Philip W. Landfield

Aging is the leading risk factor for idiopathic Alzheimer’s disease (AD), indicating that normal aging processes promote AD and likely are present in the neurons in which AD pathogenesis originates. In AD, neurofibrillary tangles (NFTs) appear first

Hippocampal overexpression of FK506-binding protein 12.6/1b (FKBP1b), a negative regulator of ryanodine receptor Ca²⁺release, reverses aging-induced memory impairment and neuronal Ca²⁺dysregulation. Here, we tested the hypothesis thatFKBP1balso can protect downstream transcriptional networks from aging-induced dysregulation. We gave hippocampal microinjections ofFKBP1b-expressing viral vector to male rats at either 13 months of age (long-term, LT) or 19 months of age (short-term, ST) and tested memory performance in the Morris water maze at 21 months of age. Aged rats treated ST or LT withFKBP1bsubstantially outperformed age-matched vector controls and performed similarly to each other and young controls (YCs). Transcriptional profiling in the same animals identified 2342 genes with hippocampal expression that was upregulated/downregulated in aged controls (ACs) compared with YCs (the aging effect). Of these aging-dependent genes, 876 (37%) also showed altered expression in agedFKBP1b-treated rats compared with ACs, withFKBP1brestoring expression of essentially all such genes (872/876, 99.5%) in the direction opposite the aging effect and closer to levels in YCs. This inverse relationship between the aging andFKBP1beffects suggests that the aging effects arise fromFKBP1bdeficiency. Functional category analysis revealed that genes downregulated with aging and restored byFKBP1bwere associated predominantly with diverse brain structure categories, including cytoskeleton, membrane channels, and extracellular region. Conversely, genes upregulated with aging but not restored byFKBP1bassociated primarily with glial–neuroinflammatory, ribosomal, and lysosomal categories. Immunohistochemistry confirmed aging-induced rarefaction andFKBP1b-mediated restoration of neuronal microtubular structure. Therefore, a previously unrecognized genomic network modulating diverse brain structural processes is dysregulated by aging and restored byFKBP1boverexpression.SIGNIFICANCE STATEMENTPreviously, we found that hippocampal overexpression of FK506-binding protein 12.6/1b (FKBP1b), a negative regulator of intracellular Ca²⁺responses, reverses both aging-related Ca²⁺dysregulation and cognitive impairment. Here, we tested whether hippocampalFKBP1boverexpression also counteracts aging changes in gene transcriptional networks. In addition to reducing memory deficits in aged rats,FKBP1bselectively counteracted aging-induced expression changes in 37% of aging-dependent genes, with cytoskeletal and extracellular structure categories highly associated with theFKBP1b-rescued genes. Our results indicate that, in parallel with cognitive processes, a novel transcriptional network coordinating brain structural organization is dysregulated with aging and restored byFKBP1b.

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 Alzheimer9s 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, Alzheimer9s 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 Alzheimer9s disease.

Although glucocorticoids (GCs) are known to exert numerous effects in the hippocampus, their chronic regulatory functions remain poorly understood. Moreover, evidence is inconsistent regarding the long-standing hypothesis that chronic GC exposure promotes brain aging/Alzheimer disease. Here, we adrenalectomized male F344 rats at 15 months of age, maintained them for 3 months with implanted corticosterone (CORT) pellets producing low or intermediate (glucocorticoid receptor–activating) blood levels of CORT, and performed microarray/pathway analyses in hippocampal CA1. We defined the chronic GC-dependent transcriptome as 393 genes that exhibited differential expression between intermediate and low CORT groups. Short-term CORT (4 days) did not recapitulate this transcriptome. Functional processes/pathways overrepresented by chronic CORT–up-regulated genes included learning/plasticity, differentiation, glucose metabolism, and cholesterol biosynthesis, whereas processes overrepresented by CORT–down-regulated genes included inflammatory/immune/glial responses and extracellular structure. These profiles indicate that GCs chronically activate neuronal/metabolic processes while coordinately repressing a glial axis of reactivity/inflammation. We then compared the GC transcriptome with a previously defined hippocampal aging transcriptome, revealing a high proportion of common genes. Although CORT and aging moved expression of some common genes in the same direction, the majority were shifted in opposite directions by CORT and aging (eg, glial inflammatory genes down-regulated by CORT are up-regulated with aging). These results contradict the hypothesis that GCs simply promote brain aging and also suggest that the opposite direction shifts during aging reflect resistance to CORT regulation. Therefore, we propose a new model in which aging-related GC resistance develops in some target pathways, whereas GC overstimulation develops in others, together generating much of the brain aging phenotype.

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 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 Ca²⁺release. Moreover, we recently found that hippocampalFkbp1bexpression is downregulated, whereasRyr2andFrap1/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 ofFkbp1bwith interfering RNAin vitro(96 h) enhanced voltage-gated Ca²⁺current in cultured neurons, whereasin vivo Fkbp1bknockdown 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 knockdownin vivowas 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.

Multiple hippocampal processes and cognitive functions change with aging or Alzheimer's disease, but the potential triggers of these aging cascades are not well understood. Here, we quantified hippocampal expression profiles and behavior across the adult lifespan to identify early aging changes and changes that coincide with subsequent onset of cognitive impairment. Well powered microarray analyses (N= 49 arrays), immunohistochemistry, and Morris spatial maze learning were used to study male F344 rats at five age points. Genes that changed with aging (by ANOVA) were assigned to one of four onset age ranges based on template pattern matching; functional pathways represented by these genes were identified statistically (Gene Ontology). In the earliest onset age range (3–6 months old), upregulation began for genes in lipid/protein catabolic and lysosomal pathways, indicating a shift in metabolic substrates, whereas downregulation began for lipid synthesis, GTP/ATP-dependent signaling, and neural development genes. By 6–9 months of age, upregulation of immune/inflammatory cytokines was pronounced. Cognitive impairment first appeared in the midlife range (9–12 months) and coincided and correlated primarily with midlife upregulation of genes associated with cholesterol trafficking (apolipoprotein E), myelinogenic, and proteolytic/major histocompatibility complex antigen-presenting pathways. Immunolabeling revealed that cholesterol trafficking proteins were substantially increased in astrocytes and that myelination increased with aging. Together, our data suggest a novel sequential model in which an early-adult metabolic shift, favoring lipid/ketone body oxidation, triggers inflammatory degradation of myelin and resultant excess cholesterol that, by midlife, activates cholesterol transport from astrocytes to remyelinating oligodendrocytes. These processes may damage structure and compete with neuronal pathways for bioenergetic resources, thereby impairing cognitive function.

The original glucocorticoid (GC) hypothesis of brain aging and Alzheimers disease proposed that chronic exposure to GCs promotes hippocampal aging and AD. This proposition arose from a study correlating increasing plasma corticosterone with hippocampal astrocyte reactivity in aging rats. Numerous subsequent studies have found evidence consistent with this hypothesis, in animal models and in humans. However, several results emerged that were inconsistent with the hypothesis, highlighting the need for a more definitive test with a broader panel of biomarkers. We used microarray analyses to identify a panel of hippocampal gene expression changes that were aging-dependent, and also corticosterone- dependent. These data enabled us to test a key prediction of the GC hypothesis, namely, that the expression of most target biomarkers of brain aging should be regulated in the same direction (increased or decreased) by both GCs and aging. This prediction was decisively contradicted, as a majority of biomarker genes were regulated in opposite directions by aging and GCs, particularly inflammatory and astrocyte-specific genes. Thus, the initial hypothesis of simple positive cooperativity between GCs and aging must be rejected. Instead, our microarray data suggest that in the brain GCs and aging interact in more complex ways that depend on the cell type. Therefore, we propose a new version of the GC-brain aging hypothesis; its main premise is that aging selectively increases GC efficacy in some cell types (e.g., neurons), enhancing catabolic processes, whereas aging selectively decreases GC efficacy in other cell types (e.g., astrocytes), weakening GC anti-inflammatory activity. We also propose that changes in GC efficacy might be mediated in part by cell type specific shifts in the antagonistic balance between GC and insulin actions, which may be of relevance for Alzheimers disease pathogenesis.

Summary: Evidence accumulated over more than two decades has implicated Ca²⁺ dysregulation in brain aging and Alzheimer's disease (AD), giving rise to the Ca²⁺ hypothesis of brain aging and dementia. Electrophysiological, imaging, and behavioral studies in hippocampal or cortical neurons of rodents and rabbits have revealed aging‐related increases in the slow afterhyperpolarization, Ca²⁺ spikes and currents, Ca²⁺ transients, and L‐type voltage‐gated Ca²⁺ channel (L‐VGCC) activity. Several of these changes have been associated with age‐related deficits in learning or memory. Consequently, one version of the Ca²⁺ hypothesis has been that increased L‐VGCC activity drives many of the other Ca²⁺‐related biomarkers of hippocampal aging. In addition, other studies have reported aging‐ or AD model‐related alterations in Ca²⁺ release from ryanodine receptors (RyR) on intracellular stores. The Ca²⁺‐sensitive RyR channels amplify plasmalemmal Ca²⁺ influx by the mechanism of Ca²⁺‐induced Ca²⁺ release (CICR). Considerable evidence indicates that a preferred functional link is present between L‐VGCCs and RyRs which operate in series in heart and some brain cells. Here, we review studies implicating RyRs in altered Ca²⁺ regulation in cell toxicity, aging, and AD. A recent study from our laboratory showed that increased CICR plays a necessary role in the emergence of Ca²⁺‐related biomarkers of aging. Consequently, we propose an expanded L‐VGCC/Ca²⁺ hypothesis, in which aging/pathological changes occur in both L‐type Ca²⁺ channels and RyRs, and interact to abnormally amplify Ca²⁺ transients. In turn, the increased transients result in dysregulation of multiple Ca²⁺‐dependent processes and, through somewhat different pathways, in accelerated functional decline during aging and AD.