Deletion of equilibrative nucleoside transporter 2 disturbs energy metabolism and exacerbates disease progression in an experimental model of Huntington's disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease, characterized by motor dysfunction and abnormal energy metabolism. Equilibrative nucleoside transporter 1 (ENT1) and ENT2 are the major nucleoside transporters in cellular plasma membrane of the brain. Yet, unlike ENT1 whose function has been better investigated in HD, the role of ENT2 in HD remains unclear. The present study aimed to investigate the impacts of ENT2 deletion on HD using a well-characterized mouse model (R6/2). Microarray analysis, quantitative real-time polymerase chain reaction, and immunostaining of ENT2 in postmortem human brain tissues were conducted. R6/2 mice with or without genetic deletion of ENT2 were generated. Motor functions, including rotarod performance and limb-clasping test, were examined at the age of 7 to 12 weeks. Biochemical changes were evaluated by immunofluorescence staining and immunoblotting at the age of 12 to 13 weeks. In regard to energy metabolism, levels of striatal metabolites were determined by liquid chromatography coupled with the fluorescence detector or quadrupole time-of-flight mass spectrometer. Mitochondrial bioenergetics was assessed by the Seahorse assay. The results showed that ENT2 protein was detected in the neurons and astrocytes of human brains and the levels in the postmortem brain tended to be higher in patients with HD. In mice, ENT2 deletion did not alter the phenotype of the non-HD controls. Yet, ENT2 deletion deteriorated motor function and increased the number of aggregated mutant huntingtin in the striatum of R6/2 mice. Notably, disturbed energy metabolism with decreased ATP level and increased AMP/ ATP ratio was observed in R6/2-Ent2-/- mice, compared with R6/2-Ent2+/+ mice, resulting in the activation of AMPK in the late disease stage. Furthermore, ENT2 deletion reduced the NAD+/NADH ratio and impaired mitochondrial respiration in the striatum of R6/2 mice. Taken together, these findings indicate the crucial role of ENT2 in energy homeostasis, in which ENT2 deletion further impairs mitochondrial bioenergetics and deteriorates motor function in R6/2 mice.

Equilibrative nucleoside transporter 1 inhibition rescues energy dysfunction and pathology in a model of tauopathy.

Tau pathology is instrumental in the gradual loss of neuronal functions and cognitive decline in tauopathies, including Alzheimer's disease (AD). Earlier reports showed that adenosine metabolism is abnormal in the brain of AD patients while consequences remained ill-defined. Herein, we aimed at investigating whether manipulation of adenosine tone would impact Tau pathology, associated molecular alterations and subsequent neurodegeneration. We demonstrated that treatment with an inhibitor (J4) of equilibrative nucleoside transporter 1 (ENT1) exerted beneficial effects in a mouse model of Tauopathy. Treatment with J4 not only reduced Tau hyperphosphorylation but also rescued memory deficits, mitochondrial dysfunction, synaptic loss, and abnormal expression of immune-related gene signatures. These beneficial effects were particularly ascribed to the ability of J4 to suppress the overactivation of AMPK (an energy reduction sensor), suggesting that normalization of energy dysfunction mitigates neuronal dysfunctions in Tauopathy. Collectively, these data highlight that targeting adenosine metabolism is a novel strategy for tauopathies.

Enhanced Na+ -K+ -2Cl- cotransporter 1 underlies motor dysfunction in huntington's disease.

BACKGROUND: Altered γ-aminobutyric acid signaling is believed to disrupt the excitation/inhibition balance in the striatum, which may account for the motor symptoms of Huntington's disease. Na-K-2Cl cotransporter-1 is a key molecule that controls γ-aminobutyric acid-ergic signaling. However, the role of Na-K-2Cl cotransporter-1 and efficacy of γ-aminobutyric acid-ergic transmission remain unknown in Huntington's disease. METHODS: We determined the levels of Na-K-2Cl cotransporter-1 in brain tissue from Huntington's disease mice and patients by real-time quantitative polymerase chain reaction, western blot, and immunocytochemistry. Gramicidin-perforated patch-clamp recordings were used to measure the Eγ-aminobutyric acid in striatal brain slices. To inhibit Na-K-2Cl cotransporter-1 activity, R6/2 mice were treated with an intraperitoneal injection of bumetanide or adeno-associated virus-mediated delivery of Na-K-2Cl cotransporter-1 short-hairpin RNA into the striatum. Motor behavior assays were employed. RESULTS: Expression of Na-K-2Cl cotransporter-1 was elevated in the striatum of R6/2 and Hdh150Q/7Q mouse models. An increase in Na-K-2Cl cotransporter-1 transcripts was also found in the caudate nucleus of Huntington's disease patients. Accordingly, a depolarizing shift of Eγ-aminobutyric acid was detected in the striatum of R6/2 mice. Expression of the mutant huntingtin in astrocytes and neuroinflammation were necessary for enhanced expression of Na-K-2Cl cotransporter-1 in HD mice. Notably, pharmacological or genetic inhibition of Na-K-2Cl cotransporter-1 rescued the motor deficits of R6/2 mice. CONCLUSIONS: Our findings demonstrate that aberrant γ-aminobutyric acid-ergic signaling and enhanced Na-K-2Cl cotransporter-1 contribute to the pathogenesis of Huntington's disease and identify a new therapeutic target for the potential rescue of motor dysfunction in patients with Huntington's disease. © 2019 International Parkinson and Movement Disorder Society.

Insights into GABAAergic system alteration in Huntington's disease.

Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disease that is characterized by a triad of motor, psychiatric and cognitive impairments. There is still no effective therapy to delay or halt the disease progress. The striatum and cortex are two particularly affected brain regions that exhibit dense reciprocal excitatory glutamate and inhibitory gamma-amino butyric acid (GABA) connections. Imbalance between excitatory and inhibitory signalling is known to greatly affect motor and cognitive processes. Emerging evidence supports the hypothesis that disrupted GABAergic circuits underlie HD pathogenesis. In the present review, we focused on the multiple defects recently found in the GABAergic inhibitory system, including altered GABA level and synthesis, abnormal subunit composition and distribution of GABAA receptors and aberrant GABAA receptor-mediated signalling. In particular, the important role of cation-chloride cotransporters (i.e. NKCC1 and KCC2) is discussed. Recent studies also suggest that neuroinflammation contributes significantly to the abnormal GABAergic inhibition in HD. Thus, GABAA receptors and cation-chloride cotransporters are potential therapeutic targets for HD. Given the limited availability of therapeutic treatments for HD, a better understanding of GABAergic dysfunction in HD could provide novel therapeutic opportunities.

Exogenous C8-Ceramide Induces Apoptosis by Overproduction of ROS and the Switch of Superoxide Dismutases SOD1 to SOD2 in Human Lung Cancer Cells.

Ceramides, abundant sphingolipids on the cell membrane, can act as signaling molecules to regulate cellular functions including cell viability. Exogenous ceramide has been shown to exert potent anti-proliferative effects against cancer cells, but little is known about how it affects reactive oxygen species (ROS) in lung cancer cells. In this study, we investigated the effect of N-octanoyl-D-erythro-sphingosine (C8-ceramide) on human non-small-cell lung cancer H1299 cells. Flow cytometry-based assays indicated that C8-ceramide increased the level of endogenous ROS in H1299 cells. Interestingly, the ratio of superoxide dismutases (SODs) SOD1 and SOD2 seem to be regulated by C8-ceramide treatment. Furthermore, the accumulation of cell cycle G1 phase and apoptotic populations in C8-ceramide-treated H1299 cells was observed. The results of the Western blot showed that C8-ceramide causes a dramatically increased protein level of cyclin D1, a critical regulator of cell cycle G1/S transition. These results suggest that C8-ceramide acts as a potent chemotherapeutic agent and may increase the endogenous ROS level by regulating the switch of SOD1 and SOD2, causing the anti-proliferation, and consequently triggering the apoptosis of NSCLC H1299 cells. Accordingly, our works may give a promising strategy for lung cancer treatment in the future.

Altered behavioral responses to gamma-aminobutyric acid pharmacological agents in a mouse model of Huntington's disease.

BACKGROUND: Disruptions in gamma-aminobutyric (GABA) acid signaling are believed to be involved in Huntington's disease pathogenesis, but the regulation of GABAergic signaling remains elusive. Here we evaluated GABAergic signaling by examining the function of GABAergic drugs in Huntington's disease and the expression of GABAergic molecules using mouse models and human brain tissues from Huntington's disease. METHODS: We treated wild-type and R6/2 mice (a transgenic Huntington's disease mouse model) acutely with vehicle, diazepam, or gaboxadol (drugs that selectively target synaptic or extrasynaptic GABAA receptors) and monitored their locomotor activity. The expression levels of GABAA receptors and a major neuron-specific chloride extruder (potassium-chloride cotransporter-2) were analyzed by real-time quantitative polymerase chain reaction, Western blot, and immunocytochemistry. RESULTS: The R6/2 mice were less sensitive to the sedative effects of both drugs, suggesting reduced function of GABAA receptors. Consistently, the expression levels of α1/α2 and δ subunits were lower in the cortex and striatum of R6/2 mice. Similar results were also found in 2 other mouse models of Huntington's disease and in Huntington's disease patients. Moreover, the interaction and expression levels of potassium-chloride cotransporter-2 and its activator (brain-type creatine kinase) were decreased in Huntington's disease neurons. These findings collectively suggest impaired chloride homeostasis, which further dampens GABAA receptor-mediated inhibitory signaling in Huntington's disease brains. CONCLUSIONS: The dysregulated GABAergic responses and altered expression levels of GABAA receptors and potassium-chloride cotransporter-2 in Huntington's disease mice appear to be authentic and may contribute to the clinical manifestations of Huntington's disease patients. © 2017 International Parkinson and Movement Disorder Society.