Reversal of Aging-Related Neuronal Ca2+ Dysregulation and Cognitive Impairment by Delivery of a Transgene Encoding FK506-Binding Protein 12.6/1b to the Hippocampus.

Brain Ca2+ regulatory processes are altered during aging, disrupting neuronal, and cognitive functions. In hippocampal pyramidal neurons, the Ca2+ -dependent slow afterhyperpolarization (sAHP) exhibits an increase with aging, which correlates with memory impairment. The increased sAHP results from elevated L-type Ca2+ channel activity and ryanodine receptor (RyR)-mediated Ca2+ release, but underlying molecular mechanisms are poorly understood. Previously, we found that expression of the gene encoding FK506-binding protein 12.6/1b (FKBP1b), a small immunophilin that stabilizes RyR-mediated Ca2+ release in cardiomyocytes, declines in hippocampus of aged rats and Alzheimer's disease subjects. Additionally, knockdown/disruption of hippocampal FKBP1b in young rats augments neuronal Ca2+ responses. Here, we test the hypothesis that declining FKBP1b underlies aging-related hippocampal Ca2+ dysregulation. Using microinjection of adeno-associated viral vector bearing a transgene encoding FKBP1b into the hippocampus of aged male rats, we assessed the critical prediction that overexpressing FKBP1b should reverse Ca2+ -mediated manifestations of brain aging. Immunohistochemistry and qRT-PCR confirmed hippocampal FKBP1b overexpression 4-6 weeks after injection. Compared to aged vector controls, aged rats overexpressing FKBP1b showed dramatic enhancement of spatial memory, which correlated with marked reduction of sAHP magnitude. Furthermore, simultaneous electrophysiological recording and Ca2+ imaging in hippocampal neurons revealed that the sAHP reduction was associated with a decrease in parallel RyR-mediated Ca2+ transients. Thus, hippocampal FKBP1b overexpression reversed key aspects of Ca2+ dysregulation and cognitive impairment in aging rats, supporting the novel hypothesis that declining FKBP1b is a molecular mechanism underlying aging-related Ca2+ dysregulation and unhealthy brain aging and pointing to FKBP1b as a potential therapeutic target. SIGNIFICANCE STATEMENT: This paper reports critical tests of a novel hypothesis that proposes a molecular mechanism of unhealthy brain aging and possibly, Alzheimer's disease. For more than 30 years, evidence has been accumulating that brain aging is associated with dysregulation of calcium in neurons. Recently, we found that FK506-binding protein 12.6/1b (FKBP1b), a small protein that regulates calcium, declines with aging in the hippocampus, a brain region important for memory. Here we used gene therapy approaches and found that raising FKBP1b reversed calcium dysregulation and memory impairment in aging rats, allowing them to perform a memory task as well as young rats. These studies identify a potential molecular mechanism of brain aging and may also have implications for treatment of Alzheimer's disease.

Microarray analyses of laser-captured hippocampus reveal distinct gray and white matter signatures associated with incipient Alzheimer's disease.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder that threatens to reach epidemic proportions as our population ages. Although much research has examined molecular pathways associated with AD, relatively few such studies have focused on the disease's critical early stages. In a prior microarray study we correlated gene expression in hippocampus with degree of Alzheimer's disease and found close associations between upregulation of apparent glial transcription factor/epigenetic/tumor suppressor genes and incipient AD. The results suggested a new model in which AD pathology spreads along myelinated axons (Blalock et al., 2004). However, the microarray analyses were performed on RNA extracted from frozen hand-dissected hippocampal CA1 tissue blocks containing both gray and white matter, limiting the confidence with which transcriptional changes in gray matter could be distinguished from those in white matter. Here, we used laser capture microdissection (LCM) to exclude major white matter tracts while selectively collecting CA1 hippocampal gray matter from formalin-fixed, paraffin-embedded (FFPE) hippocampal sections of the same subjects assessed in our prior study. Microarray analyses of this gray matter-enriched tissue revealed many transcriptional changes similar to those seen in our past study and in studies by others, particularly for downregulated neuron-related genes. Additionally, the present analyses identified several previously undetected pathway alterations, including downregulation of molecules that stabilize ryanodine receptor Ca2+ release and upregulation of vasculature development. Conversely, we found a striking paucity of the upregulated changes in the putative glial and growth-related genes that had been strongly overrepresented in the prior mixed-tissue study. We conclude that FFPE tissue can be a reliable resource for microarray studies of brain tissue, that upregulation of growth-related epigenetic/transcription factors during incipient AD is predominantly localized in and around white matter (supporting our prior findings and model), and that novel alterations in vascular and ryanodine receptor-related pathways in gray matter are closely associated with incipient AD.

Hippocampal 'zipper' slice studies reveal a necessary role for calcineurin in the increased activity of L-type Ca(2+) channels with aging.

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.

Estradiol reverses a calcium-related biomarker of brain aging in female rats.

An increase in L-type voltage-gated calcium channel (LTCC) current is a prominent biomarker of brain aging and is believed to contribute to cognitive decline and vulnerability to neuropathologies. Studies examining age-related changes in LTCCs have focused primarily on males, although estrogen (17beta-estradiol, E2) affects calcium-dependent activities associated with cognition. Therefore, to better understand brain aging in females, the effects of chronic E2 replacement on LTCC current activity in hippocampal neurons of young and aged ovariectomized rats were determined. The zipper slice preparation was used to expose cornu ammonis 1 (CA1) pyramidal neurons for recording LTCC currents using the cell-attached patch-clamp technique. We found that an age-related increase in LTCC current in neurons from control animals was prevented by E2 treatment. In addition, in situ hybridization revealed that within stratum pyramidale of the CA1 area, mRNA expression of the Ca(v)1.2 LTCC subunit, but not the Ca(v)1.3 subunit, was decreased in aged E2-treated rats. Thus, the reported benefits of E2 on cognition and neuronal health may be attributed, at least in part, to its age-related decrease in LTCC current.

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.

Incipient Alzheimer's disease: microarray correlation analyses reveal major transcriptional and tumor suppressor responses.

The pathogenesis of incipient Alzheimer's disease (AD) has been resistant to analysis because of the complexity of AD and the overlap of its early-stage markers with normal aging. Gene microarrays provide new tools for addressing complexity because they allow overviews of the simultaneous activity of multiple cellular pathways. However, microarray data interpretation is often hindered by low statistical power, high false positives or false negatives, and by uncertain relevance to functional endpoints. Here, we analyzed hippocampal gene expression of nine control and 22 AD subjects of varying severity on 31 separate microarrays. We then tested the correlation of each gene's expression with MiniMental Status Examination (MMSE) and neurofibrillary tangle (NFT) scores across all 31 subjects regardless of diagnosis. These well powered tests revealed a major transcriptional response comprising thousands of genes significantly correlated with AD markers. Several hundred of these genes were also correlated with AD markers across only control and incipient AD subjects (MMSE > 20). Biological process categories associated with incipient AD-correlated genes were identified statistically (ease program) and revealed up-regulation of many transcription factor/signaling genes regulating proliferation and differentiation, including tumor suppressors, oligodendrocyte growth factors, and protein kinase A modulators. In addition, up-regulation of adhesion, apoptosis, lipid metabolism, and initial inflammation processes occurred, and down-regulation of protein folding/metabolism/transport and some energy metabolism and signaling pathways took place. These findings suggest a new model of AD pathogenesis in which a genomically orchestrated up-regulation of tumor suppressor-mediated differentiation and involution processes induces the spread of pathology along myelinated axons.