Christopher M. Norris

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 Alzheimers 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.

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 Ca2+ dysregulation, both of which are prominent features of Alzheimers 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-Aα 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-β (Aβ(1-42)) levels in postmortem hippocampus, and oligomeric Aβ, in particular, robustly stimulated NFAT activation in primary rat astrocyte cultures. Oligomeric Aβ 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 Aβ-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 Aβ-mediated neurodegeneration.

Targeting Astrocytes Ameliorates Neurologic Changes in a Mouse Model of Alzheimer's Disease

Astrocytes are the most abundant cell type in the brain and play a critical role in maintaining healthy nervous tissue. In Alzheimers 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.

Harnessing the power of gene microarrays for the study of brain aging and Alzheimer's disease: Statistical reliability and functional correlation

During normal brain aging, numerous alterations develop in the physiology, biochemistry and structure of neurons and glia. Aging changes occur in most brain regions and, in the hippocampus, have been linked to declining cognitive performance in both humans and animals. Age-related changes in hippocampal regions also may be harbingers of more severe decrements to come from neurodegenerative disorders such as Alzheimers disease (AD). However, unraveling the mechanisms underlying brain aging, AD and impaired function has been difficult because of the complexity of the networks that drive these aging-related changes. Gene microarray technology allows massively parallel analysis of most genes expressed in a tissue, and therefore is an important new research tool that potentially can provide the investigative power needed to address the complexity of brain aging/neurodegenerative processes. However, along with this new analytic power, microarrays bring several major bioinformatics and resource problems that frequently hinder the optimal application of this technology. In particular, microarray analyses generate extremely large and unwieldy data sets and are subject to high false positive and false negative rates. Concerns also have been raised regarding their accuracy and uniformity. Furthermore, microarray analyses can result in long lists of altered genes, most of which may be difficult to evaluate for functional relevance. These and other problems have led to some skepticism regarding the reliability and functional usefulness of microarray data and to a general view that microarray data should be validated by an independent method. Given recent progress, however, we suggest that the major problem for current microarray research is no longer validity of expression measurements, but rather, the reliability of inferences from the data, an issue more appropriately redressed by statistical approaches than by validation with a separate method. If tested using statistically defined criteria for reliability/significance, microarray data do not appear a priori to require more independent validation than data obtained by any other method. In fact, because of added confidence from co-regulation, they may require less. In this article we also discuss our strategy of statistically correlating individual gene expression with biologically important endpoints designed to address the problem of evaluating functional relevance. We also review how work by ourselves and others with this powerful technology is leading to new insights into the complex processes of brain aging and AD, and to novel, more comprehensive models of aging-related brain change.

Interleukin-1β-dependent Signaling between Astrocytes and Neurons Depends Critically on Astrocytic Calcineurin/NFAT Activity

Interleukin-1β (IL-1β) and the Ca2+/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-1β (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 Ca2+ channels. Adenovirus-mediated delivery of the NFAT inhibitor, VIVIT, suppressed the IL-1β-dependent induction of several inflammatory mediators and/or markers of astrocyte activation, including tumor necrosis factor α, 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-1β 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-1β also caused an NFAT-dependent reduction in excitatory amino acid transporter levels, indicating a possible mechanism for IL-1β-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.

Calcineurin enhances L-type Ca2+ channel activity in hippocampal neurons: Increased effect with age in culture

The Ca(2+)/calmodulin-dependent protein phosphatase, calcineurin, modulates a number of key Ca(2+) signaling pathways in neurons, and has been implicated in Ca(2+)-dependent negative feedback inactivation of N-methyl-D-aspartate receptors and voltage-sensitive Ca(2+) channels. In contrast, we report here that three mechanistically disparate calcineurin inhibitors, FK-506, cyclosporin A, and the calcineurin autoinhibitory peptide, inhibited high-voltage-activated Ca(2+) channel currents by up to 40% in cultured hippocampal neurons, suggesting that calcineurin acts to enhance Ca(2+) currents. This effect occurred with Ba(2+) or Ca(2+) as charge carrier, and with or without intracellular Ca(2+) buffered by EGTA. Ca(2+)-dependent inactivation of Ca(2+) channels was not affected by FK-506. The immunosuppressant, rapamycin, and the protein phosphatase 1/2A inhibitor, okadaic acid, did not decrease Ca(2+) channel current, showing specificity for effects on calcineurin. Blockade of L-type Ca(2+) channels with nimodipine fully negated the effect of FK-506 on Ca(2+) channel current, while blockade of N-, and P-/Q-type Ca(2+) channels enhanced FK-506-mediated inhibition of the remaining L-type-enriched current. FK-506 also inhibited substantially more Ca(2+) channel current in 4-week-old vs. 2-week-old cultures, an effect paralleled by an increase in calcineurin A mRNA levels. These studies provide the first evidence that calcineurin selectively enhances L-type Ca(2+) channel activity in neurons. Moreover, this action appears to be increased concomitantly with the well-characterized increase in L-type Ca(2+) channel availability in hippocampal neurons with age-in-culture.

Early Stage Drug Treatment That Normalizes Proinflammatory Cytokine Production Attenuates Synaptic Dysfunction in a Mouse Model That Exhibits Age-Dependent Progression of Alzheimer's Disease-Related Pathology

Overproduction of proinflammatory cytokines in the CNS has been implicated as a key contributor to pathophysiology progression in Alzheimers 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.

Recovery of afferent function and synaptic strength in hippocampal CA1 following traumatic brain injury.

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.

Calcium dysregulation and neuroinflammation: Discrete and integrated mechanisms for age-related synaptic dysfunction

Some of the best biomarkers of age-related cognitive decline are closely linked to synaptic function and plasticity. This review highlights several age-related synaptic alterations as they relate to Ca(2+) dyshomeostasis, through elevation of intracellular Ca(2+), and neuroinflammation, through production of pro-inflammatory cytokines including interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α). Though distinct in many ways, Ca(2+) and neuroinflammatory signaling mechanisms exhibit extensive cross-talk and bidirectional interactions. For instance, cytokine production in glial cells is strongly dependent on the Ca(2+) dependent protein phosphatase calcineurin, which shows elevated activity in animal models of aging and disease. In turn, pro-inflammatory cytokines, such as TNF, can augment the expression/activity of L-type voltage sensitive Ca(2+) channels in neurons, leading to Ca(2+) dysregulation, hyperactive calcineurin activity, and synaptic depression. Thus, in addition to discussing unique contributions of Ca(2+) dyshomeostasis and neuroinflammation, this review emphasizes how these processes interact to hasten age-related synaptic changes.

Disrupting Function of FK506-Binding Protein 1b/12.6 Induces the Ca2+-Dysregulation Aging Phenotype in Hippocampal Neurons

With aging, multiple Ca2+-associated electrophysiological processes exhibit increased magnitude in hippocampal pyramidal neurons, including the Ca2+-dependent slow afterhyperpolarization (sAHP), L-type voltage-gated Ca2+ channel (L-VGCC) activity, Ca2+-induced Ca2+ release (CICR) from ryanodine receptors (RyRs), and Ca2+ transients. This pattern of Ca2+ dysregulation correlates with reduced neuronal excitability/plasticity and impaired learning/memory and has been proposed to contribute to unhealthy brain aging and Alzheimers 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 Ca2+ 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 Ca2+ homeostasis in hippocampal neurons and is sufficient to induce the aging phenotype of Ca2+ dysregulation in young animals. Selective knockdown of Fkbp1b with interfering RNA in vitro (96 h) enhanced voltage-gated Ca2+ 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 Ca2+-dysregulation aging phenotype in young rat hippocampus, supporting a novel hypothesis that declining FKBP function plays a major role in unhealthy brain aging.