TAR DNA-binding protein-43 (TDP-43) is a nuclear RNA-binding protein involved in many cellular pathways. TDP-43-positive inclusions are a hallmark of amyotrophic lateral sclerosis (ALS). The major clinical presentation of ALS is muscle weakness due to the degeneration of motor neurons. Mislocalization of TDP-43 from the nucleus to the cytoplasm is an early event of ALS. In this study, we demonstrate that cytoplasmic mislocalization of TDP-43 was accompanied by increased activation of AMP-activated protein kinase (AMPK) in motor neurons of ALS patients. The activation of AMPK in a motor neuron cell line (NSC34) or mouse spinal cords induced the mislocalization of TDP-43, recapitulating this characteristic of ALS. Down-regulation of AMPK-α1 or exogenous expression of a dominant-negative AMPK-α1 mutant reduced TDP-43 mislocalization. Suppression of AMPK activity using cAMP-simulating agents rescued the mislocalization of TDP-43 in NSC34 cells and delayed disease progression in TDP-43 transgenic mice. Our findings demonstrate that activation of AMPK-α1 plays a critical role in TDP-43 mislocalization and the development of ALS; thus, AMPK-α1 may be a potential drug target for this devastating disease.

Blockage of the p53 tumor suppressor has been found to impair nerve growth factor (NGF)-induced neurite outgrowth in PC-12 cells. We report herein that such impairment could be rescued by stimulation of the A 2A adenosine receptor (A 2A -R), a G protein-coupled receptor implicated in neuronal plasticity. The A 2A -R-mediated rescue occurred in the presence of protein kinase C (PKC) inhibitors or protein kinase A (PKA) inhibitors and in a PKA-deficient PC-12 variant. Thus, neither PKA nor PKC was involved. In contrast, expression of a truncated A 2A -R mutant harboring the seventh transmembrane domain and its C terminus reduced the rescue effect of A 2A -R. Using the cytoplasmic tail of the A 2A -R as bait, a novel-A 2A -R-interacting protein [translin-associated protein X (TRAX)] was identified in a yeast two-hybrid screen. The authenticity of this interaction was verified by pull-down experiments, coimmunoprecipitation, and colocalization of these two molecules in the brain. It is noteworthy that reduction of TRAX using an antisense construct suppressed the rescue effect of A 2A -R, whereas overexpression of TRAX alone caused the same rescue effect as did A 2A -R activation. Results of [ 3 H]thymidine and bromodeoxyuridine incorporation suggested that A 2A -R stimulation inhibited cell proliferation in a TRAX-dependent manner. Because the antimitotic activity is crucial for NGF function, the A 2A -R might exert its rescue effect through a TRAX-mediated antiproliferative signal. This antimitotic activity of the A 2A -R also enables a mitogenic factor (epidermal growth factor) to induce neurite outgrowth. We demonstrate that the A 2A -R modulates the differentiation ability of trophic factors through a novel interacting protein, TRAX.

Previous results from our laboratory have shown that phosphorylation of type VI adenylyl cyclase (ACVI) by protein kinase C (PKC) caused suppression of adenylyl cyclase activity. In the present study, we investigated the role of the N terminus cytosolic domain of ACVI in this PKC-mediated inhibition of ACVI. Removal of amino acids 1 to 86 of ACVI or mutation of Ser(10) (a potential PKC phosphorylation site) into alanine significantly relieved the PKC-mediated inhibition and markedly reduced the PKC-evoked protein phosphorylation. PKC also effectively phosphorylated a recombinant N terminus cytosolic domain (amino acids 1-160) protein of ACVI and a synthetic peptide representing Ser(10). In addition, the amino acids 1 to 86 truncated mutant exhibited kinetic properties similar to those of the wild type. Taken together, these data demonstrate that the highly variable N terminus cytoplasmic domain of ACVI is a regulatory domain with a critical role in PKC-mediated suppression, which is a hallmark of this adenylyl cyclase isozyme. In addition, Ser(10) was found to serve as an acceptor for the PKC-mediated phosphorylating transfer of ACVI.

Huntington's disease (HD) is a neurodegenerative disease caused by the expansion of a CAG repeat in the Huntingtin (HTT) gene. Abnormal regulation of the cyclic AMP (cAMP)/protein kinase A (PKA) pathway occurs during HD progression. Here we found that lower PKA activity was associated with proteasome impairment in the striatum for two HD mouse models (R6/2 and N171-82Q) and in mutant HTT (mHTT)-expressing striatal cells. Because PKA regulatory subunits (PKA-Rs) are proteasome substrates, the mHTT-evoked proteasome impairment caused accumulation of PKA-Rs and subsequently inhibited PKA activity. Conversely, activation of PKA enhanced the phosphorylation of Rpt6 (a component of the proteasome), rescued the impaired proteasome activity, and reduced mHTT aggregates. The dominant-negative Rpt6 mutant (Rpt6 S120A ) blocked the ability of a cAMP-elevating reagent to enhance proteasome activity, whereas the phosphomimetic Rpt6 mutant (Rpt6 S120D ) increased proteasome activity, reduced HTT aggregates, and ameliorated motor impairment. Collectively, our data demonstrated that positive feedback regulation between PKA and the proteasome is critical for HD pathogenesis. Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by expansion of a CAG repeat in the Huntingtin (HTT) gene. The normal HTT gene has 35 or fewer CAG repeats in its N-terminal region, whereas expansion of the repeat beyond 35 units confers toxicity to the Huntingtin protein (HTT) and evokes HD pathogenesis (1). The major clinical presentations of HD include chorea, cognitive decline, psychiatric symptoms, and a systemic metabolic defect (2). Selective neuronal loss occurs in multiple brain regions, particularly in the striatum and cortex. Since a great number of cellular machineries (including transcription, vesicle transport, molecular chaperones, and the ubiquitin-proteasome system [UPS]) are compromised by the expression of mutant HTT (mHTT) (3), effective removal of mHTT has become one of the most challenging aspects of drug development for HD.The UPS, which degrades misfolded and/or damaged proteins in eukaryotes, is the major regulatory proteolytic pathway. It regulates many essential cellular processes, including transcription, translation, DNA repair, chromatin remodeling, cell survival, signal transduction, protein trafficking, and synaptic plasticity (4). A recent study demonstrated that AAA-ATPase subunits are critical for cell-type-specific regulation of UPS activity (5). Interestingly, UPS activity is under metabolic control. Posttranslational modifications, such as O-GlcNacylation and phosphorylation of 19S subunits, affect the proteolytic activity of the UPS (6). In addition, impaired functioning of the UPS is believed to contribute to the pathogenic processes of many neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease (PD), and HD. Recent studies revealed that the UPS might be impaired in HD mouse models and patients (7,8). Intriguingly, although the degradation of polyubiquitin-tagg...

In the present study, we used the N terminus (amino acids 1ϳ160) of type VI adenylyl cyclase (ACVI) as bait to screen a mouse brain cDNA library and identified Snapin as a novel ACVI-interacting molecule. Snapin is a binding protein of SNAP25, a component of the SNARE complex. Co-immunoprecipitation analyses confirmed the interaction between Snapin and full-length ACVI. Mutational analysis revealed that the interaction domains of ACVI and Snapin were located within amino acids 1ϳ86 of ACVI and 33-51 of Snapin, respectively. Co-localization of ACVI and Snapin was observed in primary hippocampal neurons. Moreover, expression of Snapin specifically eliminated protein kinase C (PKC)-mediated suppression of ACVI, but not that of cAMP-dependent protein kinase (PKA) or calcium. Mutation of the potential PKC and PKA phosphorylation sites of Snapin did not affect the ability of Snapin to reverse the PKC inhibitory effect on ACVI. Phosphorylation of Snapin by PKC or PKA therefore might not be crucial for Snapin action on ACVI. In contrast, Snapin ⌬33-51 , which harbors an internal deletion of amino acids 33-51 did not affect PKC-mediated inhibition of ACVI, supporting that amino acids 33-51 of Snapin comprises the ACVI-interacting region. Consistently, Snapin exerted no effect on PKC-mediated inhibition of an ACVI mutant (ACVI-⌬A87), which lacked the Snapin-interacting region (amino acids 1-86). Snapin thus reverses its action via direct interaction with the N terminus of ACVI. Collectively, we demonstrate herein that in addition to its association with the SNARE complex, Snapin also functions as a regulator of an important cAMP synthesis enzyme in the brain. Adenylyl cyclases (ACs)1 are a family of enzymes that produce cyclic AMP (cAMP) from ATP upon extracellular stimulation. To date, at least 9 membrane-bound ACs have been isolated and characterized (1). These enzymes are capable of integrating positive and negative signals that act directly through stimulation of G protein-coupled receptors (GPCRs) or indirectly via intracellular signaling molecules in isozyme-specific patterns. In addition, the regulatory properties and expression patterns of different AC isoforms greatly diverge and may account for the distinctive cell-and tissue-specific responsiveness of ACs. Recently, several different proteins, including RGS2 and the protein associated with Myc (PAM), have been shown to interact and modulate activity of different AC isozymes (2, 3), adding additional dimensions to the isozymespecific regulation of the AC superfamily.Except for the newly identified soluble AC, all membranebound AC members share a primary structure consisting of 12 transmembrane regions and 3 large cytoplasmic domains (N, C1a/b, and C2). The C1a and C2 domains, which form the catalytic core complex, are highly conserved and are homologous to each other. The N-terminal domains of ACs, in contrast, are variable among ACs, and have been demonstrated to play mostly regulatory roles (4, 5). Among the AC isozymes, ACVI is of particular interest, because...

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by cognitive impairment and synaptic dysfunction. Adenosine is an important homeostatic modulator that controls the bioenergetic network in the brain through regulating receptor-evoked signaling pathways, bioenergetic machineries, and epigenetic-mediated gene regulation. Equilibrative nucleoside transporter 1 (ENT1) is a major adenosine transporter that recycles adenosine from the extracellular space. In the present study, we report that a small adenosine analogue (designated J4) that inhibited ENT1 prevented the decline in spatial memory in an AD mouse model (APP/PS1). Electrophysiological and biochemical analyses further demonstrated that chronic treatment with J4 normalized the impaired basal synaptic transmission and long-term potentiation (LTP) at Schaffer collateral synapses as well as the aberrant expression of synaptic proteins (e.g., NR2A and NR2B), abnormal neuronal plasticity-related signaling pathways (e.g., PKA and GSK3β), and detrimental elevation in astrocytic AR expression in the hippocampus and cortex of APP/PS1 mice. In conclusion, our findings suggest that modulation of adenosine homeostasis by J4 is beneficial in a mouse model of AD. Our study provides a potential therapeutic strategy to delay the progression of AD.

BackgroundHuntington's disease (HD) is a neurodegenerative disease caused by a CAG trinucleotide expansion in the Huntingtin (Htt) gene. The expanded CAG repeats are translated into polyglutamine (polyQ), causing aberrant functions as well as aggregate formation of mutant Htt. Effective treatments for HD are yet to be developed.Methodology/Principal FindingsHere, we report a novel dual-function compound, N 6-(4-hydroxybenzyl)adenine riboside (designated T1-11) which activates the A2AR and a major adenosine transporter (ENT1). T1-11 was originally isolated from a Chinese medicinal herb. Molecular modeling analyses showed that T1-11 binds to the adenosine pockets of the A2AR and ENT1. Introduction of T1-11 into the striatum significantly enhanced the level of striatal adenosine as determined by a microdialysis technique, demonstrating that T1-11 inhibited adenosine uptake in vivo. A single intraperitoneal injection of T1-11 in wildtype mice, but not in A2AR knockout mice, increased cAMP level in the brain. Thus, T1-11 enters the brain and elevates cAMP via activation of the A2AR in vivo. Most importantly, addition of T1-11 (0.05 mg/ml) to the drinking water of a transgenic mouse model of HD (R6/2) ameliorated the progressive deterioration in motor coordination, reduced the formation of striatal Htt aggregates, elevated proteasome activity, and increased the level of an important neurotrophic factor (brain derived neurotrophic factor) in the brain. These results demonstrate the therapeutic potential of T1-11 for treating HD.Conclusions/SignificanceThe dual functions of T1-11 enable T1-11 to effectively activate the adenosinergic system and subsequently delay the progression of HD. This is a novel therapeutic strategy for HD. Similar dual-function drugs aimed at a particular neurotransmitter system as proposed herein may be applicable to other neurotransmitter systems (e.g., the dopamine receptor/dopamine transporter and the serotonin receptor/serotonin transporter) and may facilitate the development of new drugs for other neurodegenerative diseases.

Huntington's disease (HD) is caused by an abnormal CAG expansion in the exon 1 of huntingtin gene. The treatment of HD is an unmet medical need. Given the important role of adenosine in modulating brain activity, in this study, levels of adenosine and adenine nucleotides in the cerebral spinal fluid of patients with HD and in the brain of two mouse models of HD (R6/2 and Hdh150Q) were analysed. The expression and activity of ENT1 in the striatum of mice with HD were measured. Targeting adenosine tone for treating HD was examined in R6/2 mice by genetic removal of ENT1 and by giving an ENT1 inhibitor, respectively. The results showed that the adenosine homeostasis is dysregulated in the brain of patients and mice with HD. In patients, the ratio of adenosine/ATP in the cerebral spinal fluid was negatively correlated with the disease duration, and tended to have a positive correlation with independence scale and functional capacity. In comparison to controls, mRNA level of ENT1 was higher in the striatum of R6/2 and Hdh150Q mice. Intrastriatal administration of ENT1 inhibitors increased extracellular level of adenosine in the striatum of R6/2 mice to a much higher level than controls. Chronic inhibition of ENT1 or by genetic removal of ENT1 enhanced the survival of R6/2 mice. Collectively, adenosine homeostasis and ENT1 expression are altered in HD. The inhibition of ENT1 can enhance extracellular adenosine level and be a potential therapeutic approach for treating HD.