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ABSTRACT: Astrocytes are the most abundant cell type in the brain and play a critical role in maintaining healthy nervous tissue. In Alzheimer's disease (AD) and most other neurodegenerative disorders, many astrocytes convert to a chronically "activated" phenotype characterized by morphologic and biochemical changes that appear to compromise protective properties and/or promote harmful neuroinflammatory processes. Activated astrocytes emerge early in the course of AD and become increasingly prominent as clinical and pathological symptoms progress, but few studies have tested the potential of astrocyte-targeted therapeutics in an intact animal model of AD. Here, we used adeno-associated virus (AAV) vectors containing the astrocyte-specific Gfa2 promoter to target hippocampal astrocytes in APP/PS1 mice. AAV-Gfa2 vectors drove the expression of VIVIT, a peptide that interferes with the immune/inflammatory calcineurin/NFAT (nuclear factor of activated T-cells) signaling pathway, shown by our laboratory and others to orchestrate biochemical cascades leading to astrocyte activation. After several months of treatment with Gfa2-VIVIT, APP/PS1 mice exhibited improved cognitive and synaptic function, reduced glial activation, and lower amyloid levels. The results confirm a deleterious role for activated astrocytes in AD and lay the groundwork for exploration of other novel astrocyte-based therapies.
Journal of Neuroscience 11/2012; 32(46):16129-16140. · 7.11 Impact Factor
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ABSTRACT: Overproduction of proinflammatory cytokines in the CNS has been implicated as a key contributor to pathophysiology progression in Alzheimer's disease (AD), and extensive studies with animal models have shown that selective suppression of excessive glial proinflammatory cytokines can improve neurologic outcomes. The prior art, therefore, raises the logical postulation that intervention with drugs targeting dysregulated glial proinflammatory cytokine production might be effective disease-modifying therapeutics if used in the appropriate biological time window. To test the hypothesis that early stage intervention with such drugs might be therapeutically beneficial, we examined the impact of intervention with MW01-2-151SRM (MW-151), an experimental therapeutic that selectively attenuates proinflammatory cytokine production at low doses. MW-151 was tested in an APP/PS1 knock-in mouse model that exhibits increases in AD-relevant pathology progression with age, including increases in proinflammatory cytokine levels. Drug was administered during two distinct but overlapping therapeutic time windows of early stage pathology development. MW-151 treatment attenuated the increase in microglial and astrocyte activation and proinflammatory cytokine production in the cortex and yielded improvement in neurologic outcomes, such as protection against synaptic protein loss and synaptic plasticity impairment. The results also demonstrate that the therapeutic time window is an important consideration in efficacy studies of drugs that modulate glia biological responses involved in pathology progression and suggest that such paradigms should be considered in the development of new therapeutic regimens that seek to delay the onset or slow the progression of AD.
Journal of Neuroscience 07/2012; 32(30):10201-10. · 7.11 Impact Factor
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ABSTRACT: Increased function of neuronal L-type voltage-sensitive Ca(2+) channels (L-VSCCs) is strongly linked to impaired memory and altered hippocampal synaptic plasticity in aged rats. However, no studies have directly assessed L-VSCC function in any of the common mouse models of Alzheimer's disease where neurologic deficits are typically more robust. Here, we used cell-attached patch-clamp recording techniques to measure L-VSCC activity in CA1 pyramidal neurons of partially dissociated hippocampal "zipper" slices prepared from 14-month-old wild-type mice and memory-impaired APP/PS1 double knock-in mice. Surprisingly, the functional channel density of L-VSCCs was significantly reduced in the APP/PS1 group. No differences in voltage dependency and unitary conductance of L-VSCCs were observed. The results suggest that mechanisms for Ca(2+) dysregulation can differ substantially between animal models of normal aging and models of pathological aging.
Biochimica et Biophysica Acta 04/2012; 1822(4):546-9. · 4.66 Impact Factor
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ABSTRACT: The role of tumor necrosis factor α (TNF) in neural function has been investigated extensively in several neurodegenerative conditions, but rarely in brain aging, where cognitive and physiologic changes are milder and more variable. Here, we show that protein levels for TNF receptor 1 (TNFR1) are significantly elevated in the hippocampus relative to TNF receptor 2 (TNFR2) in aged (22 months) but not young adult (6 months) Fischer 344 rats. To determine if altered TNF/TNFR1 interactions contribute to key brain aging biomarkers, aged rats received chronic (4-6 week) intracranial infusions of XPro1595: a soluble dominant negative TNF that preferentially inhibits TNFR1 signaling. Aged rats treated with XPro1595 showed improved Morris Water Maze performance, reduced microglial activation, reduced susceptibility to hippocampal long-term depression, increased protein levels for the GluR1 type glutamate receptor, and lower L-type voltage sensitive Ca(2+) channel (VSCC) activity in hippocampal CA1 neurons. The results suggest that diverse functional changes associated with brain aging may arise, in part, from selective alterations in TNF signaling.
PLoS ONE 01/2012; 7(5):e38170. · 4.09 Impact Factor
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ABSTRACT: With aging, multiple Ca(2+)-associated electrophysiological processes exhibit increased magnitude in hippocampal pyramidal neurons, including the Ca(2+)-dependent slow afterhyperpolarization (sAHP), L-type voltage-gated Ca(2+) channel (L-VGCC) activity, Ca(2+)-induced Ca(2+) release (CICR) from ryanodine receptors (RyRs), and Ca(2+) transients. This pattern of Ca(2+) dysregulation correlates with reduced neuronal excitability/plasticity and impaired learning/memory and has been proposed to contribute to unhealthy brain aging and Alzheimer's disease. However, little is known about the underlying molecular mechanisms. In cardiomyocytes, FK506-binding protein 1b/12.6 (FKBP1b) binds and stabilizes RyR2 in the closed state, inhibiting RyR-mediated Ca(2+) release. Moreover, we recently found that hippocampal Fkbp1b expression is downregulated, whereas Ryr2 and Frap1/Mtor (mammalian target of rapamycin) expression is upregulated with aging in rats. Here, we tested the hypothesis that disrupting FKBP1b function also destabilizes Ca(2+) homeostasis in hippocampal neurons and is sufficient to induce the aging phenotype of Ca(2+) dysregulation in young animals. Selective knockdown of Fkbp1b with interfering RNA in vitro (96 h) enhanced voltage-gated Ca(2+) current in cultured neurons, whereas in vivo Fkbp1b knockdown by microinjection of viral vector (3-4 weeks) dramatically increased the sAHP in hippocampal slice neurons from young-adult rats. Rapamycin, which displaces FKBP1b from RyRs in myocytes, similarly enhanced VGCC current and the sAHP and also increased CICR. Moreover, FKBP1b knockdown in vivo was associated with upregulation of RyR2 and mTOR protein expression. Thus, disruption of FKBP1b recapitulated much of the Ca(2+)-dysregulation aging phenotype in young rat hippocampus, supporting a novel hypothesis that declining FKBP function plays a major role in unhealthy brain aging.
Journal of Neuroscience 02/2011; 31(5):1693-703. · 7.11 Impact Factor
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ABSTRACT: The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid. Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments. The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory. Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds. This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)(1-3). Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research. Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity. Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment. Here, we discuss the steps taken during the dissection procedure to minimize these problems. Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments. The possible impact of other methodological factors on synaptic function in these animal models (e.g. recording solution components, stimulation parameters) are also discussed. While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models.
Journal of Visualized Experiments 01/2011;
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ABSTRACT: Recent reports demonstrate that the activation and interaction of the protease calpain (CP) and the protein phosphatase calcineurin (CN) are elevated in the late stages of Alzheimer's disease (AD). However, the extent to which CPs and CN interact during earlier stages of disease progression remains unknown. Here, we investigated CP and CN protein levels in cytosolic, nuclear, and membrane fractions prepared from human postmortem hippocampal tissue from aged non-demented subjects, and subjects diagnosed with mild cognitive impairment (MCI). The results revealed a parallel increase in CP I and the 48 kDa CN-Aα (ΔCN-Aα48) proteolytic fragment in cytosolic fractions during MCI. In primary rat hippocampal cultures, CP-dependent proteolysis and activation of CN was stimulated by application of oligomeric Aβ((1-42)) peptides. Deleterious effects of Aβ on neuronal morphology were reduced by blockade of either CP or CN. NMDA-type glutamate receptors, which help regulate cognition and neuronal viability, and are modulated by CPs and CN, were also investigated in human hippocampus. Relative to controls, MCI subjects showed significantly greater proteolytic levels of the NR2B subunit. Within subjects, the extent of NR2B proteolysis was strongly correlated with the generation of ΔCN-Aα48 in the cytosol. A similar proteolytic pattern for NR2B was also observed in primary rat hippocampal cultures treated with oligomeric Aβ and prevented by inhibition of CP or CN. Together, the results demonstrate that the activation and interaction of CPs and CN are increased early in cognitive decline associated with AD and may help drive other pathologic processes during disease progression.
Aging cell 10/2010; 10(1):103-13. · 7.55 Impact Factor
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ABSTRACT: Nuclear factor of activated T cells (NFAT) is a transcription factor that translocates from cytosol to nucleus following dephosphorylation by the Ca2+/calmodulin dependent protein phosphatase calcineurin (CN). In nervous tissue, aberrant CN signaling is increasingly linked to a variety of pathologic features associated with Alzheimer’s disease (AD), including synaptic dysfunction, glial activation, and neuronal death. Consistent with this linkage, our recent work on postmortem human hippocampal tissue discovered increased nuclear accumulation of select NFAT isoforms at different stages of AD. Some of these changes occurred at the early stages of the disease process and/or paralleled diminishing cognitive status. In addition, inhibition of astrocytic NFAT activity in primary cultures of neurons and glia dampened glutamate levels and alleviated neuronal death in response to pathogenic amyloid- peptides. In this article, we discuss our recent findings and expand upon the possible isoform specific contributions of NFATs to the progression of AD. We also consider the possible benefits of using NFAT inhibitors to treat AD and other neurodegenerative disorders, as well.
Molecular and Cellular Pharmacology. 01/2010;
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ABSTRACT: Nuclear factor of activated T cells (NFAT) is a transcription factor that translocates from cytosol to nucleus following dephosphorylation by the Ca(2+)/calmodulin dependent protein phosphatase calcineurin (CN). In nervous tissue, aberrant CN signaling is increasingly linked to a variety of pathologic features associated with Alzheimer's disease (AD), including synaptic dysfunction, glial activation, and neuronal death. Consistent with this linkage, our recent work on postmortem human hippocampal tissue discovered increased nuclear accumulation of select NFAT isoforms at different stages of AD. Some of these changes occurred at the early stages of the disease process and/or paralleled diminishing cognitive status. In addition, inhibition of astrocytic NFAT activity in primary cultures of neurons and glia dampened glutamate levels and alleviated neuronal death in response to pathogenic amyloid-β peptides. In this article, we discuss our recent findings and expand upon the possible isoform specific contributions of NFATs to the progression of AD. We also consider the possible benefits of using NFAT inhibitors to treat AD and other neurodegenerative disorders, as well.
Molecular and Cellular Pharmacology 01/2010; 2(1):7-14.
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ABSTRACT: In astrocytes, the Ca(2+)-dependent protein phosphatase calcineurin (CN) strongly regulates neuro-immune/inflammatory cascades through activation of the transcription factor, nuclear factor of activated T cells (NFAT). While primary cell cultures provide a useful model system for investigating astrocytic CN/NFAT signaling, variable results may arise both within and across labs because of differences in culture conditions. Here, we determined the extent to which serum and cell confluency affect basal and evoked astrocytic NFAT activity in primary cortical astrocyte cultures. Cells were grown to either approximately 50% or >90% confluency, pre-loaded with an NFAT-luciferase reporter construct, and maintained for 16 h in medium with or without 10% fetal bovine serum (FBS). NFAT-dependent luciferase expression was then measured 5h after treatment with vehicle alone to assess basal NFAT activity, or with Ca(2+) mobilizers and IL-1 beta to assess evoked activity. The results revealed significantly higher levels of basal NFAT activity in FBS-containing medium, regardless of cell confluency. Conversely, evoked NFAT activation was significantly lower in serum-containing medium, with an even greater inhibition observed in confluent cultures. Application of 10% FBS to serum-free astrocyte cultures quickly evoked a roughly seven-fold increase in NFAT activity that was significantly reduced by co-delivery of neutralizing agents for IL-1 beta, TNFalpha, and/or IFN gamma, suggesting that serum occludes evoked NFAT activation through a cytokine-based mechanism. Together, the results demonstrate that the presence of serum and cell confluency have a major impact on CN/NFAT signaling in primary astrocyte cultures and therefore must be taken into consideration when using this model system.
Neuroscience Letters 12/2009; 469(3):365-9. · 2.11 Impact Factor
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Hafiz Mohmmad Abdul,
Michelle A Sama,
Jennifer L Furman,
Diana M Mathis,
Tina L Beckett,
Adam M Weidner,
Ela S Patel,
Irfan Baig,
M Paul Murphy,
Harry LeVine,
Susan D Kraner, Christopher M Norris
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ABSTRACT: 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.
Journal of Neuroscience 10/2009; 29(41):12957-69. · 7.11 Impact Factor
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ABSTRACT: Cortical contusion injury can result in the partial loss of ipsilateral CA3 neurons within 48 h, leading to a proportional reduction in the number of afferent fibers to CA1 stratum radiatum. While the loss of afferent input to CA1 exhibits a remarkable, albeit incomplete, recovery over the next few weeks, little is known about the functional status of presynaptic afferents during the depletion and recovery phases following injury. Here, we prepared hippocampal slices from adult Sprague Dawley rats at 2, 7, and 14 days after lateral cortical contusion injury and measured fiber volley (FV) amplitudes extracellularly in CA1 stratum radiatum. Field excitatory post-synaptic potentials (EPSPs) were also measured and plotted as a function of FV amplitude to assess relative synaptic strength of residual and/or regenerated synaptic contacts. At 2 days post-injury, FV amplitude and synaptic strength were markedly reduced in the ipsilateral, relative to the contralateral, hippocampus. FV amplitude in ipsilateral CA1 showed a complete recovery by 7 days, indicative of a post-injury sprouting response. Synaptic strength in ipsilateral CA1 also showed a dramatic recovery over this time; however, EPSP-to-FV curves remained slightly suppressed at both the 7 and 14 day time points. Despite these deficits, ipsilateral slices retained the capacity to express long-term potentiation, indicating that at least some mechanisms for synaptic plasticity remain intact, or are compensated for. These results are in agreement with anatomical evidence showing a profound deafferentation, followed by a remarkable re-enervation, of ipsilateral CA1 in the first few weeks after traumatic brain injury. Although plasticity mechanisms appear to remain intact, synaptic strength deficits in CA1 could limit information throughput in the hippocampus, leading to persistent memory dysfunction.
Journal of neurotrauma 08/2009; 26(12):2269-78. · 4.25 Impact Factor
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ABSTRACT: Interleukin-1beta (IL-1beta) and the Ca(2+)/calmodulin-dependent protein phosphatase, calcineurin, have each been shown to play an important role in neuroinflammation. However, whether these signaling molecules interact to coordinate immune/inflammatory processes and neurodegeneration has not been investigated. Here, we show that exogenous application of IL-1beta (10 ng/ml) recruited calcineurin/NFAT (nuclear factor of activated T cells) activation in primary astrocyte-enriched cultures within minutes, through a pathway involving IL-1 receptors and L-type Ca(2+) channels. Adenovirus-mediated delivery of the NFAT inhibitor, VIVIT, suppressed the IL-1beta-dependent induction of several inflammatory mediators and/or markers of astrocyte activation, including tumor necrosis factor alpha, granulocyte/macrophage colony-stimulating factor, and vimentin. Expression of an activated form of calcineurin in one set of astrocyte cultures also triggered the release of factors that, in turn, stimulated NFAT activity in a second set of "naive" astrocytes. This effect was prevented when calcineurin-expressing cultures co-expressed VIVIT, suggesting that the calcineurin/NFAT pathway coordinates positive feedback signaling between astrocytes. In the presence of astrocytes and neurons, 48-h delivery of IL-1beta was associated with several excitotoxic effects, including NMDA receptor-dependent neuronal death, elevated extracellular glutamate, and hyperexcitable synaptic activity. Each of these effects were reversed or ameliorated by targeted delivery of VIVIT to astrocytes. IL-1beta also caused an NFAT-dependent reduction in excitatory amino acid transporter levels, indicating a possible mechanism for IL-1beta-mediated excitotoxicity. Taken together, the results have potentially important implications for the propagation and maintenance of neuroinflammatory signaling processes associated with many neurodegenerative conditions and diseases.
Journal of Biological Chemistry 07/2008; 283(32):21953-64. · 4.77 Impact Factor
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ABSTRACT: Previous studies have shown that inhibition of the Ca(2+)-/calmodulin-dependent protein phosphatase calcineurin (CN) blocks L-type voltage sensitive Ca(2+) channel (L-VSCC) activity in cultured hippocampal neurons. However, it is not known whether CN contributes to the increase in hippocampal L-VSCC activity that occurs with aging in at least some mammalian species. It is also unclear whether CN's necessary role in VSCC activity is simply permissive or is directly enhancing. To resolve these questions, we used partially dissociated hippocampal "zipper" slices to conduct cell-attached patch recording and RT-PCR on largely intact single neurons from young-adult, mid-aged, and aged rats. Further, we tested for direct CN enhancement of L-VSCCs using virally mediated infection of cultured neurons with an activated form of CN. Similar to previous work, L-VSCC activity was elevated in CA1 neurons of mid-aged and aged rats relative to young adults. The CN inhibitor, FK-506 (5muM) completely blocked the aging-related increase in VSCC activity, reducing the activity level in aged rat neurons to that in younger rat neurons. However, aging was not associated with an increase in neuronal CN mRNA expression, nor was CN expression correlated with VSCC activity. Delivery of activated CN to primary hippocampal cultures induced an increase in neuronal L-VSCC activity but did not elevate L-VSCC protein levels. Together, the results provide the first evidence that CN activity, but not increased expression, plays a selective and necessary role in the aging-related increase in available L-VSCCs, possibly by direct activation. Thus, these studies point to altered CN function as a novel and potentially key factor in aging-dependent neuronal Ca(2+) dysregulation.
Neurobiology of aging 06/2008; 31(2):328-38. · 5.94 Impact Factor
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ABSTRACT: Although Ca(2+)-dependent signaling pathways are important for skeletal muscle plasticity, the sources of Ca(2+) that activate these signaling pathways are not completely understood. Influx of Ca(2+) through surface membrane Ca(2+) channels may activate these pathways. We examined expression of two L-type Ca(2+) channels in adult skeletal muscle, the Ca(V) 1.1 and Ca(V) 1.2, with isoform-specific antibodies in Western blots and immunocytochemistry assays. Consistent with a large body of work, expression of the Ca(V) 1.1 was restricted to skeletal muscle where it was expressed in T-tubules. Ca(V) 1.2 was also expressed in skeletal muscle, in the sarcolemma of type I and IIa myofibers. Exercise-induced alterations in muscle fiber types cause a concomitant increase in the number of both Ca(V) 1.2 and type IIa-positive fibers. Taken together, these data suggest that the Ca(V) 1.2 Ca(2+) channel is expressed in adult skeletal muscle in a fiber type-specific manner, which may help to maintain oxidative muscle phenotype.
Muscle & Nerve 11/2007; 36(4):482-90. · 2.37 Impact Factor
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ABSTRACT: 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.
Journal of Neuroscience 06/2005; 25(18):4649-58. · 7.11 Impact Factor
