A novel small molecule, N-(4-(2-pyridyl)(1,3-thiazol-2-yl))-2-(2,4,6-trimethylphenoxy) acetamide, selectively protects against oxidative stress-induced cell death by activating the Nrf2-ARE pathway: therapeutic implications for ALS.

Antioxidant defense is crucial in restoring cellular redox homeostasis. Recent findings have suggested that oxidative stress plays pivotal roles in the pathogenesis of many neurodegenerative diseases. Thus, an anti-oxidative stress remedy might be a promising means for the treatment of such disorders. In this study, we employed a novel ligand-based virtual screening system and identified a novel small molecule, N-(4-(2-pyridyl)(1,3-thiazol-2-yl))-2-(2,4,6-trimethylphenoxy) acetamide (CPN-9), which selectively suppressed oxidative stress-induced cell death in a cell-type-independent manner. CPN-9 upregulates NF-E2-related factor 2 (Nrf2), a key transcriptional regulator of the expression of phase II detoxification enzymes and antioxidant proteins, and Nrf2-regulated factors such as heme oxygenase-1 (HO-1), NAD(P)H quinone oxidoreductase 1 (NQO1), and glutamate-cysteine ligase modifier subunit (GCLM). The CPN-9-mediated upregulation of HO-1, NQO1, and GCLM was abolished by Nrf2 knockdown. Moreover, the antioxidant N-acetylcysteine reduced the protective effect of CPN-9 against oxidative stress-induced cell death with concomitant diminishing of Nrf2 nuclear translocation. These results indicate that CPN-9 exerts its activity via the reactive oxygen species-dependent activation of the Nrf2 signaling pathway in cultured cells. It is noteworthy that the postonset systemic administration of CPN-9 to a transgenic ALS mouse model carrying the H46R mutation in the human Cu/Zn superoxide dismutase (SOD1) gene sustained motor functions and delayed disease progression after onset. Collectively, CPN-9 is a novel Nrf2 activator and a neuroprotective candidate for the treatment of neurodegenerative diseases, including ALS.

Bromocriptine methylate suppresses glial inflammation and moderates disease progression in a mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by a selective loss of upper and lower motor neurons. Since oxidative stress plays a crucial role in the progression of motor neuron loss observed in ALS, anti-oxidative agents could be an important therapeutic means for the ALS treatment. We have previously developed a drug screening system allowing the identification of small chemical compounds that upregulate endogenous neuronal apoptosis inhibitory protein (NAIP), an oxidative stress-induced cell death suppressor. Using this system, we identified the dopamine D2 receptor agonist bromocriptine (BRC) as one of NAIP-upregulating compounds. In this study, to prove the efficacy of BRC in ALS, we conducted a set of preclinical studies using a transgenic ALS mouse model carrying the H46R mutation in the human Cu/Zn superoxide dismutase (SOD1) gene ALS(SOD1(H46R)) by the post-onset administration of BRC. ALS(SOD1(H46R)) mice receiving BRC showed sustained motor functions and modest prolonged survival after onset. Further, BRC treatment delayed anterior horn cell loss, and reduced the number of reactive astrocytes and the level of inflammatory factors such as inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF)-α in the spinal cord of late symptomatic mice. In vitro study showed the reduced level of extracellular TNF-α after lipopolysaccharide (LPS) exposure in BRC-treated mouse astrocytes. BRC-treated ALS(SOD1(H46R)) mice also showed a reduced level of oxidative damage in the spinal cord. Notably, BRC treatment resulted in an upregulation of anti-oxidative stress genes, activating transcription factor 3 (ATF3) and heme oxygenase-1 (HO-1), and the generation of a glutathione (GSH) in SH-SY5Y cultured neuronal cells in a dopamine receptor-independent manner. These results imply that BRC protects motor neurons from the oxidative injury via suppression of astrogliosis in the spinal cord of ALS(SOD1(H46R)) mice. Thus, BRC might be a promising therapeutic agent for the treatment of ALS.

Defective relocalization of ALS2/alsin missense mutants to Rac1-induced macropinosomes accounts for loss of their cellular function and leads to disturbed amphisome formation.

Loss of ALS2/alsin function accounts for several recessive motor neuron diseases. ALS2 is a Rab5 activator and its endosomal localization is regulated by Rac1 via macropinocytosis. Here, we show that the pathogenic missense ALS2 mutants fail to be localized to Rac1-induced macropinosomes as well as endosomes, which leads to loss of the ALS2 function as a Rab5 activator on endosomes. Further, these mutants lose the competence to enhance the formation of amphisomes, the hybrid-organelle formed upon fusion between autophagosomes and endosomes. Thus, Rac1-induced relocalization of ALS2 might be crucial to exert the ALS2 function associated with the autophagy-endolysosomal degradative pathway.

Morphodynamics of ovarian follicles during oogenesis in mice.

In the mouse, oogonia enter the prophase of the first meiotic division and differentiate into oocyte while developing in the fetal ovary. Shortly after birth, all oocytes are arrested in the dictyate stage of late prophase in the developing follicles; a small number of follicles reach the ovulatory stage; the rest are lost by apoptosis. The resumption of meiotic division and nuclear progression to metaphase II (oocyte maturation) occur in the ovulatory follicles. In this article we review recent morphological data that have clarified how cytokines and glycosaminoglycans (GAGs) are involved in mouse follicular development, atresia, and maturation during oogenesis, as exogenous/endogenous factors. (1) Microvascular networks and angiogenic factors (epidermal growth factor; GAGs) are deeply involved in selective mouse oocyte growth beyond approximately 20-30 microm in diameter. (2) Gonadotropin-inducible neuronal apoptosis inhibitory protein may indirectly affect oocyte survival as a result of the inhibition of apoptotic granulosa-cell death during folliculogenesis. (3) The pattern of oocyte degeneration depends on follicle and oocyte developmental stages, and follicle stimulating hormone accelerates the process of degeneration of oocytes. (4) The process of degeneration of mouse oocytes/eggs is modulated by tumor necrosis factor-alpha that is accumulated in the expanded cumulus during oocyte maturation. (5) A colloidal iron-positive substance was detected in the intercellular spaces of follicular tissue, especially in the cumulus mass. Cells located where the cumulus mass and granulosa cell layer interwound became enlarged during the resumption of oocyte meiosis. Colloidal iron-positive substances accumulated extensively within the intercellular spaces of the enlarged cells.

HIP14, a novel ankyrin domain-containing protein, links huntingtin to intracellular trafficking and endocytosis.

Huntington disease (HD) is caused by polyglutamine [poly(Q)] expansion in the protein huntingtin (htt). Although the exact mechanism of disease progression remains to be elucidated, altered interactions of mutant htt with its protein partners could contribute to the disease. Using the yeast two-hybrid system, we have isolated a novel htt interacting protein, HIP14. HIP14's interaction with htt is inversely correlated to the poly(Q) length in htt. mRNAs of 9 and 6 bp are transcribed from the HIP14 gene, with the 6 kb transcript being predominantly expressed in the brain. HIP14 protein is enriched in the brain, shows partial co-localization with htt in the striatum, and is found in medium spiny projection neurons, the subset of neurons affected in HD. HIP14 localizes to the Golgi, and to vesicles in the cytoplasm. The HIP14 protein has sequence similarity to Akr1p, a protein essential for endocytosis in Saccharomyces cerevisiae. Expression of human HIP14 results in rescue of the temperature-sensitive lethality in akr1 Delta yeast cells and, furthermore, restores their defect in endocytosis, demonstrating a role for HIP14 in intracellular trafficking. Our findings suggest that decreased interaction between htt and HIP14 could contribute to the neuronal dysfunction in HD by perturbing normal intracellular transport pathways in neurons.

Inhibition of cyclin-dependent kinases improves CA1 neuronal survival and behavioral performance after global ischemia in the rat.

Increasing evidence suggests that cyclin-dependent kinases participate in neuronal death induced by multiple stresses in vitro. However, their role in cell death paradigms in vivo is not well characterized. Accordingly, the authors examined whether cyclin-dependent kinase inhibition resulted in functionally relevant and sustained neuroprotection in a model of global ischemia. Intracerebroventricular administration of the cyclin-dependent kinase inhibitor flavopiridol, immediately or at 4 hours postreperfusion after a global insult, reduced injury in the CA1 of the hippocampus when examined 7 days after reperfusion. No significant protection was observed when flavopiridol was administered 8 hours after reperfusion. The tumor-suppressor retinoblastoma protein, a substrate of cyclin-dependent kinase, was phosphorylated on a cyclin-dependent kinase consensus site after the global insult; this phosphorylation was inhibited by flavopiridol administration. Importantly, flavopiridol had no effect on core body temperature, suggesting that the mechanism of neuroprotection was through cyclin-dependent kinase inhibition but not through hypothermia. Furthermore, inhibition of cyclin-dependent kinases improved spatial learning behavior as assessed by the Morris water maze 7 to 9 days after reperfusion. However, the histologic protection observed at day 7 was absent 28 days after reperfusion. These results indicate that cyclin-dependent kinase inhibition provides an extended period of morphologic and functional neuroprotection that may allow time for other neuroprotective modalities to be introduced.