Targeting Astrocyte Signaling Alleviates Cerebrovascular and Synaptic Function Deficits in a Diet-Based Mouse Model of Small Cerebral Vessel Disease.

Despite the indispensable role that astrocytes play in the neurovascular unit, few studies have investigated the functional impact of astrocyte signaling in cognitive decline and dementia related to vascular pathology. Diet-mediated induction of hyperhomocysteinemia (HHcy) recapitulates numerous features of vascular contributions to cognitive impairment and dementia (VCID). Here, we used astrocyte targeting approaches to evaluate astrocyte Ca2+ dysregulation and the impact of aberrant astrocyte signaling on cerebrovascular dysfunction and synapse impairment in male and female HHcy diet mice. Two-photon imaging conducted in fully awake mice revealed activity-dependent Ca2+ dysregulation in barrel cortex astrocytes under HHcy. Stimulation of contralateral whiskers elicited larger Ca2+ transients in individual astrocytes of HHcy diet mice compared with control diet mice. However, evoked Ca2+ signaling across astrocyte networks was impaired in HHcy mice. HHcy also was associated with increased activation of the Ca2+/calcineurin-dependent transcription factor NFAT4, which has been linked previously to the reactive astrocyte phenotype and synapse dysfunction in amyloid and brain injury models. Targeting the NFAT inhibitor VIVIT to astrocytes, using adeno-associated virus vectors, led to reduced GFAP promoter activity in HHcy diet mice and improved functional hyperemia in arterioles and capillaries. VIVIT expression in astrocytes also preserved CA1 synaptic function and improved spontaneous alternation performance on the Y maze. Together, the results demonstrate that aberrant astrocyte signaling can impair the major functional properties of the neurovascular unit (i.e., cerebral vessel regulation and synaptic regulation) and may therefore represent a promising drug target for treating VCID and possibly Alzheimer's disease and other related dementias.SIGNIFICANCE STATEMENT The impact of reactive astrocytes in Alzheimer's disease and related dementias is poorly understood. Here, we evaluated Ca2+ responses and signaling in barrel cortex astrocytes of mice fed with a B-vitamin deficient diet that induces hyperhomocysteinemia (HHcy), cerebral vessel disease, and cognitive decline. Multiphoton imaging in awake mice with HHcy revealed augmented Ca2+ responses in individual astrocytes, but impaired signaling across astrocyte networks. Stimulation-evoked arteriole dilation and elevated red blood cell velocity in capillaries were also impaired in cortex of awake HHcy mice. Astrocyte-specific inhibition of the Ca2+-dependent transcription factor, NFAT, normalized cerebrovascular function in HHcy mice, improved synaptic properties in brain slices, and stabilized cognition. Results suggest that astrocytes are a mechanism and possible therapeutic target for vascular-related dementia.

Calcineurin/NFAT Signaling in Activated Astrocytes Drives Network Hyperexcitability in Aß-Bearing Mice.

Hyperexcitable neuronal networks are mechanistically linked to the pathologic and clinical features of Alzheimer's disease (AD). Astrocytes are a primary defense against hyperexcitability, but their functional phenotype during AD is poorly understood. Here, we found that activated astrocytes in the 5xFAD mouse model were strongly associated with proteolysis of the protein phosphatase calcineurin (CN) and the elevated expression of the CN-dependent transcription factor nuclear factor of activated T cells 4 (NFAT4). Intrahippocampal injections of adeno-associated virus vectors containing the astrocyte-specific promoter Gfa2 and the NFAT inhibitory peptide VIVIT reduced signs of glutamate-mediated hyperexcitability in 5xFAD mice, measured in vivo with microelectrode arrays and ex vivo brain slices, using whole-cell voltage clamp. VIVIT treatment in 5xFAD mice led to increased expression of the astrocytic glutamate transporter GLT-1 and to attenuated changes in dendrite morphology, synaptic strength, and NMDAR-dependent responses. The results reveal astrocytic CN/NFAT4 as a key pathologic mechanism for driving glutamate dysregulation and neuronal hyperactivity during AD.SIGNIFICANCE STATEMENT Neuronal hyperexcitability and excitotoxicity are increasingly recognized as important mechanisms for neurodegeneration and dementia associated with Alzheimer's disease (AD). Astrocytes are profoundly activated during AD and may lose their capacity to regulate excitotoxic glutamate levels. Here, we show that a highly active calcineurin (CN) phosphatase fragment and its substrate transcription factor, nuclear factor of activated T cells (NFAT4), appear in astrocytes in direct proportion to the extent of astrocyte activation. The blockade of astrocytic CN/NFAT signaling in a common mouse model of AD, using adeno-associated virus vectors normalized glutamate signaling dynamics, increased astrocytic glutamate transporter levels and alleviated multiple signs of neuronal hyperexcitability. The results suggest that astrocyte activation drives hyperexcitability during AD through a mechanism involving aberrant CN/NFAT signaling and impaired glutamate transport.

Blockade of Astrocytic Calcineurin/NFAT Signaling Helps to Normalize Hippocampal Synaptic Function and Plasticity in a Rat Model of Traumatic Brain Injury.

Increasing evidence suggests that the calcineurin (CN)-dependent transcription factor NFAT (Nuclear Factor of Activated T cells) mediates deleterious effects of astrocytes in progressive neurodegenerative conditions. However, the impact of astrocytic CN/NFAT signaling on neural function/recovery after acute injury has not been investigated extensively. Using a controlled cortical impact (CCI) procedure in rats, we show that traumatic brain injury is associated with an increase in the activities of NFATs 1 and 4 in the hippocampus at 7 d after injury. NFAT4, but not NFAT1, exhibited extensive labeling in astrocytes and was found throughout the axon/dendrite layers of CA1 and the dentate gyrus. Blockade of the astrocytic CN/NFAT pathway in rats using adeno-associated virus (AAV) vectors expressing the astrocyte-specific promoter Gfa2 and the NFAT-inhibitory peptide VIVIT prevented the injury-related loss of basal CA1 synaptic strength and key synaptic proteins and reduced the susceptibility to induction of long-term depression. In conjunction with these seemingly beneficial effects, VIVIT treatment elicited a marked increase in the expression of the prosynaptogenic factor SPARCL1 (hevin), especially in hippocampal tissue ipsilateral to the CCI injury. However, in contrast to previous work on Alzheimer's mouse models, AAV-Gfa2-VIVIT had no effects on the levels of GFAP and Iba1, suggesting that synaptic benefits of VIVIT were not attributable to a reduction in glial activation per se. Together, the results implicate the astrocytic CN/NFAT4 pathway as a key mechanism for disrupting synaptic remodeling and homeostasis in the hippocampus after acute injury. SIGNIFICANCE STATEMENT: Similar to microglia, astrocytes become strongly "activated" with neural damage and exhibit numerous morphologic/biochemical changes, including an increase in the expression/activity of the protein phosphatase calcineurin. Using adeno-associated virus (AAV) to inhibit the calcineurin-dependent activation of the transcription factor NFAT (Nuclear Factor of Activated T cells) selectively, we have shown that activated astrocytes contribute to neural dysfunction in animal models characterized by progressive/chronic neuropathology. Here, we show that the suppression of astrocytic calcineurin/NFATs helps to protect synaptic function and plasticity in an animal model in which pathology arises from a single traumatic brain injury. The findings suggest that at least some astrocyte functions impair recovery after trauma and may provide druggable targets for treating victims of acute nervous system injury.

Calcineurin proteolysis in astrocytes: Implications for impaired synaptic function.

Mounting evidence suggests that astrocyte activation, found in most forms of neural injury and disease, is linked to the hyperactivation of the protein phosphatase calcineurin. In many tissues and cell types, calcineurin hyperactivity is the direct result of limited proteolysis. However, little is known about the proteolytic status of calcineurin in activated astrocytes. Here, we developed a polyclonal antibody to a high activity calcineurin proteolytic fragment in the 45-48kDa range (ΔCN) for use in immunohistochemical applications. When applied to postmortem human brain sections, the ΔCN antibody intensely labeled cell clusters in close juxtaposition to amyloid deposits and microinfarcts. Many of these cells exhibited clear activated astrocyte morphology. The expression of ΔCN in astrocytes near areas of pathology was further confirmed using confocal microscopy. Multiple NeuN-positive cells, particularly those within microinfarct core regions, also labeled positively for ΔCN. This observation suggests that calcineurin proteolysis can also occur within damaged or dying neurons, as reported in other studies. When a similar ΔCN fragment was selectively expressed in hippocampal astrocytes of intact rats (using adeno-associated virus), we observed a significant reduction in the strength of CA3-CA1 excitatory synapses, indicating that the hyperactivation of astrocytic calcineurin is sufficient for disrupting synaptic function. Together, these results suggest that proteolytic activation of calcineurin in activated astrocytes may be a central mechanism for driving and/or exacerbating neural dysfunction during neurodegenerative disease and injury.

Vitamin D prevents cognitive decline and enhances hippocampal synaptic function in aging rats.

Vitamin D is an important calcium-regulating hormone with diverse functions in numerous tissues, including the brain. Increasing evidence suggests that vitamin D may play a role in maintaining cognitive function and that vitamin D deficiency may accelerate age-related cognitive decline. Using aging rodents, we attempted to model the range of human serum vitamin D levels, from deficient to sufficient, to test whether vitamin D could preserve or improve cognitive function with aging. For 5-6 mo, middle-aged F344 rats were fed diets containing low, medium (typical amount), or high (100, 1,000, or 10,000 international units/kg diet, respectively) vitamin D3, and hippocampal-dependent learning and memory were then tested in the Morris water maze. Rats on high vitamin D achieved the highest blood levels (in the sufficient range) and significantly outperformed low and medium groups on maze reversal, a particularly challenging task that detects more subtle changes in memory. In addition to calcium-related processes, hippocampal gene expression microarrays identified pathways pertaining to synaptic transmission, cell communication, and G protein function as being up-regulated with high vitamin D. Basal synaptic transmission also was enhanced, corroborating observed effects on gene expression and learning and memory. Our studies demonstrate a causal relationship between vitamin D status and cognitive function, and they suggest that vitamin D-mediated changes in hippocampal gene expression may improve the likelihood of successful brain aging.

Elevated calcineurin activity in primary astrocytes leads to the dephosphorylation of connexin 43 in conjunction with increased membrane permeability.

Hyperactivation of the Ca2+/calmodulin-dependent phosphatase calcineurin (CN) is observed in reactive astrocytes associated with neuroinflammation and progressive degenerative diseases, like Alzheimer's disease. Apart from key transcription factors (e.g. nuclear factor of activated t cells and nuclear factor-κB) very few other CN-dependent pathways have been studied in astrocytes. The hemichannel protein, connexin 43 (Cx43) is found at high levels in astrocytes and contains a CN-sensitive Ser residue near its carboxy terminus. CN-dependent dephosphorylation of Cx43 has been reported in primary astrocytes treated with injurious stimuli, but much remains unknown about CN/Cx43 interactions in the context of neuroinflammation and disease. Western blots were used to assess total Cx43 and dephosphorylated Cx43 subtypes in rat embryonic primary astrocytes treated with a hyperactive CN fragment (ΔCN, via adenovirus), or with a proinflammatory cytokine cocktail. Under similar treatment conditions, an ethidium bromide (EtBr) uptake assay was used to assess membrane permeability. Effects of ΔCN and cytokines were tested in the presence or absence of the CN inhibitor, cyclosporin A. A connexin inhibitor, carbenoxolone was also used in EtBr assays to assess the involvement of connexins in membrane permeability. Treatment with ΔCN or cytokines increased dephosphorylated Cx43 levels in conjunction with increased membrane permeability (elevated EtBr uptake). Effects of ΔCN or cytokine treatment were blocked by cyclosporine A. Treatment-induced changes in EtBr uptake were also inhibited by carbenoxolone. The results suggest that Cx43 hemichannels could be an important mechanism through which astrocytic CN disrupts neurologic function associated with neurodegenerative disease.

Development of a monoclonal antibody specific for a calpain-generated ∆48 kDa calcineurin fragment, a marker of distressed astrocytes.

BACKGROUND: Calcineurin (CN) is a Ca2+/calmodulin-dependent protein phosphatase. In healthy tissue, CN exists mainly as a full-length (∼60 kDa) highly-regulated protein phosphatase involved in essential cellular functions. However, in diseased or injured tissue, CN is proteolytically converted to a constitutively active fragment that has been causatively-linked to numerous pathophysiologic processes. These calpain-cleaved CN fragments (∆CN) appear at high levels in human brain at early stages of cognitive decline associated with Alzheimer's disease (AD). NEW METHOD: We developed a monoclonal antibody to ∆CN, using an immunizing peptide corresponding to the C-terminal end of the ∆CN fragment. RESULTS: We obtained a mouse monoclonal antibody, designated 26A6, that selectively detects ∆CN in Western analysis of calpain-cleaved recombinant human CN. Using this antibody, we screened both pathological and normal human brain sections provided by the University of Kentucky's Alzheimer's Disease Research Center. 26A6 showed low reactivity towards normal brain tissue, but detected astrocytes both surrounding AD amyloid plaques and throughout AD brain tissue. In brain tissue with infarcts, there was considerable concentration of 26A6-positive astrocytes within/around infarcts, suggesting a link with anoxic/ischemia pathways. COMPARISON WITH EXISTING METHOD: The results obtained with the new monoclonal are similar to those obtained with a polyclonal we had previously developed. However, the monoclonal is an abundant tool available to the dementia research community. CONCLUSIONS: The new monoclonal 26A6 antibody is highly selective for the ∆CN proteolytic fragment and labels a subset of astrocytes, and could be a useful tool for marking insidious brain pathology and identifying novel astrocyte phenotypes.

Upregulation of the CaV 1.1-ryanodine receptor complex in a rat model of critical illness myopathy.

The processes that trigger severe muscle atrophy and loss of myosin in critical illness myopathy (CIM) are poorly understood. It has been reported that muscle disuse alters Ca(2+) handling by the sarcoplasmic reticulum. Since inactivity is an important contributor to CIM, this finding raises the possibility that elevated levels of the proteins involved in Ca(2+) handling might contribute to development of CIM. CIM was induced in 3- to 5-mo-old rats by sciatic nerve lesion and infusion of dexamethasone for 1 wk. Western blot analysis revealed increased levels of ryanodine receptor (RYR) isoforms-1 and -2 as well as the dihydropyridine receptor/voltage-gated calcium channel type 1.1 (DHPR/Ca(V) 1.1). Immunostaining revealed a subset of fibers with elevation of RYR1 and Ca(V) 1.1 that had severe atrophy and disorganization of sarcomeres. These findings suggest increased Ca(2+) release from the sarcoplasmic reticulum may be an important contributor to development of CIM. To assess the endogenous functional effects of increased intracellular Ca(2+) in CIM, proteolysis of α-fodrin, a well-known target substrate of Ca(2+)-activated proteases, was measured and found to be 50% greater in CIM. There was also selective degradation of myosin heavy chain relative to actin in CIM muscle. Taken together, our findings suggest that increased Ca(2+) release from the sarcoplasmic reticulum may contribute to pathology in CIM.

Q134R: Small chemical compound with NFAT inhibitory properties improves behavioral performance and synapse function in mouse models of amyloid pathology.

Inhibition of the protein phosphatase calcineurin (CN) ameliorates pathophysiologic and cognitive changes in aging rodents and mice with aging-related Alzheimer's disease (AD)-like pathology. However, concerns over adverse effects have slowed the transition of common CN-inhibiting drugs to the clinic for the treatment of AD and AD-related disorders. Targeting substrates of CN, like the nuclear factor of activated T cells (NFATs), has been suggested as an alternative, safer approach to CN inhibitors. However, small chemical inhibitors of NFATs have only rarely been described. Here, we investigate a newly developed neuroprotective hydroxyquinoline derivative (Q134R) that suppresses NFAT signaling, without inhibiting CN activity. Q134R partially inhibited NFAT activity in primary rat astrocytes, but did not prevent CN-mediated dephosphorylation of a non-NFAT target, either in vivo, or in vitro. Acute (≤1 week) oral delivery of Q134R to APP/PS1 (12 months old) or wild-type mice (3-4 months old) infused with oligomeric Aß peptides led to improved Y maze performance. Chronic (≥3 months) oral delivery of Q134R appeared to be safe, and, in fact, promoted survival in wild-type (WT) mice when given for many months beyond middle age. Finally, chronic delivery of Q134R to APP/PS1 mice during the early stages of amyloid pathology (i.e., between 6 and 9 months) tended to reduce signs of glial reactivity, prevented the upregulation of astrocytic NFAT4, and ameliorated deficits in synaptic strength and plasticity, without noticeably altering parenchymal Aß plaque pathology. The results suggest that Q134R is a promising drug for treating AD and aging-related disorders.

Cognitive decline in Alzheimer's disease is associated with selective changes in calcineurin/NFAT signaling.

Upon activation by calcineurin, the nuclear factor of activated T-cells (NFAT) translocates to the nucleus and guides the transcription of numerous molecules involved in inflammation and Ca(2+) dysregulation, both of which are prominent features of Alzheimer's disease (AD). However, NFAT signaling in AD remains relatively uninvestigated. Using isolated cytosolic and nuclear fractions prepared from rapid-autopsy postmortem human brain tissue, we show that NFATs 1 and 3 shifted to nuclear compartments in the hippocampus at different stages of neuropathology and cognitive decline, whereas NFAT2 remained unchanged. NFAT1 exhibited greater association with isolated nuclear fractions in subjects with mild cognitive impairment (MCI), whereas NFAT3 showed a strong nuclear bias in subjects with severe dementia and AD. Similar to NFAT1, calcineurin-Aalpha also exhibited a nuclear bias in the early stages of cognitive decline. But, unlike NFAT1 and similar to NFAT3, the nuclear bias for calcineurin became more pronounced as cognition worsened. Changes in calcineurin/NFAT3 were directly correlated to soluble amyloid-beta (Abeta((1-42))) levels in postmortem hippocampus, and oligomeric Abeta, in particular, robustly stimulated NFAT activation in primary rat astrocyte cultures. Oligomeric Abeta also caused a significant reduction in excitatory amino acid transporter 2 (EAAT2) protein levels in astrocyte cultures, which was blocked by NFAT inhibition. Moreover, inhibition of astrocytic NFAT activity in mixed cultures ameliorated Abeta-dependent elevations in glutamate and neuronal death. The results suggest that NFAT signaling is selectively altered in AD and may play an important role in driving Abeta-mediated neurodegeneration.

Basic helix-loop-helix factors recruit nuclear factor I to enhance expression of the NaV 1.4 Na+ channel gene.

We have previously shown that the basic helix-loop-helix (bHLH) transcription factors coordinate Na(V) 1.4 Na(+) channel gene expression in skeletal muscle, but the identity of the co-factors they direct is unknown. Using C2C12 muscle cells as a model system, we test the hypothesis that the bHLH factors counteract negative regulation exerted through a repressor E box (-90/-85) by recruiting positive-acting transcription factors to the nucleotides (-135/-57) surrounding the repressor E box. We used electrophoretic mobility shift assays to identify candidate factors that bound the repressor E box or these adjacent regions. Repressor E box-binding factors included the known transcription factor, ZEB/AREB6, and a novel repressor E box-binding factor designated REB. Mutations of the repressor E box that interfere with the binding of these factors prevented repression. The transcription factor, nuclear factor I (NFI), bound immediately upstream and downstream of the repressor E box. Mutation of the NFI-binding sites diminished the ability of myogenin and MRF4 to counteract repression. Based on these observations we suggest that bHLH factors recruit NFI to enhance skeletal muscle Na(+) channel expression.

Astrocyte Activation and the Calcineurin/NFAT Pathway in Cerebrovascular Disease.

Calcineurin (CN) is a Ca2+/calmodulin-dependent protein phosphatase with high abundance in nervous tissue. Though enriched in neurons, CN can become strongly induced in subsets of activated astrocytes under different pathological conditions where it interacts extensively with the nuclear factor of activated T cells (NFATs). Recent work has shown that regions of small vessel damage are associated with the upregulation of a proteolized, highly active form of CN in nearby astrocytes, suggesting a link between the CN/NFAT pathway and chronic cerebrovascular disease. In this Mini Review article, we discuss CN/NFAT signaling properties in the context of vascular disease and use previous cell type-specific intervention studies in Alzheimer's disease and traumatic brain injury models as a framework to understand how astrocytic CN/NFATs may couple vascular pathology to neurodegeneration and cognitive loss.

NFATc2 Modulates Microglial Activation in the AßPP/PS1 Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) brains are characterized by fibrillar amyloid-ß (Aß) peptide containing plaques and associated reactive microglia. The proinflammatory phenotype of the microglia suggests that they may negatively affect disease course and contribute to behavioral decline. This hypothesis predicts that attenuating microglial activation may provide benefit against disease. Prior work from our laboratory and others has characterized a role for the transcription factor, nuclear factor of activated T cells (NFAT), in regulating microglial phenotype in response to different stimuli, including Aß peptide. We observed that the NFATc2 isoform was the most highly expressed in murine microglia cultures, and inhibition or deletion of NFATc2 was sufficient to attenuate the ability of the microglia to secrete cytokines. In order to determine whether the NFATc2 isoform, in particular, was a valid immunomodulatory target in vivo, we crossed an NFATc2-/- line to a well-known AD mouse model, an AßPP/PS1 mouse line. As expected, the AßPP/PS1 x NFATc2-/- mice had attenuated cytokine levels compared to AßPP/PS1 mice as well as reduced microgliosis and astrogliosis with no effect on plaque load. Although some species differences in relative isoform expression may exist between murine and human microglia, it appears that microglial NFAT activity is a viable target for modulating the proinflammatory changes that occur during AD.

Calcineurin triggers reactive/inflammatory processes in astrocytes and is upregulated in aging and Alzheimer's models.

Astrocyte reactivity (i.e., activation) and associated neuroinflammation are increasingly thought to contribute to neurodegenerative disease. However, the mechanisms that trigger astrocyte activation are poorly understood. Here, we studied the Ca2+-dependent phosphatase calcineurin, which regulates inflammatory signaling pathways in immune cells, for a role in astrogliosis and brain neuroinflammation. Adenoviral transfer of activated calcineurin to primary rat hippocampal cultures resulted in pronounced thickening of astrocyte somata and processes compared with uninfected or virus control cultures, closely mimicking the activated hypertrophic phenotype. This effect was blocked by the calcineurin inhibitor cyclosporin A. Parallel microarray studies, validated by extensive statistical analyses, showed that calcineurin overexpression also induced genes and cellular pathways representing most major markers associated with astrocyte activation and recapitulated numerous changes in gene expression found previously in the hippocampus of normally aging rats or in Alzheimer's disease (AD). No genomic or morphologic evidence of apoptosis or damage to neurons was seen, indicating that the calcineurin effect was mediated by direct actions on astrocytes. Moreover, immunocytochemical studies of the hippocampus/neocortex in normal aging and AD model mice revealed intense calcineurin immunostaining that was highly selective for activated astrocytes. Together, these studies show that calcineurin overexpression is sufficient to trigger essentially the full genomic and phenotypic profiles associated with astrocyte activation and that hypertrophic astrocytes in aging and AD models exhibit dramatic upregulation of calcineurin. Thus, the data identify calcineurin upregulation in astrocytes as a novel candidate for an intracellular trigger of astrogliosis, particularly in aging and AD brain.

A selective role for MRF4 in innervated adult skeletal muscle: Na(V) 1.4 Na+ channel expression is reduced in MRF4-null mice.

The factors that regulate transcription and spatial expression of the adult skeletal muscle Na+ channel, Na(V) 1.4, are poorly understood. Here we tested the role of the transcription factor MRF4, one of four basic helix-loop-helix (bHLH) factors expressed in skeletal muscle, in regulation of the Na(V) 1.4 Na+ channel. Overexpression of MRF4 in C2C12 muscle cells dramatically elevated Na(V) 1.4 reporter gene expression, indicating that MRF4 is more efficacious than the other bHLH factors expressed at high levels endogenously in these cells. In vivo, MRF4 protein was found both in extrajunctional and subsynaptic muscle nuclei. To test the importance of MRF4 in Na(V) 1.4 gene regulation in vivo, we examined Na+ channel expression in MRF4-null mice using several techniques, including Western blotting, immunocytochemistry, and electrophysiological recording. By all methods, we found that expression of the Na(V) 1.4 Na+ channel was substantially reduced in MRF4-null mice, both in the surface membrane and at neuromuscular junctions. In contrast, expression of the acetylcholine receptor, and in particular its alpha subunit, was unchanged, indicating that MRF4 regulation of Na+ channel expression was selective. Expression of the bHLH factors myf-5, MyoD, and myogenin was increased in MRF4-null mice, but these factors were not able to fully maintain Na(V) 1.4 Na+ channel expression either in the extrajunctional membrane or at the synapse. Thus, MRF4 appears to play a novel and selective role in adult muscle.

The metastasis suppressor NME1 regulates expression of genes linked to metastasis and patient outcome in melanoma and breast carcinoma.

NME1 is a well-documented metastasis suppressor gene, with suppressor activity demonstrated across a wide spectrum of human cancers including melanoma and carcinomas of the breast, stomach and thyroid. A primary aim of the current study was to identify profiles of genes whose expression is regulated by NME1 in cell lines of melanoma and thyroid carcinoma origin. Impact of NME1 was determined by forcing its expression transiently in cell lines using a novel Ad5-based adenoviral vector (Ad5-NME1), followed 48 h later by analysis of RNA expression profiles using the U133A microarray chip. Robust NME1 expression was achieved following infection with the Ad5-NME1 adenovirus in the human metastasis-derived cell lines WM1158 (melanoma) and WRO82 (follicular thyroid carcinoma), resulting in wide-ranging effects on gene expression in both settings. A substantial proportion of the NME1-regulated genes identified in the analyses were of clear potential relevance to metastasis, such as matrix metalloproteinase-1 (MMP1), angiopoietin-2 (ANGPT2), SERPINB9 and colony stimulating factor receptor-2B (CSFR2B). Nine genes were identified (false discovery rate <0.1) that were regulated by NME1 in both the WM1158 and WRO82 cell lines, each possessing one or more such metastasis-relevant activities as stress fiber formation and focal adhesion (PPM1E, ZYX, PFN1), chemotaxis (CCR1) epithelial-mesenchymal signaling (WNT6), differentiation and morphogenesis (TBX4, ZFP36L2), and G protein modulation (GPR52 and PFN1). In addition, a number of the NME1-regulated genes were shown to be of prognostic value for distant disease-free survival and overall survival in melanoma and breast cancer. The combined expression of three NME1-regulated genes CSFR2B, MSF4A1 and SERPINB9 provided a strongly synergistic correlation with distant disease-free survival in the basal subtype of breast cancer (p<3.5e(-5), hazard ratio=0.33). Our study demonstrates that analysis of NME1-dependent gene expression is a powerful approach for identifying potential modulators of metastatic potential in multiple cancer types, which in turn may represent useful therapeutic targets. The study also highlights NME1-dependent genes as potential prognostic/diagnostic indices, which are profoundly lacking at present in melanoma.

Functional ryanodine receptors in the membranes of neurohypophysial secretory granules.

Highly localized Ca(2+) release events have been characterized in several neuronal preparations. In mouse neurohypophysial terminals (NHTs), such events, called Ca(2+) syntillas, appear to emanate from a ryanodine-sensitive intracellular Ca(2+) pool. Traditional sources of intracellular Ca(2+) appear to be lacking in NHTs. Thus, we have tested the hypothesis that large dense core vesicles (LDCVs), which contain a substantial amount of calcium, represent the source of these syntillas. Here, using fluorescence immunolabeling and immunogold-labeled electron micrographs of NHTs, we show that type 2 ryanodine receptors (RyRs) are localized specifically to LDCVs. Furthermore, a large conductance nonspecific cation channel, which was identified previously in the vesicle membrane and has biophysical properties similar to that of an RyR, is pharmacologically affected in a manner characteristic of an RyR: it is activated in the presence of the RyR agonist ryanodine (at low concentrations) and blocked by the RyR antagonist ruthenium red. Additionally, neuropeptide release experiments show that these same RyR agonists and antagonists modulate Ca(2+)-elicited neuropeptide release from permeabilized NHTs. Furthermore, amperometric recording of spontaneous release events from artificial transmitter-loaded terminals corroborated these ryanodine effects. Collectively, our findings suggest that RyR-dependent syntillas could represent mobilization of Ca(2+) from vesicular stores. Such localized vesicular Ca(2+) release events at the precise location of exocytosis could provide a Ca(2+) amplification mechanism capable of modulating neuropeptide release physiologically.

Obesity and diabetes cause cognitive dysfunction in the absence of accelerated ß-amyloid deposition in a novel murine model of mixed or vascular dementia.

Mid-life obesity and type 2 diabetes mellitus (T2DM) confer a modest, increased risk for Alzheimer's disease (AD), though the underlying mechanisms are unknown. We have created a novel mouse model that recapitulates features of T2DM and AD by crossing morbidly obese and diabetic db/db mice with APPΔNL/ΔNLx PS1P264L/P264L knock-in mice. These mice (db/AD) retain many features of the parental lines (e.g. extreme obesity, diabetes, and parenchymal deposition of ß-amyloid (Aß)). The combination of the two diseases led to additional pathologies-perhaps most striking of which was the presence of severe cerebrovascular pathology, including aneurysms and small strokes. Cortical Aß deposition was not significantly increased in the diabetic mice, though overall expression of presenilin was elevated. Surprisingly, Aß was not deposited in the vasculature or removed to the plasma, and there was no stimulation of activity or expression of major Aß-clearing enzymes (neprilysin, insulin degrading enzyme, or endothelin-converting enzyme). The db/AD mice displayed marked cognitive impairment in the Morris Water Maze, compared to either db/db or APPΔNLx PS1P264L mice. We conclude that the diabetes and/or obesity in these mice leads to a destabilization of the vasculature, leading to strokes and that this, in turn, leads to a profound cognitive impairment and that this is unlikely to be directly dependent on Aß deposition. This model of mixed or vascular dementia provides an exciting new avenue of research into the mechanisms underlying the obesity-related risk for age-related dementia, and will provide a useful tool for the future development of therapeutics.

Altered sodium channel-protein associations in critical illness myopathy.

BACKGROUND: During the acute phase of critical illness myopathy (CIM) there is inexcitability of skeletal muscle. In a rat model of CIM, muscle inexcitability is due to inactivation of sodium channels. A major contributor to this sodium channel inactivation is a hyperpolarized shift in the voltage dependence of sodium channel inactivation. The goal of the current study was to find a biochemical correlate of the hyperpolarized shift in sodium channel inactivation. METHODS: The rat model of CIM was generated by cutting the sciatic nerve and subsequent injections of dexamethasone for 7 days. Skeletal muscle membranes were prepared from gastrocnemius muscles, and purification and biochemical analyses carried out. Immunoprecipitations were performed with a pan-sodium channel antibody, and the resulting complexes probed in Western blots with various antibodies. RESULTS: We carried out analyses of sodium channel glycosylation, phosphorylation, and association with other proteins. Although there was some loss of channel glycosylation in the disease, as assessed by size analysis of glycosylated and de-glycosylated protein in control and CIM samples, previous work by other investigators suggest that such loss would most likely shift channel inactivation gating in a depolarizing direction; thus such loss was viewed as compensatory rather than causative of the disease. A phosphorylation site at serine 487 was identified on the NaV 1.4 sodium channel α subunit, but there was no clear evidence of altered phosphorylation in the disease. Co-immunoprecipitation experiments carried out with a pan-sodium channel antibody confirmed that the sodium channel was associated with proteins of the dystrophin associated protein complex (DAPC). This complex differed between control and CIM samples. Syntrophin, dystrophin, and plectin associated strongly with sodium channels in both control and disease conditions, while ß-dystroglycan and neuronal nitric oxide synthase (nNOS) associated strongly with the sodium channel only in CIM. Recording of action potentials revealed that denervated muscle in mice lacking nNOS was more excitable than control denervated muscle. CONCLUSION: Taken together, these data suggest that the conformation/protein association of the sodium channel complex differs in control and critical illness myopathy muscle membranes; and suggest that nitric oxide signaling plays a role in development of muscle inexcitability.

Hippocampal CA1 transcriptional profile of sleep deprivation: relation to aging and stress.

BACKGROUND: Many aging changes seem similar to those elicited by sleep-deprivation and psychosocial stress. Further, sleep architecture changes with age suggest an age-related loss of sleep. Here, we hypothesized that sleep deprivation in young subjects would elicit both stress and aging-like transcriptional responses. METHODOLOGY/PRINCIPAL FINDINGS: F344 rats were divided into control and sleep deprivation groups. Body weight, adrenal weight, corticosterone level and hippocampal CA1 transcriptional profiles were measured. A second group of animals was exposed to novel environment stress (NES), and their hippocampal transcriptional profiles measured. A third cohort exposed to control or SD was used to validate transcriptional results with Western blots. Microarray results were statistically contrasted with prior transcriptional studies. Microarray results pointed to sleep pressure signaling and macromolecular synthesis disruptions in the hippocampal CA1 region. Animals exposed to NES recapitulated nearly one third of the SD transcriptional profile. However, the SD-aging relationship was more complex. Compared to aging, SD profiles influenced a significant subset of genes. mRNA associated with neurogenesis and energy pathways showed agreement between aging and SD, while immune, glial, and macromolecular synthesis pathways showed SD profiles that opposed those seen in aging. CONCLUSIONS/SIGNIFICANCE: We conclude that although NES and SD exert similar transcriptional changes, selective presynaptic release machinery and Homer1 expression changes are seen in SD. Among other changes, the marked decrease in Homer1 expression with age may represent an important divergence between young and aged brain response to SD. Based on this, it seems reasonable to conclude that therapeutic strategies designed to promote sleep in young subjects may have off-target effects in the aged. Finally, this work identifies presynaptic vesicular release and intercellular adhesion molecular signatures as novel therapeutic targets to counter effects of SD in young subjects.