Three-Year Follow-Up of High-Dose Ubiquinol Supplementation in a Case of Familial Multiple System Atrophy with Compound Heterozygous COQ2 Mutations.

We report a 3-year follow-up of high-dose ubiquinol supplementation in a case of familial multiple system atrophy (MSA) with compound heterozygous nonsense (R387X) and missense (V393A) mutations in COQ2. A high-dose ubiquinol supplementation substantially increased total coenzyme Q10 levels in cerebrospinal fluid as well as in plasma. The patient was at the advanced stage of MSA, and the various scores of clinical rating scales remained stable without changes during the 3 years. The cerebral metabolic ratio of oxygen measured by 15O2 PET, however, increased by approximately 30% after administration of ubiquinol, suggesting that ubiquinol can improve mitochondrial oxidative metabolism in the brain. It also suggests the therapeutic potential of ubiquinol for patients with MSA with COQ2 mutations. Further clinical trials of administration of high-dose ubiquinol to MSA patients are warranted.

TIM-3 is expressed on activated human CD4+ T cells and regulates Th1 and Th17 cytokines.

TIM-3 is a molecule selectively expressed on a subset of murine IFN-gamma-secreting T helper 1 (Th1) cells but not Th2 cells, and regulates Th1 immunity and tolerance in vivo. At this time little is known about the role of TIM-3 on human T cells. To determine if TIM-3 similarly identifies and regulates Th1 cells in humans, we generated a panel of mAb specific for human TIM-3. We report that TIM-3 is expressed by a subset of activated CD4(+) cells, and that anti-CD3/anti-CD28 stimulation increases both the level of expression as well as the number of TIM-3(+) T cells. We also find that TIM-3 is expressed at high levels on in vitro polarized Th1 cells, and is expressed at lower levels on Th17 cells. In addition, human CD4(+) T cells secreted elevated levels of IFN-gamma, IL-17, IL-2, and IL-6, but not IL-10, IL-4, or TNF-alpha, when stimulated with anti-CD3/anti-CD28 in the presence of TIM-3-specific, putative antagonistic antibodies. This was not mediated by differences in proliferation or cell death, but rather by induction of cytokines at the transcriptional level. These results suggest that TIM-3 is a negative regulator of human T cells and regulates Th1 and Th17 cytokine secretion.

Dysregulated T cell expression of TIM3 in multiple sclerosis.

T cell immunoglobulin- and mucin domain-containing molecule (TIM)3 is a T helper cell (Th)1-associated cell surface molecule that regulates Th1 responses and promotes tolerance in mice, but its expression and function in human T cells is unknown. We generated 104 T cell clones from the cerebrospinal fluid (CSF) of six patients with multiple sclerosis (MS) (n = 72) and four control subjects (n = 32) and assessed their cytokine profiles and expression levels of TIM3 and related molecules. MS CSF clones secreted higher amounts of interferon (IFN)-gamma than did those from control subjects, but paradoxically expressed lower levels of TIM3 and T-bet. Interleukin 12-mediated polarization of CSF clones induced substantially higher amounts of IFN-gamma secretion but lower levels of TIM3 in MS clones relative to control clones, demonstrating that TIM3 expression is dysregulated in MS CSF clones. Reduced levels of TIM3 on MS CSF clones correlated with resistance to tolerance induced by costimulatory blockade. Finally, reduction of TIM3 on ex vivo CD4+ T cells using small interfering (si)RNA enhanced proliferation and IFN-gamma secretion, directly demonstrating that TIM3 expression on human T cells regulates proliferation and IFN-gamma secretion. Failure to up-regulate T cell expression of TIM3 in inflammatory sites may represent a novel, intrinsic defect that contributes to the pathogenesis of MS and other human autoimmune diseases.

Induction of cyclooxygenase-2 in reactive glial cells by the CD40 pathway: relevance to amyotrophic lateral sclerosis.

An inflammatory process in association with reactive gliosis has been suggested to play an important role in the pathogenesis of amyotrophic lateral sclerosis (ALS). One of the key findings is a marked increase in the level of cyclooxygenase-2 (COX-2), a therapeutic target of ALS. We investigated the expression of CD40 in the spinal cord of a transgenic mouse model of ALS (G93A mice), and its relevance to COX-2 upregulation. CD40 was predominantly expressed in neurons in normal spinal cord and upregulated in reactive glial cells in spinal cord injury. In the spinal cord of G93A mice, the expression of CD40 was increased in both reactive microglia and astrocytes, where COX-2 was especially increased. The level of COX-2 was upregulated in microglia and astrocytes by CD40 stimulation in vitro. CD40 stimulation in primary spinal cord cultures caused motor neuron loss that was protected by selective COX-2 inhibitor. These results suggest that CD40, which is upregulated in reactive glial cells in ALS, participates in motor neuron loss via induction of COX-2.

Microglial cell cycle-associated proteins control microglial proliferation in vivo and in vitro and are regulated by GM-CSF and density-dependent inhibition.

Hyperplasia of microglia is one characteristic of reactive gliosis, which is observed in various pathologic conditions in the central nervous system (CNS). To investigate the regulation mechanisms of microglial proliferation, the effect of granulocyte macrophage colony-stimulating factor (GM-CSF) on the expression of cell cycle-associated proteins was examined in the microglial cell line GMI-M6-3. After GM-CSF administration, cyclins D1, E, and A and cyclin-dependent kinase inhibitor p21Cip1 were increased, and another cyclin-dependent kinase inhibitor, p27Kip1, was decreased with morphologic transformation into ameboid form. By contrast, downregulation of these cyclins and p21Cip1, and strong upregulation of p27Kip1 accompanied by ramification were observed with GM-CSF deprivation. We also found that GMI-M6-3 exhibited homotypic contact inhibition of proliferation without any morphologic transformation. The increase of p27Kip1 and the decrease of cyclin A were suggested to play an important role in microglial contact inhibition. In addition, the direct effect of p27Kip1 to inhibit microglial proliferation was demonstrated both in vitro and in vivo by overexpression of p27Kip1.

Modulation of astrocyte proliferation by cyclin-dependent kinase inhibitor p27(Kip1).

We previously demonstrated that type 1 astrocytes exhibited homotypic cell contact-dependent inhibition of proliferation with increased expression of cyclin-dependent kinase inhibitor p27(Kip1). Here, we investigated the functional role of p27 in contact-dependent inhibition of astrocytes and reactive gliosis in vitro and in vivo. An increase in the number of proliferating cells was detected in high-density cultures of astrocytes derived from mice carrying a targeted deletion in the p27 gene compared to astrocytes from wild-type mice. Overexpression of p27 by adenovirus vectors inhibited astrocyte proliferation, which was accompanied by downregulation of cyclin A. In a gliosis model in vitro, a transient decrease in the p27 level and an increase in the proliferation rate were observed. Astrocyte proliferation following cortical injury lasted longer in p27-deficient mice than in wild-type mice. Forced expression of p27 in both in vitro and in vivo models of gliosis effectively suppressed astrocyte proliferation. In summary, we demonstrated that p27 contributed to the cell contact-dependent inhibition of astrocyte proliferation and to the cessation of proliferation in reactive astrocytosis. p27 may be used to modulate reactive astrocytosis.