Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by a CAG trinucleotide expansion in exon 1 of the Huntingtin (Htt) gene. We show herein that in an HD transgenic mouse model (R6/2), daily administration of CGS21680 (CGS), an A 2A adenosine receptor (A 2A -R)-selective agonist, delayed the progressive deterioration of motor performance and prevented a reduction in brain weight. 3D-lMRI analysis revealed that CGS reversed the enlarged ventricle-to-brain ratio of R6/2 mice, with particular improvements in the left and right ventricles. 1 H-MRS showed that CGS significantly reduced the increased choline levels in the striatum. Immunohistochemical analyses further demonstrated that CGS reduced the size of ubiquitin-positive neuronal intranuclear inclusions (NIIs) in the striatum of R6/2 mice and ameliorated mutant Htt aggregation in a striatal progenitor cell line overexpressing mutant Htt with expanded polyQ. Moreover, chronic CGS treatment normalized the elevated blood glucose levels and reduced the overactivation of a major metabolic sensor [5¢AMP-activated protein kinase (AMPK)] in the striatum of R6/2 mice. Since AMPK is a master switch for energy metabolism, modulation of energy dysfunction caused by the mutant Htt might contribute to the beneficial effects of CGS. Collectively, CGS is a potential drug candidate for the treatment of HD.

Huntington's disease (HD) is a hereditary neurological disease caused by expended CAG repeats in the HD gene, which codes for a protein called Huntingtin (Htt). The resultant mutant Huntingtin (mHtt) forms aggregates in neurons and causes neuronal dysfunction. In astrocytes, the largest population of brain cells, mHtt also exists. We report herein that astrocyte-conditioned medium (ACM) collected from astrocytes of R6/2 mice (a mouse model of HD) caused primary cortical neurons to grow less-mature neurites, migrate more slowly, and exhibit lower calcium influx after depolarization than those maintained in wild-type (WT) ACM. Using a cytokine antibody array and ELISA assays, we demonstrated that the amount of a chemokine [chemokine (C-C motif) ligand 5 (CCL5)/regulated on activation normal T cell expressed and secreted (RANTES)] released by R6/2 astrocytes was much less than that by WT astrocytes. When cortical neurons were treated with the indicated ACM, supplementation with recombinant CCL5/RANTES ameliorated the neuronal deficiency caused by HD-ACM, whereas removing CCL5/RANTES from WT-ACM using an anti-CCL5/RANTES antibody mimicked the effects evoked by HD-ACM. Quantitative PCR and promoter analyses demonstrated that mHtt hindered the activation of the CCL5/RANTES promoter by reducing the availability of nuclear factor B-p65 and, hence, reduced the transcript level of CCL5/RANTES. Moreover, ELISA assays and immunocytochemical staining revealed that mHtt retained the residual CCL5/RANTES inside R6/2 astrocytes. In line with the above findings, elevated cytosolic CCL5/RANTES levels were also observed in the brains of two mouse models of HD [R6/2 and Hdh (CAG)150 ] and human HD patients. These findings suggest that mHtt hinders one major trophic function of astrocytes which might contribute to the neuronal dysfunction of HD.

Mitogen-activated protein kinases (MAP kinases) are active only when phosphorylated. Here we exmine whether the activation of Xenopus p42 MAP kinase might involve changes in its association with other proteins as well as changes in its phosphorylation state. We find that when p42 MAP kinase is phosphorylated and active, it is monomeric, and that when p42 MAP kinase is nonphosphorylated and inactive, about half of it is monomeric and half is a component of a 110-kDa complex. We identify Rsk, an 82-kDa protein kinase that can be phosphorylated and partially activated by p42 MAP kinase, as being specifically associated with inactive p42 MAP kinase. It is possible that the complex of inactive p42 MAP kinase and inactive Rsk acts as a single signal reception particle and that the activation of the two kinases may be better described as a fork in a bifurcating signal transuction pathway than as successive levels in a kinase cascade. Gel Filtration Chromatography. Lysates were further clarified by centrifugation at 50,000 rpm for 1 hr in a Beckman TLA 100.3 rotor at 40C followed by filtration through a 0.45-gum-pore membrane. Samples (100 sl, containing roughly 2.5 mg oftotal protein) were subjected to gel filtration chromatography at a flow rate of 0.4 ml/min on a Superose 12 column (HR10/30, Pharmacia) equilibrated with lysis buffer. Molecular size standards were thyroglobulin (670 kDa), 'y-globulin (158 kDa), bovine serum albumin (68 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and cyanocobalamin (1.4 kDa). MitogenSucrose Gradient Centrifugation. Lysates were layered on linear 5-20%o sucrose gradients (5.5 ml) made up in lysis buffer. Samples were centrifuged at 50,000 rpm in a Beckman SW50.1 rotor for 12 hr at 40C. Sedimentation standards were -globulin (7.2 S) and ovalbumin (3.5 S).Antibodies. p42 MAP kinase was detected with a polyclonal antiserum (X15) raised against a 12-amino acid peptide from the Xenopus p42 MAP kinase sequence (IFEETAEFQPGY) conjugated through an amino-terminal cysteine residue to bovine serum albumin. Tyrosine-phosphorylated p42 MAP kinase was detected with a polyclonal anti-phosphotyrosine antiserum (11). Rsk was detected with a polyclonal antiserum provided by E. Erikson and J. Maller (17 5480The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

BackgroundTraumatic brain injury (TBI) is a global health concern that typically causes emotional disturbances and cognitive dysfunction. Secondary pathologies following TBI may be associated with chronic neurodegenerative disorders and an enhanced likelihood of developing dementia-like disease in later life. There are currently no approved drugs for mitigating the acute or chronic effects of TBI.MethodsThe effects of the drug pomalidomide (Pom), an FDA-approved immunomodulatory agent, were evaluated in a rat model of moderate to severe TBI induced by controlled cortical impact. Post-TBI intravenous administration of Pom (0.5 mg/kg at 5 or 7 h and 0.1 mg/kg at 5 h) was evaluated on functional and histological measures that included motor function, fine more coordination, somatosensory function, lesion volume, cortical neurodegeneration, neuronal apoptosis, and the induction of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6).ResultsPom 0.5 mg/kg administration at 5 h, but not at 7 h post-TBI, significantly mitigated the TBI-induced injury volume and functional impairments, neurodegeneration, neuronal apoptosis, and cytokine mRNA and protein induction. To evaluate underlying mechanisms, the actions of Pom on neuronal survival, microglial activation, and the induction of TNF-α were assessed in mixed cortical cultures following a glutamate challenge. Pom dose-dependently ameliorated glutamate-mediated cytotoxic effects on cell viability and reduced microglial cell activation, significantly attenuating the induction of TNF-α.ConclusionsPost-injury treatment with a single Pom dose within 5 h significantly reduced functional impairments in a well-characterized animal model of TBI. Pom decreased the injury lesion volume, augmented neuronal survival, and provided anti-inflammatory properties. These findings strongly support the further evaluation and optimization of Pom for potential use in clinical TBI.

Chloride intracellular channel 4 (CLIC4) is a mammalian homologue of EXC-4 whose mutation is associated with cystic excretory canals in nematodes. Here we show that CLIC4-null mouse embryos exhibit impaired renal tubulogenesis. In both developing and developed kidneys, CLIC4 is specifically enriched in the proximal tubule epithelial cells, in which CLIC4 is important for luminal delivery, microvillus morphogenesis, and endolysosomal biogenesis. Adult CLIC4-null proximal tubules display aberrant dilation. In MDCK 3D cultures, CLIC4 is expressed on early endosome, recycling endosome and apical transport carriers before reaching its steady-state apical membrane localization in mature lumen. CLIC4 suppression causes impaired apical vesicle coalescence and central lumen formation, a phenotype that can be rescued by Rab8 and Cdc42. Furthermore, we show that retromer- and branched actin-mediated trafficking on early endosome regulates apical delivery during early luminogenesis. CLIC4 selectively modulates retromer-mediated apical transport by negatively regulating the formation of branched actin on early endosomes.

Traumatic brain injury leads to major brain anatomopathological damages underlined by neuroinflammation, oxidative stress and progressive neurodegeneration, ultimately leading to motor and cognitive deterioration. The multiple pathological events resulting from traumatic brain injury can be addressed not by a single therapeutic approach, but rather by a synergistic biotherapy capable of activating a complementary set of signaling pathways and providing synergistic neuroprotective, anti-inflammatory, antioxidative, and neurorestorative activities. Human platelet lysate might fulfill these requirements as it is comprised of a plethora of biomolecules readily accessible as a traumatic brain injury biotherapy. In the present study, we have tested the therapeutic potential of human platelet lysate using in vitro and in vivo models of traumatic brain injury. We first prepared and characterized platelet lysate from clinical-grade human platelet concentrates. Platelets were pelletized, lysed by three freeze-thaw cycles, and centrifuged. The supernatant was purified by 56 °C-30 minutes heat-treatment and spun to obtain the heat-treated platelet pellet lysate that was characterized by ELISA and proteomic analyses. Two mouse models were used to investigate platelet lysate neuroprotective potential. The injury was induced by an in-house manual controlled scratching of the animals' cortex or by controlled cortical impact injury. The platelet lysate treatment was performed by topical application of 60 µL in the lesioned area, followed by daily 60 µL intranasal administration from day 1 to 6 post-injury. Platelet lysate proteomics identified over 1000 proteins including growth factors, neurotrophins, and antioxidants. ELISA detected several neurotrophic and angiogenic factors at ca. 1–50 ng/mL levels. We demonstrate, using the two mouse models of traumatic brain injury that topical application and intranasal platelet lysate consistently improved mice motor function in the beam and rotarod tests, mitigated cortical neuroinflammation, and oxidative stress in the injury area, as revealed by downregulation of pro-inflammatory genes and the reduction in reactive oxygen species levels. Moreover, platelet lysate treatment reduced the loss of cortical synaptic proteins. Unbiased proteomic analyses revealed that HPPL reversed several pathways promoted by both CCI and CBS and related to transport, post-synaptic density, mitochondria or lipid metabolism. The present data strongly support for the first time that human platelet lysate is a reliable and effective therapeutic source of neurorestorative factors. Therefore, brain administration of platelet lysate is a therapeutical strategy that deserves serious and urgent consideration for universal brain trauma treatment.

SUMMARY Chloride intracellular channel (CLIC) 4 has diverse functions in membrane trafficking, apoptosis, angiogenesis and cell differentiation. CLIC4 is abundantly expressed in macrophages, but its role in innate immune functions is unclear. Here we show that primary murine macrophages expressed increased amounts of CLIC4 after exposure to bacterial lipopolysaccharide (LPS). The endogenous CLIC4 level is significantly elevated in the brain, heart, lung, kidney, liver and spleen after LPS injection of mice. Stable macrophage lines overexpressing CLIC4 produced more TNF, IL-6, IL-12 and CCL5 than mock transfectants when exposed to LPS. To explore the role of CLIC4 in vivo, we generated CLIC4-null mice. These mice were protected from LPS-induced death, had reduced serum levels of inflammatory cytokines. Upon infection with Listeria monocytogenes, CLIC4-deficient mice were impaired in their ability to clear infection, and their macrophages responded to Listeria by producing less inflammatory cytokines and chemokines than the wild type controls. When challenged with LPS in vitro, deletion of clic4 gene had little effect in MAPK and NF-κB activation, but led to a reduced accumulation of phosphorylated IRF3 within macrophages. Conversely, overexpression of CLIC4 enhanced LPS-mediated IRF3. Thus, CLIC4 is an LPS-induced product that can serve as a positive regulator of LPS signaling.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by a CAG trinucleotide expansion in the Huntingtin (Htt) gene. The resultant mutant Htt protein (mHtt) forms aggregates in the brain and several peripheral tissues (e.g. the liver) and causes devastating neuronal degeneration. Metabolic defects resulting from Htt aggregates in peripheral tissues also contribute to HD pathogenesis. Simultaneous improvement of defects in both the CNS and peripheral tissues is thus the most effective therapeutic strategy and is highly desirable. We earlier showed that an agonist of the A(2A) adenosine receptor (A(2A) receptor), CGS21680 (CGS), attenuates neuronal symptoms of HD. We found herein that the A(2A) receptor also exists in the liver, and that CGS ameliorated the urea cycle deficiency by reducing mHtt aggregates in the liver. By suppressing aggregate formation, CGS slowed the hijacking of a crucial transcription factor (HSF1) and two protein chaperons (Hsp27 and Hsp70) into hepatic Htt aggregates. Moreover, the abnormally high levels of high-molecular-mass ubiquitin conjugates in the liver of an HD mouse model (R6/2) were also ameliorated by CGS. The protective effect of CGS against mHtt-induced aggregate formation was reproduced in two cells lines and was prevented by an antagonist of the A(2A) receptor and a protein kinase A (PKA) inhibitor. Most importantly, the mHtt-induced suppression of proteasome activity was also normalized by CGS through PKA. Our findings reveal a novel therapeutic pathway of A(2A) receptors in HD and further strengthen the concept that the A(2A) receptor can be a drug target in treating HD.