Beclin1 controls the levels of p53 by regulating the deubiquitination activity of USP10 and USP13.

Autophagy is an important intracellular catabolic mechanism that mediates the degradation of cytoplasmic proteins and organelles. We report a potent small molecule inhibitor of autophagy named "spautin-1" for specific and potent autophagy inhibitor-1. Spautin-1 promotes the degradation of Vps34 PI3 kinase complexes by inhibiting two ubiquitin-specific peptidases, USP10 and USP13, that target the Beclin1 subunit of Vps34 complexes. Beclin1 is a tumor suppressor and frequently monoallelically lost in human cancers. Interestingly, Beclin1 also controls the protein stabilities of USP10 and USP13 by regulating their deubiquitinating activities. Since USP10 mediates the deubiquitination of p53, regulating deubiquitination activity of USP10 and USP13 by Beclin1 provides a mechanism for Beclin1 to control the levels of p53. Our study provides a molecular mechanism involving protein deubiquitination that connects two important tumor suppressors, p53 and Beclin1, and a potent small molecule inhibitor of autophagy as a possible lead compound for developing anticancer drugs.

Negative regulation of Vps34 by Cdk mediated phosphorylation.

Vacuolar protein sorting 34 (Vps34) complexes, the class III PtdIns3 kinase, specifically phosphorylate the D3 position of PtdIns to produce PtdIns3P. Vps34 is involved in the control of multiple key intracellular membrane trafficking pathways including endocytic sorting and autophagy. In mammalian cells, Vps34 interacts with Beclin 1, an ortholog of Atg6 in yeast, to regulate the production of PtdIns3P and autophagy. We show that Vps34 is phosphorylated on Thr159 by Cdk1, which negatively regulates its interaction with Beclin 1 during mitosis. Cdk5/p25, a neuronal Cdk shown to play a role in Alzheimer's disease, can also phosphorylate Thr159 of Vps34. Phosphorylation of Vps34 on Thr159 inhibits its interaction with Beclin 1. We propose that phosphorylation of Thr159 in Vps34 is a key regulatory mechanism that controls the class III PtdIns3 kinase activity in cell-cycle progression, development, and human diseases including neurodegeneration and cancers.

Cerebral infarction developing in a patient without cancer with a markedly elevated level of mucinous tumor marker.

Previous studies have shown the possible role of mucin in cerebral infarction associated with coagulation abnormalities in patients with cancer, particularly adenocarcinoma. We report a 42-year-old woman who developed motor aphasia and cerebral infarction in the left frontal lobe and right parietal lobe. A mucinous tumor marker, CA125 level, was markedly elevated at 1750 U/mL (normal, <36 U/mL), and the D-dimer level was 6.0 µg/mL (normal, <1 µg/mL). She had adenomyosis and no malignancy was revealed. The CA125 and the D-dimer levels became normal after treatment of adenomyosis. Our findings suggest for the first time that marked elevation of mucinous tumor marker level may cause cerebral infarction even in benign conditions.

Gradient echo T2*-weighted magnetic resonance imaging revealing cerebral microbleeds in a patient with microscopic polyangiitis complicated by cerebrovascular disease.

A 67-year-old woman developed alveolar hemorrhage and was positive for the myeloperoxidase antineutrophil cytoplasmic antibody (MPO-ANCA). She was diagnosed as having probable microscopic polyangiitis (MPA) in accordance with the Japanese criteria. Brain computed tomographic and magnetic resonance imaging (MRI) scans revealed asymptomatic chronic cerebral hemorrhage and white matter lesions. She also later developed thalamic infarction. Gradient echo T2-weighted MRI showed cerebral microbleeds. Cerebral microbleeds in our patient represent bleeding-prone necrotizing vasculitis, which is a characteristic pathologic feature of MPA. Although cerebrovascular disease is not major complication of MPA, cerebral hemorrhage occurs more frequently than ischemic infarction, and it can be critical and fatal. Brain MRIs, including gradient echo T2-weighted imaging, should be performed on patients diagnosed as or suspected of having MPA to assess the risk of cerebral hemorrhage.

Generation of mouse macrophages expressing membrane-bound TNF variants with selectivity for TNFR1 or TNFR2.

Tumor necrosis factor-alpha (TNF) is expressed on the cell surface as a transmembrane form (tmTNF), that can be released as a soluble form (solTNF) via proteolytic cleavage. These two types of TNF exert their biological functions by binding to one of two TNF receptors, TNFR1 or TNFR2. However, the biological function of tmTNF through these two receptors remains to be determined. Here, we generated macrophages that expressed tmTNF mutants with selectivity for either TNFR1 or TNRF2 as a tool to evaluate signaling through these receptors. Wild-type TNF (wtTNF), TNFR1-selective mutant TNF (mutTNF-R1) or TNFR2-selective mutant TNF (mutTNF-R2) were individually expressed on the TNFR1(-/-)R2(-/-) mouse macrophages (Mphi) as the tmTNF forms. tm-mutTNF-R1-expressing Mphi exhibited significant selectivity for binding to TNFR1, whereas tm-mutTNF-R2-expressing Mphi only showed a slight selectivity for binding to TNFR2. Signaling by tm-mutTNF-R1-expressing Mphi through the hTNFR2 was weaker than that of tm-wtTNF-expressing Mphi, suggesting that the binding selectivity correlated with functional selectivity. Interestingly, signaling by tm-mutTNF-R2-expressing Mphi through TNFR2 was much stronger than signaling by tm-wtTNF-expressing Mphi, whereas signaling by the corresponding soluble form was weaker than that mediated by wtTNF. These results indicate tmTNF variants might prove useful for the functional analysis of signaling through TNF receptors.

Alteration in the differentiation-related molecular expression in the subventricular zone in a mouse model of Parkinson's disease.

Enhancement of neurogenesis could be a suitable treatment approach to up-regulate dopaminergic neurons in Parkinson's disease (PD). In the present study, we focused on the kinetics of the subventricular zone (SVZ) in a mouse model of PD induced by MPTP injection. We showed recently the proliferation potential of neuronal stem cells (NSCs) prepared from the olfactory bulb of an animal model of PD [Hayakawa, H., Hayashita-Kinoh, H., Nihira, T., Seki, T., Mizuno, Y., Mochizuki, H., 2007. The isolation of neural stem cells from the OB of Parkinson's disease model. Neurosci. Res.]. In this study, we examined the relationship between proliferation and differentiation of NSCs in SVZ of both acute and chronic PD models. Only acute MPTP treatment significantly increased the areas of glial fibrillary acidic protein (GFAP)-expressing cells and decreased the areas of polysialylated neural cell adhesion molecule (PSA-NCAM)-expressing cells in the SVZ. In the case of caspase-11 knockout mice, MPTP did not induce alteration in the areas of GFAP-expressing cells and PSA-NCAM-expressing cells. Our results suggest that neuroinflammation related to the caspase-11 cascade in the striatum regulates differentiation of neural stem cells in the SVZ of our mouse model of PD.

Caspase-11 mediates inflammatory dopaminergic cell death in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease.

The present study was designed to elucidate the inflammatory and apoptotic mechanisms of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity in a model of Parkinson's disease. Our results showed that mutant mice lacking the caspase-11 gene were significantly more resistant to the effects of acute treatment with MPTP than their wild-type mice. Thus, the neurotoxicity of MPTP seems to be mediated by the induction of both mitochondrial dysfunction and free radical generation. Previously, we showed that overexpression of the Apaf-1 dominant-negative inhibitor inhibited the mitochondrial apoptotic cascade in chronic MPTP treatment but not in acute MPTP treatment. The present results indicate that MPTP neurotoxicity may be mediated via activation of the caspase-11 cascade and inflammatory cascade, as well as the mitochondrial apoptotic cascade.

Establishment of modified chimeric mice using GFP bone marrow as a model for neurological disorders.

Chimeric mice stably reconstituted with bone marrow cells represent a good model for analysis of the mechanism of bone marrow cell infiltration in the brain. However, in preparing chimeric mice, irradiation of the recipient mice is necessary to kill their own bone marrow before transplantation, which induces gliosis and inflammatory response by activation of astrocytes and microglia in the brain. Here, we determined the most suitable dose of irradiation associated with the least brain damage before transplantation for reconstitution of chimeric mice, using FACS analysis. Our mouse model of 10 Gy body/5 Gy head irradiation should be useful for investigating the mechanism(s) of microglial activation in various neurological disorders such as stroke, Alzheimer's disease and Parkinson's disease.

[A 65-year-old woman with progressive loss of vision and visual field defects].

We report a 65-year-old Japanese lady who suffered from progressive loss of vision and visual field defect. She was well until her 61 years of the age in November of 1999, when she was found to have bitemporal hemianopsia. A small enhancing mass lesion was found in the chiasmatic region. She was treated with steroid and she noted marked improvement in her visual field defects. In August of 2000, she noted disturbance of gait. Cranial MRI revealed a mass in the right midbrain extending into the hypothalamic and thalamic regions. She was again treated with steroid with marked improvement. However, in November of 2001, she started to show somnolence and diabetes insipidus. She was treated with steroid, nasal desmopressin, and insulin for her steroid induced diabetes mellitus. Cranial CT scan showed a large enhancing lesion involving the entire midbrain, hypothalamus, and the thalamic regions. She developed respiratory arrest on July 15, 2001 and was pronounced dead. She was discussed in a neurological CPC and the chief discussant arrived at the conclusion that the patient had a primary malignant lymphoma of the brain. Clinical diagnosis in the early stage of her disease was neurosarcoidosis. Post-mortem examination revealed a mass continuously involving the pons, midbrain, hypothalamus, thalamus, and the putamen. The optic chiasm was enlarged. By histologic examination, the mass consisted of dense medium sized tumor cells. Immunohistologic observation revealed that the tumor cells were B-cell type malignant lymphoma. No tumor cells were found in the systemic organs.

Reversible focal cerebral cortical lesions in a patient with heat stroke.

We herein report the rare case of a 56-year-old man who suffered from heat stroke. Although he was in a coma with convulsions on arrival and developed multiorgan failure, he recovered after two weeks of successful treatments. Hyperintense signals on the right temporoparietooccipital cortex, which disappeared within one week, were demonstrated on diffusion-weighted magnetic resonance images. A diagnosis of transient cortical injury caused by heat stroke was suggested. Although the cerebellum is most susceptible to lesion formation, the mechanisms underlying heat stroke are multifactorial and may result in a variety of brain lesions.

Anti-aquaporin-4 antibody-positive definite neuromyelitis optica in a patient with thymectomy for myasthenia gravis.

BACKGROUND: Since serum anti-aquaporin-4 (AQP4) antibody/neuromyelitis optica (NMO)-IgG has been detected as a specific biomarker for NMO), new diagnostic criteria have been proposed. Recently, a rare coexistence of NMO and myasthenia gravis (MG) has been documented. Here, we provide further evidence of the association of these 2 immunologic disorders and review the literature. CASE REPORT: A 60-year-old woman, who had a history of optic neuritis at age 43, developed longitudinally extended transverse myelitis (LETM). She had repeated optic neuritis and longitudinally extended transverse myelitis attacks, which have been treated by corticosteroid pulse therapy. She was diagnosed as having definite NMO, because serum anti-aquaporin-4 antibody was positive. She had also been suffering from generalized MG since age 30 and underwent thymectomy at age 40. There have been 14 patients with NMO associated with MG, most of whom developed NMO years after thymectomy. Our patient is the seventh patient with seropositive NMO who underwent thymectomy for MG. CONCLUSIONS: The coexistence of MG with thymectomy and seropositive NMO suggests an etiopathogenic link between these 2 disorders but remains underrecognized by neurologists. Neurologists should consider this possible association when encountering atypical presentations of either MG or NMO.

Convulsive movements in bilateral paramedian thalamic and midbrain infarction.

Although some previous reports have described convulsive movements in bilateral paramedian thalamic and midbrain infarction, little is known about their nature. A 71-year-old man presented with impaired consciousness and clonic movements of both arms. Each series of movements lasted 10 to 20 s and occurred at 2-to 3-min intervals, which disappeared after intravenous administration of diazepam and phenytoin. Magnetic resonance imaging showed acute bilateral paramedian thalamic and midbrain infarction. A review of the literature revealed that convulsive movements were observed mostly at the onset of infarction. Clonic movements appeared frequently in the limbs, particularly in both arms. Clinical observations and results of animal experiments suggest that these seizures might originate from the mesencephalic reticular formation. Physicians should recognize this condition, because not only seizure control but also early management of ischemic stroke is required.

Possible neuro-Sweet disease mimicking brain tumor in the medulla oblongata--case report.

A 62-year-old male presented with a rare case of possible neuro-Sweet Disease (NSD) mimicking brain tumor in the medulla oblongata, manifesting as numbness in the bilateral upper and lower extremities, gait disturbance, dysarthria, and swallowing disturbance which gradually deteriorated over 3 months. Magnetic resonance imaging showed a mass lesion in the medulla oblongata, extending to the upper cervical cord with rim enhancement by gadolinium. The preoperative diagnosis was brain tumor, such as glioma, or inflammatory disease. His neurological symptoms gradually deteriorated, so biopsy was performed through the midline suboccipital approach. Histological examination showed infiltration of inflammatory cells, mainly lymphocytes and macrophages. Human leukocyte antigen typing showed Cw1 and B54 which strongly suggested possible NSD. Steroid pulse therapy was started after surgery and the clinical symptoms improved. Neurosurgeons should be aware of inflammatory disorders such as NSD mimicking brain tumor.

[Cryptococcal meningitis successfully treated with liposomal amphotericin B and voriconazole in an elderly patient].

Abstract An 81-year-old woman who had microscopic polyangiitis that was being treated with corticosteroids for 2 months was admitted to our department because of fever and clouding of consciousness. Neurological examination showed disturbance of consciousness and nuchal stiffness. Analysis of cerebrospinal fluid (CSF) revealed pleocytosis, low glucose level, and elevated protein levels. On the basis of the presence of Cryptococcus neoformans in CSF, the patient was diagnosed with cryptococcal meningitis. On the basis of established practice guidelines, liposomal amphotericin B (L-AMB) was administered to avoid the possible nephrotoxicity of amphotericin B. After the treatment was started, the patient's condition gradually improved. The results of CSF analysis also showed a gradual recovery. Because the cryptococcal antigen in CSF did not disappear completely, voriconazole (VRCZ) was administered orally; subsequently, the CSF cryptococcal antigen titer gradually decreased. During the course of the treatment with L-AMB and VRCZ, there were no severe side effects that required a change in treatment. To the best of our knowledge, in Japan, the combination of L-AMB and VRCZ has rarely been reported to be effective for the treatment of cryptococcal meningitis. The recovery of our patient indicates that the administration of L-AMB and VRCZ to elderly patients with cryptococcal meningitis and renal insufficiency is safe and leads to a successful outcome.

Regulation of intracellular accumulation of mutant Huntingtin by Beclin 1.

Intracellular accumulation of mutant Huntingtin with expanded polyglutamine provides a context-dependent cytotoxicity critical for the pathogenesis of Huntington disease (Everett, C. M., and Wood, N. W. (2004) Brain 127, 2385-2405). Here we demonstrate that the accumulation of mutant Huntingtin is highly sensitive to the expression of beclin 1, a gene essential for autophagy. Moreover, we show that the accumulated mutant Huntingtin recruits Beclin 1 and impairs the Beclin 1-mediated long lived protein turnover. Thus, sequestration of Beclin 1 in the vulnerable neuronal population of Huntington disease patients might further reduce Beclin 1 function and autophagic degradation of mutant Huntingtin. Finally, we demonstrate that the expression of beclin 1 decreases in an age-dependent fashion in human brains. Because beclin 1 gene is haploid insufficient in regulating autophagosome function (Qu, X., Yu, J., Bhagat, G., Furuya, N., Hibshoosh, H., Troxel, A., Rosen, J., Eskelinen, E. L., Mizushima, N., Ohsumi, Y., Cattoretti, G., and Levine, B. (2003) J. Clin. Invest. 112, 1809-1820; Yue, Z., Jin, S., Yang, C., Levine, A. J., and Heintz, N. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 15077-15082), we propose that the age-dependent decrease of beclin 1 expression may lead to a reduction of autophagic activity during aging, which in turn promotes the accumulation of mutant Htt and the progression of the disease.

Recombinant human granulocyte colony-stimulating factor protects against MPTP-induced dopaminergic cell death in mice by altering Bcl-2/Bax expression levels.

Granulocyte colony-stimulating factor (G-CSF) has been used for the treatment of neutropenia in hematologic disorders. The neuroprotective effects of G-CSF were reported in neurological disease models. In the present study, we examined whether G-CSF can protect dopaminergic neurons against MPTP-induced cell death in a mouse model of Parkinson's disease. Mice of one group were injected intraperitoneally with MPTP for five consecutive days, those of another group with MPTP and intraperitoneal G-CSF at 2 days and 1 day before the first MPTP injection, and 30 min before each MPTP injection, while control mice received saline injections. Immunohistochemistry, western blotting analysis, and HPLC were performed to evaluate damage of substantia nigra dopaminergic neurons and expression of Bcl-2 and Bax protein. MPTP induced dopaminergic cell death in the substantia nigra. G-CSF significantly prevented MPTP-induced loss of tyrosine hydroxylase-positive neurons (p < 0.05), increased Bcl-2 protein and decreased Bax protein expression. Our findings indicate that G-CSF provides neuroprotection against MPTP-induced cell death and this effect is mediated by increasing Bcl-2 expression levels and decreasing Bax expression levels in C57BL/6 mice.

Neurotoxic effects of lipopolysaccharide on nigral dopaminergic neurons are mediated by microglial activation, interleukin-1beta, and expression of caspase-11 in mice.

The endotoxin lipopolysaccharide (LPS), a component of the Gram-negative bacterial cell wall, selectively induces degeneration of substantia nigral (SN) dopaminergic neurons via activation of microglial cells in rats and mice. Caspase-11 plays a crucial role in LPS-induced septic shock in mice. We examined the mechanism of LPS neurotoxicity on SN dopaminergic neurons in C57BL/6 mice and caspase-11 knockout mice. Mice were stereotaxically injected with LPS into the SN on one side and vehicle into the SN of the other side. Immunohistochemistry, Western blotting analysis, enzyme-linked immunosorbent assay, and reverse transcriptase-PCR were performed to evaluate damage of SN dopaminergic neurons and activation of microglial cells. Intranigral injection of LPS at 1 or 3 microg/microl/site decreased tyrosine hydroxylase-positive neurons and increased microglial cells in the SN compared with the contralateral side injected with vehicle at days 7 and 14 post-injection in C57BL/6 mice. Intranigral injection of LPS at 3 microg/microl/site induced the expression of caspase-11 mRNA in the ventral midbrain at 6, 8, and 12 h post-injection, and the expression of caspase-11-positive cells in the SN at 8 and 12 h post-injection. Moreover, LPS at 3 microg/microl/site increased interleukin-1beta content in the ventral midbrain at 12 and 24 h post-injection. LPS failed to elicit these responses in caspase-11 knockout mice. Our results indicate that the neurotoxic effects of LPS on nigral dopaminergic neurons are mediated by microglial activation, interleukin-1beta, and caspase-11 expression in mice.