Inactivation and Secondary Structure in the D4/S4-5 Region of the SkM1 Sodium Channel

The D4/S4-5 interhelical region plays a role in sodium channel fast inactivation. Examination of S4-5 primary structure in all domains suggests a possible amphipathic helical conformation in which a conserved group of small hydrophobic residues occupies one contiguous surface with a more variable complement of nonpolar and polar residues on the opposite face. We evaluated this potential structure by replacing each residue in D4/S4-5 of the rat SkM1 skeletal muscle sodium channel with substitutions having different side chain properties. Of the 63 mutations analyzed, 44 produced functional channels. P1473 was intolerant of substitutions. Nonpolar substitutions in the conserved hydrophobic region were functionally similar to wild type, while charged mutations in this region before P1473 were nonfunctional. Charged mutations at F1466, M1469, M1470, and A1474, located on the opposite surface of the predicted helix, produced functional channels with pronounced slowing of inactivation, shifted voltage dependence of steady-state inactivation, and increased rate of recovery from inactivation. The substituted-cysteine-accessibility method was used to probe accessibility at each position. Residues L1465, F1466, A1467, M1469, M1470, L1472, A1474, and F1476C were easily accessible for modification by sulfhydryl reagents; L1464, L1468, S1471, and L1475 were not accessible within the time frame of our measurements. Molecular dynamics simulations of residues A1458 to N1477 were then used to explore energetically favorable local structures. Based on mutagenesis, substituted-cysteine-accessibility method, and modeling results, we suggest a secondary structure for the D4/S4-5 region in which the peptide chain is α-helical proximal to P1473, bends at this residue, and may continue beyond this point as a random coil. In this configuration, the entire resultant loop is amphipathic; four residues on one surface could form part of the binding site for the inactivation particle

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 AβPP/PS1 Mouse Model of Alzheimer\u27s Disease

Alzheimer’s disease (AD) brains are characterized by fibrillar amyloid-β (Aβ) 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 Aβ 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 AβPP/PS1 mouse line. As expected, the AβPP/PS1 x NFATc2–/– mice had attenuated cytokine levels compared to AβPP/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

The α\u3csub\u3e1D\u3c/sub\u3e-Adrenergic Receptor Is Expressed Intracellularly and Coupled to Increases in Intracellular Calcium and Reactive Oxygen Species in Human Aortic Smooth Muscle Cells

Background: The cellular localization of the α1D-adrenergic receptor (α1D-AR) is controversial. Studies in heterologous cell systems have shown that this receptor is expressed in intracellular compartments. Other studies show that dimerization with other ARs promotes the cell surface expression of the α1D-AR. To assess the cellular localization in vascular smooth muscle cells, we developed an adenoviral vector for the efficient expression of a GFP labeled α1D-AR. We also measured cellular localization with immunocytochemistry. Intracellular calcium levels, measurement of reactive oxygen species and contraction of the rat aorta were used as measures of functional activity. Results: The adenovirally expressed α1D-AR was expressed in intracellular compartments in human aortic smooth muscle cells. The intracellular localization of the α1D-AR was also demonstrated with immunocytochemistry using an α1D-AR specific antibody. RT-PCR analysis detected mRNA transcripts corresponding to the α1A-α1B- and α1D-ARs in these aortic smooth muscle cells. Therefore, the presence of the other α1-ARs, and the potential for dimerization with these receptors, does not alter the intracellular expression of the α1D-AR. Despite the predominant intracellular localization in vascular smooth muscle cells, the α1D-AR remained signaling competent and mediated the phenylephrine-induced increases in intracellular calcium. The α1D-AR also was coupled to the generation of reactive oxygen species in smooth muscle cells. There is evidence from heterologous systems that the α1D-AR heterodimerizes with the β2-AR and that desensitization of the β2-AR results in α1D-AR desensitization. In the rat aorta, desensitization of the β2-AR had no effect on contractile responses mediated by the α1D-AR. Conclusion: Our results suggest that the dimerization of the α1D-AR with other ARs does not alter the cellular expression or functional response characteristics of the α1D-AR

The α1D-adrenergic receptor is expressed intracellularly and coupled to increases in intracellular calcium and reactive oxygen species in human aortic smooth muscle cells

Background: The cellular localization of the α1D-adrenergic receptor (α1D-AR) is controversial. Studies in heterologous cell systems have shown that this receptor is expressed in intracellular compartments. Other studies show that dimerization with other ARs promotes the cell surface expression of the α1D-AR. To assess the cellular localization in vascular smooth muscle cells, we developed an adenoviral vector for the efficient expression of a GFP labeled α1D-AR. We also measured cellular localization with immunocytochemistry. Intracellular calcium levels, measurement of reactive oxygen species and contraction of the rat aorta were used as measures of functional activity. Results: The adenovirally expressed α1D-AR was expressed in intracellular compartments in human aortic smooth muscle cells. The intracellular localization of the α1D-AR was also demonstrated with immunocytochemistry using an α1D-AR specific antibody. RT-PCR analysis detected mRNA transcripts corresponding to the α1A-α1B- and α1D-ARs in these aortic smooth muscle cells. Therefore, the presence of the other α1-ARs, and the potential for dimerization with these receptors, does not alter the intracellular expression of the α1D-AR. Despite the predominant intracellular localization in vascular smooth muscle cells, the α1D-AR remained signaling competent and mediated the phenylephrine-induced increases in intracellular calcium. The α1D-AR also was coupled to the generation of reactive oxygen species in smooth muscle cells. There is evidence from heterologous systems that the α1D-AR heterodimerizes with the β2-AR and that desensitization of the β2-AR results in α1D-AR desensitization. In the rat aorta, desensitization of the β2-AR had no effect on contractile responses mediated by the α1D-AR. Conclusion: Our results suggest that the dimerization of the α1D-AR with other ARs does not alter the cellular expression or functional response characteristics of the α1D-AR

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

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\u27s 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

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 (= 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 A beta plaque pathology. The results suggest that Q134R is a promising drug for treating AD and aging-related disorders

Calcineurin/NFAT Signaling in Activated Astrocytes Drives Network Hyperexcitability in A\u3cem\u3eβ\u3c/em\u3e-Bearing Mice

Hyperexcitable neuronal networks are mechanistically linked to the pathologic and clinical features of Alzheimer\u27s 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

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\u27s 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 Aβ 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 Aβ plaque pathology. The results suggest that Q134R is a promising drug for treating AD and aging-related disorders