) is markedly upregulated in the cerebral cortex of HD patients. Wig1 preferentially upregulates the level of mutant Huntingtin (Htt) compared with wild-type Htt. This allele-specific characteristic of Wig1 is likely to be explained by higher affinity binding to mutant Htt transcripts than normal counterpart for the stabilization. Knockdown of Wig1 level significantly ameliorates mutant Htt-elicited cytotoxicity and aggregate formation. Together, we propose that Wig1, a key p53 downstream molecule in HD condition, play an important role in stabilizing mutant Htt mRNA and thereby accelerating HD pathology in the mHtt-p53-Wig1 positive feedback manner.

Nicotine enhances the malignant potential of human pancreatic cancer cells via activation of atypical protein kinase C.

BACKGROUND: Pancreatic cancer (PC) is the most lethal malignancy among solid tumors, and the most common risk factor for its development is cigarette smoking. Atypical protein kinase C (aPKC) isozymes function in cell polarity, proliferation, and survival, and have also been implicated in carcinogenesis. However, the involvement of aPKC in PC progression and the effect of nicotine, a major component of cigarette smoke, on the biological activities of aPKC remain to be fully elucidated. METHODS: We investigated the effects of nicotine on the proliferation, migration and invasion of the human PC cell lines Panc1 and BxPC3. We analyzed aPKC localization and activity by immunohistochemistry and in vitro kinase assays, respectively, to assess their involvement in the regulation of PC progression. Moreover, we examined the effect of nicotine on implanted peritoneal tumors of PC cells in mice. RESULTS: Nicotine enhanced cell proliferation, migration and invasion in Panc1 and BxPC3 cells. In nicotine-treated PC cells, the aPKC was significantly activated. We also found that nicotine induced phosphatidylinositol 3-kinase (PI3K) signal activation, and a specific inhibitor of the nicotine acetylcholine receptor (nAChR) as well as knockdown of nAChR prevented nicotine-mediated Akt phosphorylation and aPKC activation. In a peritoneal dissemination model of PC, nicotine-treated mice had larger tumors and increased numbers of nodules. Immunohistochemistry showed enhanced expression levels of aPKC and phosphorylated Akt in nodules from nicotine-treated mice. CONCLUSIONS AND GENERAL SIGNIFICANCE: Nicotine induces aberrant activation of aPKC via nAChR/PI3K signaling in PC cells, resulting in enhancement of cellular proliferation, migration and invasion.

Depletion of p62 reduces nuclear inclusions and paradoxically ameliorates disease phenotypes in Huntington's model mice.

Huntington's disease (HD) is a dominantly inherited genetic disease caused by mutant huntingtin (htt) protein with expanded polyglutamine (polyQ) tracts. A neuropathological hallmark of HD is the presence of neuronal inclusions of mutant htt. p62 is an important regulatory protein in selective autophagy, a process by which aggregated proteins are degraded, and it is associated with several neurodegenerative disorders including HD. Here, we investigated the effect of p62 depletion in three HD model mice: R6/2, HD190QG and HD120QG mice. We found that loss of p62 in these models led to longer life spans and reduced nuclear inclusions, although cytoplasmic inclusions increased with polyQ length. In mouse embryonic fibroblasts (MEFs) with or without p62, mutant htt with a nuclear localization signal (NLS) showed no difference in nuclear inclusion between the two MEF types. In the case of mutant htt without NLS, however, p62 depletion increased cytoplasmic inclusions. Furthermore, to examine the effect of impaired autophagy in HD model mice, we crossed R6/2 mice with Atg5 conditional knockout mice. These mice also showed decreased nuclear inclusions and increased cytoplasmic inclusions, similar to HD mice lacking p62. These data suggest that the genetic ablation of p62 in HD model mice enhances cytoplasmic inclusion formation by interrupting autophagic clearance of polyQ inclusions. This reduces polyQ nuclear influx and paradoxically ameliorates disease phenotypes by decreasing toxic nuclear inclusions.

The estrogen receptor ß-PI3K/Akt pathway mediates the cytoprotective effects of tocotrienol in a cellular Parkinson's disease model.

Tocotrienols (T3s) are members of the vitamin E family, have antioxidant properties, and are promising candidates for neuroprotection in the pathogenesis of neurodegenerative disorders such as Parkinson's disease (PD). However, whether their antioxidant capacities are required for their cytoprotective activity remains unclear. In this regard, the antioxidant-independent cytoprotective activity of T3s has received considerable attention. Here, we investigated the signaling pathways that are induced during T3-dependent cytoprotection of human neuroblastoma SH-SY5Y cells, as these cells are used to model certain elements of PD. T3s were cytoprotective against 1-methyl-4-phenylpyridinium ion (MPP(+)) and other PD-related toxicities. γT3 and δT3 treatments led to marked activation of the PI3K/Akt signaling pathway. Furthermore, we identified estrogen receptor (ER) ß as an upstream mediator of PI3K/Akt signaling following γT3/δT3 stimulation. Highly purified γT3/δT3 bound to ERß directly in vitro, and knockdown of ERß in SH-SY5Y cells abrogated both γT3/δT3-dependent cytoprotection and Akt phosphorylation. Since membrane-bound ERß was important for the signal-related cytoprotective effects of γT3/δT3, we investigated receptor-mediated caveola formation as a candidate for the early events of signal transduction. Knockdown of caveolin-1 and/or caveolin-2 prevented the cytoprotective effects of γT3/δT3, but did not affect Akt phosphorylation. This finding suggests that T3s and, in particular, γT3/δT3, exhibit not only antioxidant effects but also a receptor signal-mediated protective action following ERß/PI3K/Akt signaling. Furthermore, receptor-mediated caveola formation is an important event during the early steps following T3 treatment.

Deficiency of p62/Sequestosome 1 causes hyperphagia due to leptin resistance in the brain.

The cytoplasmic regulatory protein p62 (Sequestosome 1/A170) is known to modulate various receptor-mediated intracellular signaling pathways. p62 deficiency was shown to result in mature-onset obesity in mice, but the mechanisms underlying this abnormality remained unclear. Here we report that hyperphagia due to central leptin resistance is the cause of obesity in p62(-/-) mice. We found that these mice show hyperphagia. Restriction of food to the amount eaten by wild-type mice prevented excess body weight gain and fat accumulation, suggesting that overfeeding is the primary cause of obesity in p62(-/-) mice. Brain-specific p62 deficiency caused mature-onset obesity to the same extent as in p62(-/-) mice, further supporting a neuronal mechanism as the major cause of obesity in these mice. Immunohistochemical analysis revealed that p62 is highly expressed in hypothalamic neurons, including POMC neurons in the arcuate nucleus. Central leptin resistance was observed even in young preobese p62(-/-) mice. We found a defect in intracellular distribution of the transcription factor Stat3, which is essential for the action of leptin, in p62(-/-) mice. These results indicate that brain p62 plays an important role in bodyweight control by modulating the central leptin-signaling pathway and that lack of p62 in the brain causes leptin resistance, leading to hyperphagia. Thus, p62 could be a clinical target for treating obesity and metabolic syndrome.

Roles of Microglia in Neurodegenerative Diseases.

In recent years, microglia have attracted attention owing to their roles in various neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Microglia, which are brain-resident macrophages, not only act as immune cells but also perform other functions in the body. Interestingly, they exert contrasting effects on different neurodegenerative diseases. In addition to the previously reported M1 (toxic) and M2 (protective) types, microglia now also include disease-associated microglia owing to a more elaborate classification. Understanding this detailed classification is necessary to elucidate the association between microglia and neurodegenerative diseases. In this review, we discuss the diverse roles of microglia in neurodegenerative diseases and highlight their potential as therapeutic targets.

Parkin degrades estrogen-related receptors to limit the expression of monoamine oxidases.

Parkin, whose mutations cause Parkinson disease (PD), controls oxidative stress by limiting the expression of monoamine oxidases (MAO)--mitochondrial enzymes responsible for the oxidative de-amination of dopamine. Here, we show that parkin performed this function by increasing the ubiquitination and degradation of estrogen-related receptors (ERR), orphan nuclear receptors that play critical roles in the transcription regulation of many nuclear-encoded mitochondrial proteins. All three ERRs (α, ß and γ) increased the transcription of MAOs A and B; the effects were abolished by parkin, but not by its PD-linked mutants. Parkin bound to ERRs and increased their ubiquitination and degradation. In fibroblasts from PD patients with parkin mutations or brain slices from parkin knockout mice, degradation of ERRs was significantly attenuated. The results reveal the molecular mechanism by which parkin suppresses the transcription of MAOs to control oxidative stress induced by dopamine oxidation.

TYRO3 promotes chemoresistance via increased LC3 expression in pancreatic cancer.

Pancreatic cancer (PC) is an aggressive malignancy with few treatment options, and improved treatment strategies are urgently required. TYRO3, a member of the TAM receptor tyrosine kinase family, is a known oncogene; however, the relationship between TYRO3 expression and PC chemoresistance remains to be elucidated. We performed gain- and loss-of-function experiments on TYRO3 to examine whether it is involved in chemoresistance in PC cells. TYRO3 knockdown decreased cell viability and enhanced apoptosis following treatment of PC cells with gemcitabine and 5-fluorouracil (5-FU). In contrast, no such effects were observed in TYRO3-overexpressing PC cells. It is known that autophagy is associated with cancer chemoresistance. We then examined effects of TYRO3 on autophagy in PC cells. TYRO3 overexpression increased LC3 mRNA levels and induced LC3 puncta in PC cells. Inhibition of autophagy by chloroquine mitigated cell resistance to gemcitabine and 5-FU. In a xenograft mouse model, TYRO3 silencing significantly increased sensitivity of the cells to gemcitabine and 5-FU. To further investigate the involvement of autophagy in patients with PC, we immunohistochemically analyzed LC3 expression in the tissues of patients who underwent pancreatectomy and compared it with disease prognosis and TYRO3 expression. LC3 expression was negatively and positively correlated with prognosis and TYRO3 expression, respectively. Furthermore, LC3- and TYRO3-positive patients had a significantly worse prognosis among patients with PC who received chemotherapy after recurrence. These results indicated that the TYRO3-autophagy signaling pathway confers PC resistance to gemcitabine and 5-FU, and could be a novel therapeutic target to resolve PC chemoresistance.

High Glucose-stimulated aPKC Activation Promotes Pancreatic Cancer Cell Progression Through YAP Signaling.

BACKGROUND/AIM: Persistent hyperglycemia caused by diabetes mellitus is a risk factor for pancreatic cancer (PC). We have previously reported that aberrant activation of atypical protein kinase C (aPKC) enhances PC cell progression. However, no reports have elucidated whether hyperglycemia promotes PC cell progression and whether aPKC activation is related to PC cell progression mechanisms. MATERIALS AND METHODS: We examined whether high-glucose stimulation accelerates PC cell proliferation, migration, and invasion. Furthermore, to determine whether PC cells activate aPKC upon high-glucose stimulation, we measured the phosphorylation of aPKC at T560 in PC cells. RESULTS: High-glucose stimulation accelerated PC cell proliferation, migration, and invasion. High-glucose treatment increased aPKC's activated form, with T560 phosphorylation, in PC cells. However, aPKC knockdown attenuated these effects. aPKC reportedly induces cell transformation through Yes-associated protein (YAP) activation. YAP expression was increased in high glucose-treated PC cells but not in aPKC-knockdown cells. aPKC interacts with partitioning defective 3 (Par-3), which aids in establishing cell polarity and inhibits aPKC by binding as a substrate. In Par-3-knockdown PC cells, YAP expression increased independently of high-glucose treatment. Over-expression of Par-3 and aPKC-dominant negative mutants prevented the high glucose-stimulated nuclear localization of YAP. YAP forms a complex with the zinc finger E-box binding homeobox 1 protein (ZEB1), an activator of epithelial-mesenchymal transition. ZEB1 expression was increased by high glucose treatment or Par-3 knockdown, but aPKC knockdown suppressed this increase. CONCLUSION: High glucose-induced aPKC activation promotes PC progression by enhancing the YAP signaling pathway.

Oncogenic role of TYRO3 receptor tyrosine kinase in the progression of pancreatic cancer.

The expression and functions of TYRO3, a member of the TAM receptor tyrosine kinase family, in pancreatic cancer (PC) have not been specifically elucidated. In this study, we confirmed TYRO3 expression in five human PC cell lines (PANC-1, MIA PaCa-2, BxPC-3, AsPC-1, and PK-9) using Western blotting. TYRO3 silencing and overexpression studies have revealed that TYRO3 promotes cell proliferation and invasion in PC via phosphorylation of protein kinase B (Akt) and extracellular signal-regulated kinase (ERK). Using a mouse xenograft model, we showed that tumor growth was significantly suppressed in mice subcutaneously inoculated with TYRO3-knockdown PC cells compared with mice inoculated with control PC cells. Furthermore, TYRO3 expression was examined in PC tissues obtained from 106 patients who underwent pancreatic resection for invasive ductal carcinoma through immunohistochemical staining. TYRO3-positive patients had poor prognoses for overall survival and disease-specific survival compared with TYRO3-negative patients. Multivariate analysis revealed that TYRO3 expression is an independent prognostic factor for overall survival. Our study demonstrates the critical role of TYRO3 in PC progression through Akt and ERK activation and suggests TYRO3 as a novel promising target for therapeutic strategies against PC.

System xc- in microglia is a novel therapeutic target for post-septic neurological and psychiatric illness.

Post-septic neurological and psychiatric illness (PSNPI) including dementia and depression may be observed after sepsis. However, the etiology of PSNPI and therapeutic treatment of PSNPI are unclear. We show that glutamate produced from microglia through the activity of system xc- plays a role in PSNPI. We established a mouse model of PSNPI by lipopolysaccharide (LPS) treatment that shows a disturbance of short/working memory and depression-like hypoactivity. Glutamate receptor antagonists (MK801 and DNQX) reduced these phenotypes, and isolated microglia from LPS-treated mice released abundant glutamate. We identified system xc- as a source of the extracellular glutamate. xCT, a component of system xc-, was induced and expressed in microglia after LPS treatment. In xCT knockout mice, PSNPI were decreased compared to those in wildtype mice. Moreover, TNF-α and IL-1ß expression in wildtype mice was increased after LPS treatment, but inhibited in xCT knockout mice. Thus, system xc- in microglia may be a therapeutic target for PSNPI. The administration of sulfasalazine, an inhibitor of xCT, in symptomatic and post-symptomatic mice improved PSNPI. Our results suggest that glutamate released from microglia through system xc- plays a critical role in the manifestations of PSNPI and that system xc- may be a therapeutic target for PSNPI.

Parkin mediates neuroprotection through activation of IkappaB kinase/nuclear factor-kappaB signaling.

Mutations in the parkin gene are a major cause of autosomal recessive Parkinson's disease. Here we show that the E3 ubiquitin ligase parkin activates signaling through the IkappaB kinase (IKK)/nuclear factor kappaB (NF-kappaB) pathway. Our analysis revealed that activation of this signaling cascade is causally linked to the neuroprotective potential of parkin. Inhibition of NF-kappaB activation by an IkappaB super-repressor or a kinase-inactive IKKbeta interferes with the neuroprotective activity of parkin. Furthermore, pathogenic parkin mutants with an impaired neuroprotective capacity show a reduced ability to stimulate NF-kappaB-dependent transcription. Finally, we present evidence that parkin interacts with and promotes degradation-independent ubiquitylation of IKKgamma/NEMO (NF-kappaB essential modifier) and TRAF2 [TNF (tumor necrosis factor) receptor-associated factor 2], two critical components of the NF-kappaB pathway. Thus, our results support a direct link between the neuroprotective activity of parkin and ubiquitin signaling in the IKK/NF-kappaB pathway.

Caffeine activates the PI3K/Akt pathway and prevents apoptotic cell death in a Parkinson's disease model of SH-SY5Y cells.

Parkinson's disease (PD) is one of the most common neurodegenerative diseases. Recent epidemiological studies suggest that caffeine, one of the major components of coffee, has a protective effect against developing PD. However, the detailed mechanisms of how caffeine suppresses neuronal death have not been fully elucidated. We investigated the cytoprotective mechanisms of caffeine using human dopaminergic neuroblastoma SH-SY5Y cells as a PD model. Caffeine prevented the apoptotic cell death induced by serum/retinoic acid (RA) deprivation, MPP+, rotenone, and 6-OHDA in SH-SY5Y cells in a dose dependent manner. Caffeine lowered caspase-3 activity induced by serum/RA deprivation and 6-OHDA administration, and also decreased the number of apoptotic condensed and/or fragmented nuclei. Akt was phosphorylated 60 min after caffeine administration in a dose dependent manner; PI3K inhibitors, wortmannin and LY294002 canceled this cytoprotective effect of caffeine. On the other hand, MAPKs such as Erk1/2, p38, or JNK were not activated by caffeine. These results suggest that caffeine has a cytoprotective effect due to the activation of the PI3K/Akt pathways in SH-SY5Y cells.

Pramipexole has astrocyte-mediated neuroprotective effects against lactacystin toxicity.

Pramipexole, a dopamine D2/D3 receptor agonist used in the treatment of Parkinson's disease, has been reported to have neuroprotective potential. We investigated the effect of pramipexole against cell death induced by a proteasome inhibitor, lactacystin, using primary mecencephalic neuronal cultures and SH-SY5Y cells. In E14 rat primary mesencephalic cultures, the number of surviving tyrosine hydroxylase (TH)-positive neurons and microtubule associated protein 2 (MAP2)-positive neurons was decreased by exposure to 1-5 microM lactacystin in a dose-dependent manner. Pretreatment with 100 microM pramipexole rescued TH-positive neurons and MAP2-positive neurons from the toxicity of lactacystin. The protective effect of pramipexole was not selective for TH-positive dopaminergic neurons. However, the treatment with 100 microM pramipexole did not protect SH-SY5Y cells against lactacystin-induced cell toxicity and proteasome dysfunction. We hypothesized that the protective effect of pramipexole against the lactacystin-toxicity was not direct but a secondary effect mediated by astrocytes. Therefore, we investigated the efficacy of conditioned medium collected from mecencephalic astrocytes treated with pramipexole. The conditioned medium increased the viability of SH-SY5Y cells against the toxicity of lactacystin. Pramipexole increased the levels of brain derived neurotrophic factor (BDNF) in the conditioned medium of astrocyte cultures. These protective effects were not significantly inhibited by dopamine D2 or D3 receptor antagonists. We demonstrated that pramipexole had the protective effect against lactacystin toxicity, mediated by a neurotrophic effect of astrocyte-produced factors including BDNF.

α-Tocopherol promotes HaCaT keratinocyte wound repair through the regulation of polarity proteins leading to the polarized cell migration.

In many developed countries including Japan, how to care the bedridden elderly people with chronic wounds such as decubitus becomes one of the most concerned issues. Although antioxidant micronutrients including vitamin E, especially α-tocopherol (α-Toc), are reported to shorten a period of wound closure, the promoting effect of α-Toc on wound healing independent of its antioxidant activity remains to be fully elucidated. The aim of this study was to examine whether α-Toc affects wound-mediated HaCaT keratinocyte polarization process including the recruitment of polarity regulating proteins, leading to wound repair independently of its antioxidant activity. We investigated the effects of α-Toc and other antioxidants such as Trolox, a cell-permeable α-Toc analog on the migration, proliferation, and cell polarization of HaCaT keratinocytes after wounding. We analyzed the localization and complex formation of polarity proteins, partitioning defective 3 (Par3), and atypical protein kinase C (aPKC), and aPKC activity by immunohistochemistry, immunoprecipitation analyses, and in vitro kinase assays, respectively. α-Toc but not other antioxidants enhanced the wound closure and cell polarization in HaCaT keratinocytes after wounding. α-Toc regulated the localization and complex formation of Par3 and aPKC during wound healing. Knockdown of aPKC or Par3 abrogated α-Toc-mediated promotion of the wound closure and cell polarization in HaCaT keratinocytes. Furthermore, aPKC kinase activity was significantly increased in α-Toc-treated cells through activation of phosphatidylinositol 3-kinase/Akt signaling pathway. These results suggest that α-Toc promotes HaCaT keratinocyte wound repair by regulating the aPKC kinase activity and the formation of aPKC-Par3 complex. © 2017 BioFactors, 44(2):180-191, 2018.

Homocysteine is toxic for dopaminergic neurons in primary mesencephalic culture.

Hyperhomocysteinemia associated with L-3,4-dihydroxyphenylalanine (L-dopa) treatment has been observed in patients with Parkinson's disease. We investigated the toxicity of homocysteine (Hcy) on E14-rat-primary mesencephalic culture. Exposure to 0-5 mM Hcy decreased number of tyrosine hydroxylase (TH)-positive dopaminergic neurons and microtubule associated protein 2 (MAP2)-positive neurons in a dose-dependent manner. TH-positive neurons had vulnerability to the insult of Hcy compared with the other MAP2-positive neurons. In dopaminergic neurons, 5 microM reserpine enhanced the Hcy toxicity, whereas 50 microM alpha-methyltyrosine attenuated the toxic effect, showing that the intracellular dopamine increased the cytotoxicity of Hcy. Hcy enhanced the toxicity of 1-methyl-4-phenylpyridinium (MPP+) for dopaminergic neurons. It was suggested that the Hcy toxicity was associated with the oxidative stress. Hcy is toxic for dopaminergic neurons, and hyperhomocysteinemia may modify the clinical course of Parkinson's disease.

Diagnostic markers for diagnosing dementia with Lewy bodies: CSF and MIBG cardiac scintigraphy study.

OBJECTIVE: This study examined the diagnostic value of cerebrospinal fluid (CSF) markers and iodine-123 metaiodobenzylguanidine ((123)I-MIBG) cardiac scintigraphy in distinguishing dementia with Lewy bodies (DLB) from Alzheimer's disease (AD). METHODS: CSF levels of amyloid beta1-42 (Abeta42) and 181-Thr phosphorylated tau (p-tau) were measured using enzyme linked immunosorbent assay (ELISA) kits. (123)I-MIBG cardiac scintigraphy was performed in patients with AD and DLB, and control (CTL) subjects. RESULTS: Increased CSF levels of p-tau in AD were found compared to DLB patients and CTL subjects (P<0.01), but there was no significant difference in CSF levels of Abeta42 between AD and DLB patients. The early and delayed heart to mediastinum (H/M) ratios of (123)I-MIBG cardiac scintigraphy were significantly decreased in patients with DLB compared to AD patients and CTL subjects (P<0.01). The receiver operating characteristic (ROC) analysis revealed that the diagnostic value of (123)I-MIBG cardiac scintigraphy was superior to that of CSF markers. CONCLUSIONS: (123)I-MIBG cardiac scintigraphy may be useful for discriminating between DLB and AD.

Detection of compound heterozygous deletions in the parkin gene of fibroblasts in patients with autosomal recessive hereditary parkinsonism (PARK2).

Mutations in the parkin gene are a common cause of autosomal recessive, juvenile or early onset parkinsonism (PARK2). In this report, we use RT-PCR to detect compound heterozygous deletions of the parkin gene in fibroblasts from two cases of middle age-onset familial parkinsonism with lower extremities-dominant resting tremor and mild cogwheel rigidity. Although exonic amplification of the parkin gene showed a deletional mutation of exon 3-4, their family histories suggested that the deletional mutations were a compound heterozygous abnormality of discrete origin. Immunoblotting demonstrated that abundant Parkin protein was expressed in fibroblasts, but little expression was detected in lymphocytes. RT-PCR using RNA isolated from the patients' fibroblasts indicated a parkin mutation in this family that consisted of compound heterozygous deletions (del exon3-4/del exon3-5). These results suggest that RT-PCR using the patients' fibroblasts may be helpful for the detection of compound heterozygous abnormalities in the parkin gene.

[Anti-Yo antibody associated paraneoplastic cerebellar degeneration with gastric adenocarcinoma in a male patient: a case report].

We report a 71-year-old man presenting with paraneoplastic cerebellar degeneration (PCD) associated anti-Yo antibody after surgery for gastric adenocarcinoma. Seven months after partial gastrectomy, he deviated to the right on walking. Furthermore, a feeling of dysarthria appeared and he was unable to sit after 2 months. When he was hospitalized, he showed a disturbance of his eye movement on lower gaze, a nystagmus on lateral gaze, saccadic eye movement on smooth pursuit, cerebellar ataxia, and decreasing of muscle tonus in his extremities. However, no atrophic findings of the brainstem and cerebellum were revealed by brain MRI. He responded poorly to treatment with high-dose methylprednisolone, high-dose immunoglobulin, double filtration plasmapheresis and rehabilitation. There was a strong anti-Yo immunohistochemical staining of the cytoplasm in both the patient's tumor cells and normal cerebellar Purkinje cells. These findings suggest that PCD associated with anti-Yo antibody triggered by adenocarcinoma might occur in this male patient.