Susan D. Kraner

REST: A mammalian silencer protein that restricts sodium channel gene expression to neurons

Expression of the type II voltage-dependent sodium channel gene is restricted to neurons by a silencer element active in nonneuronal cells. We have cloned cDNA coding for a transcription factor (REST) that binds to this silencer element. Expression of a recombinant REST protein confers the ability to silence type II reporter genes in neuronal cell types lacking the native REST protein, whereas expression of a dominant negative form of REST in nonneuronal cells relieves silencing mediated by the native protein. REST transcripts in developing mouse embryos are detected ubiquitously outside of the nervous system. We propose that expression of the type II sodium channel gene in neurons reflects a default pathway that is blocked in nonneuronal cells by the presence of REST.

Disruption of TrkB-Mediated Signaling Induces Disassembly of Postsynaptic Receptor Clusters at Neuromuscular Junctions

Neurotrophins and tyrosine receptor kinase (Trk) receptors are expressed in skeletal muscle, but it is unclear what functional role Trk-mediated signaling plays during postnatal life. Full-length TrkB (trkB.FL) as well as truncated TrkB (trkB.t1) were found to be localized primarily to the postsynaptic acetylcholine receptor- (AChR-) rich membrane at neuromuscular junctions. In vivo, dominant-negative manipulation of TrkB signaling using adenovirus to overexpress trkB.t1 in mouse sternomastoid muscle fibers resulted in the disassembly of postsynaptic AChR clusters at neuromuscular junctions, similar to that observed in mutant trkB+/- mice. When TrkB-mediated signaling was disrupted in cultured myotubes in the absence of motor nerve terminals and Schwann cells, agrin-induced AChR clusters were also disassembled. These results demonstrate a novel role for neurotrophin signaling through TrkB receptors on muscle fibers in the ongoing maintenance of postsynaptic AChR regions.

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.

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.

Increased vulnerability of hippocampal neurons with age in culture: temporal association with increases in NMDA receptor current, NR2A subunit expression and recruitment of L-type calcium channels.

Excessive glutamate (Glu) stimulation of the NMDA-R is a widely recognized trigger for Ca(2+)-mediated excitotoxicity. Primary neurons typically show a large increase in vulnerability to excitotoxicity with increasing days in vitro (DIV). This enhanced vulnerability has been associated with increased expression of the NR2B subunit or increased NMDA-R current, but the detailed age-courses of these variables in primary hippocampal neurons have not been compared in the same study. Further, it is not clear whether the NMDA-R is the only source of excess Ca(2+). Here, we used primary hippocampal neurons to examine the age dependence of the increase in excitotoxic vulnerability with changes in NMDA-R current, and subunit expression. We also tested whether L-type voltage-gated Ca(2+) channels (L-VGCCs) contribute to the enhanced vulnerability. The EC(50) for Glu toxicity decreased by approximately 10-fold between 8-9 and 14-15 DIV, changing little thereafter. Parallel experiments found that during the same period both amplitude and duration of NMDA-R current increased dramatically; this was associated with an increase in protein expression of the NR1 and NR2A subunits, but not of the NR2B subunit. Compared to MK-801, ifenprodil, a selective NR2B antagonist, was less effective in protecting older than younger neurons from Glu insult. Conversely, nimodipine, an L-VGCC antagonist, protected older but not younger neurons. Our results indicate that enhanced excitotoxic vulnerability with age in culture was associated with a substantial increase in NMDA-R current, concomitant increases in NR2A and NR1 but not NR2B subunit expression, and with apparent recruitment of L-VGCCs into the excitotoxic process.

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

Significance Higher blood levels of vitamin D are associated with better health outcomes. Vitamin D deficiency, however, is common among the elderly. Despite targets in the brain, little is known about how vitamin D affects cognitive function. In aging rodents, we modeled human serum vitamin D levels ranging from deficient to sufficient and tested whether increasing dietary vitamin D could maintain or improve cognitive function. Treatment was initiated at middle age, when markers of aging emerge, and maintained for ∼6 mo. Compared with low- or normal-dietary vitamin D groups, only aging rats on higher vitamin D could perform a complex memory task and had blood levels considered in the optimal range. These results suggest that vitamin D may improve the likelihood of healthy cognitive aging. 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.

Altered Gene Expression in Steroid-Treated Denervated Muscle

In rats treated with high-dose corticosteroids, skeletal muscle that is denervated in vivo (steroid-denervated) develops electrical inexcitability similar to that seen in patients with acute quadriplegic myopathy. To determine whether changes in muscle gene transcription might underlie inexcitability of steroid-denervated muscle we performed RNase protection assays to quantitate adult (SkM1) and embryonic (SkM2) sodium channel isoforms and chloride channel (CLC-1) mRNA levels in control, denervated, steroid-innervated, and steroid-denervated skeletal muscle. While SkM1 mRNA levels were relatively unaffected by denervation or steroid treatment, SkM2 mRNA levels were increased by both. These effects were synergistic and high levels of SkM2 mRNA were expressed in denervated muscle exposed to corticosteroids. Skeletal muscle CLC-1 mRNA levels were decreased by denervation. To better understand the marked upregulation of SkM2 in steroid-denervated muscle we examined changes in myogenin and glucocorticoid receptor mRNA levels. However, changes in these mRNA levels cannot account for the upregulation of SkM2 in steroid-denervated muscle.

Obesity and diabetes cause cognitive dysfunction in the absence of accelerated β-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 β-amyloid (Aβ)). 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 Aβ deposition was not significantly increased in the diabetic mice, though overall expression of presenilin was elevated. Surprisingly, Aβ was not deposited in the vasculature or removed to the plasma, and there was no stimulation of activity or expression of major Aβ-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 Aβ 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.