Targeting Astrocyte Signaling Alleviates Cerebrovascular and Synaptic Function Deficits in a Diet-Based Mouse Model of Small Cerebral Vessel Disease.

Despite the indispensable role that astrocytes play in the neurovascular unit, few studies have investigated the functional impact of astrocyte signaling in cognitive decline and dementia related to vascular pathology. Diet-mediated induction of hyperhomocysteinemia (HHcy) recapitulates numerous features of vascular contributions to cognitive impairment and dementia (VCID). Here, we used astrocyte targeting approaches to evaluate astrocyte Ca2+ dysregulation and the impact of aberrant astrocyte signaling on cerebrovascular dysfunction and synapse impairment in male and female HHcy diet mice. Two-photon imaging conducted in fully awake mice revealed activity-dependent Ca2+ dysregulation in barrel cortex astrocytes under HHcy. Stimulation of contralateral whiskers elicited larger Ca2+ transients in individual astrocytes of HHcy diet mice compared with control diet mice. However, evoked Ca2+ signaling across astrocyte networks was impaired in HHcy mice. HHcy also was associated with increased activation of the Ca2+/calcineurin-dependent transcription factor NFAT4, which has been linked previously to the reactive astrocyte phenotype and synapse dysfunction in amyloid and brain injury models. Targeting the NFAT inhibitor VIVIT to astrocytes, using adeno-associated virus vectors, led to reduced GFAP promoter activity in HHcy diet mice and improved functional hyperemia in arterioles and capillaries. VIVIT expression in astrocytes also preserved CA1 synaptic function and improved spontaneous alternation performance on the Y maze. Together, the results demonstrate that aberrant astrocyte signaling can impair the major functional properties of the neurovascular unit (i.e., cerebral vessel regulation and synaptic regulation) and may therefore represent a promising drug target for treating VCID and possibly Alzheimer's disease and other related dementias.SIGNIFICANCE STATEMENT The impact of reactive astrocytes in Alzheimer's disease and related dementias is poorly understood. Here, we evaluated Ca2+ responses and signaling in barrel cortex astrocytes of mice fed with a B-vitamin deficient diet that induces hyperhomocysteinemia (HHcy), cerebral vessel disease, and cognitive decline. Multiphoton imaging in awake mice with HHcy revealed augmented Ca2+ responses in individual astrocytes, but impaired signaling across astrocyte networks. Stimulation-evoked arteriole dilation and elevated red blood cell velocity in capillaries were also impaired in cortex of awake HHcy mice. Astrocyte-specific inhibition of the Ca2+-dependent transcription factor, NFAT, normalized cerebrovascular function in HHcy mice, improved synaptic properties in brain slices, and stabilized cognition. Results suggest that astrocytes are a mechanism and possible therapeutic target for vascular-related dementia.

FK506-Binding Protein 12.6/1b, a Negative Regulator of [Ca2+], Rescues Memory and Restores Genomic Regulation in the Hippocampus of Aging Rats.

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 that FKBP1b also can protect downstream transcriptional networks from aging-induced dysregulation. We gave hippocampal microinjections of FKBP1b-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 with FKBP1b substantially 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 aged FKBP1b-treated rats compared with ACs, with FKBP1b restoring 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 and FKBP1b effects suggests that the aging effects arise from FKBP1b deficiency. Functional category analysis revealed that genes downregulated with aging and restored by FKBP1b were associated predominantly with diverse brain structure categories, including cytoskeleton, membrane channels, and extracellular region. Conversely, genes upregulated with aging but not restored by FKBP1b associated primarily with glial-neuroinflammatory, ribosomal, and lysosomal categories. Immunohistochemistry confirmed aging-induced rarefaction and FKBP1b-mediated restoration of neuronal microtubular structure. Therefore, a previously unrecognized genomic network modulating diverse brain structural processes is dysregulated by aging and restored by FKBP1b overexpression.SIGNIFICANCE STATEMENT Previously, 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 hippocampal FKBP1b overexpression also counteracts aging changes in gene transcriptional networks. In addition to reducing memory deficits in aged rats, FKBP1b selectively counteracted aging-induced expression changes in 37% of aging-dependent genes, with cytoskeletal and extracellular structure categories highly associated with the FKBP1b-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 by FKBP1b.

Insulin Receptor Plasma Membrane Levels Increased by the Progesterone Receptor Membrane Component 1.

The insulin receptor (IR) is a ligand-activated receptor tyrosine kinase that has a key role in metabolism, cellular survival, and proliferation. Progesterone receptor membrane component 1 (PGRMC1) promotes cellular signaling via receptor trafficking and is essential for some elements of tumor growth and metastasis. In the present study, we demonstrate that PGRMC1 coprecipitates with IR. Furthermore, we show that PGRMC1 increases plasma membrane IR levels in multiple cell lines and decreases insulin binding at the cell surface. The findings have therapeutic applications because a small-molecule PGRMC1 ligand, AG205, also decreases plasma membrane IR levels. However, PGRMC1 knockdown via short hairpin RNA expression and AG205 treatment potentiated insulin-mediated phosphorylation of the IR signaling mediator AKT. Finally, PGRMC1 also increased plasma membrane levels of two key glucose transporters, GLUT-4 and GLUT-1. Our data support a role for PGRMC1 maintaining plasma membrane pools of the receptor, modulating IR signaling and function.

In vivo imaging of prodromal hippocampus CA1 subfield oxidative stress in models of Alzheimer disease and Angelman syndrome.

Hippocampus oxidative stress is considered pathogenic in neurodegenerative diseases, such as Alzheimer disease (AD), and in neurodevelopmental disorders, such as Angelman syndrome (AS). Yet clinical benefits of antioxidant treatment for these diseases remain unclear because conventional imaging methods are unable to guide management of therapies in specific hippocampus subfields in vivo that underlie abnormal behavior. Excessive production of paramagnetic free radicals in nonhippocampus brain tissue can be measured in vivo as a greater-than-normal 1/T1 that is quenchable with antioxidant as measured by quench-assisted (Quest) MRI. Here, we further test this approach in phantoms, and we present proof-of-concept data in models of AD-like and AS hippocampus oxidative stress that also exhibit impaired spatial learning and memory. AD-like models showed an abnormal gradient along the CA1 dorsal-ventral axis of excessive free radical production as measured by Quest MRI, and redox-sensitive calcium dysregulation as measured by manganese-enhanced MRI and electrophysiology. In the AS model, abnormally high free radical levels were observed in dorsal and ventral CA1. Quest MRI is a promising in vivo paradigm for bridging brain subfield oxidative stress and behavior in animal models and in human patients to better manage antioxidant therapy in devastating neurodegenerative and neurodevelopmental diseases.-Berkowitz, B. A., Lenning, J., Khetarpal, N., Tran, C., Wu, J. Y., Berri, A. M., Dernay, K., Haacke, E. M., Shafie-Khorassani, F., Podolsky, R. H., Gant, J. C., Maimaiti, S., Thibault, O., Murphy, G. G., Bennett, B. M., Roberts, R. In vivo imaging of prodromal hippocampus CA1 subfield oxidative stress in models of Alzheimer disease and Angelman syndrome.

Calcium's role as nuanced modulator of cellular physiology in the brain.

Neuroscientists studying normal brain aging, spinal cord injury, Alzheimer's disease (AD) and other neurodegenerative diseases have focused considerable effort on carefully characterizing intracellular perturbations in calcium dynamics or levels. At the cellular level, calcium is known for controlling life and death and orchestrating most events in between. For many years, intracellular calcium has been recognized as an essential ion associated with nearly all cellular functions from cell growth to degeneration. Often the emphasis is on the negative impact of calcium dysregulation and the typical worse-case-scenario leading inevitably to cell death. However, even high amplitude calcium transients, when executed acutely, can alter neuronal communication and synaptic strength in positive ways, without necessarily killing neurons. Here, we focus on the evidence that calcium has a subtle and distinctive role in shaping and controlling synaptic events that underpin neuronal communication and that these subtle changes in aging or AD may contribute to cognitive decline. We emphasize that calcium imaging in dendritic components is ultimately necessary to directly test for the presence of age- or disease-associated alterations during periods of synaptic activation.

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

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.

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.

Reduction in neuronal L-type calcium channel activity in a double knock-in mouse model of Alzheimer's disease.

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.

Age- and sex-dependent alterations in primary somatosensory cortex neuronal calcium network dynamics during locomotion.

Over the past 30 years, the calcium (Ca2+ ) hypothesis of brain aging has provided clear evidence that hippocampal neuronal Ca2+ dysregulation is a key biomarker of aging. Age-dependent Ca2+ -mediated changes in intrinsic excitability, synaptic plasticity, and activity have helped identify some of the mechanisms engaged in memory and cognitive decline based on work done mostly at the single-cell level and in the slice preparation. Recently, our lab identified age- and Ca2+ -related neuronal network dysregulation in the cortex of the anesthetized animal. Still, investigations in the awake animal are needed to test the generalizability of the Ca2+ hypothesis of brain aging. Here, we used in vigilo two-photon imaging in ambulating mice, to image GCaMP8f in the primary somatosensory cortex (S1), during ambulation and at rest. We investigated aging- and sex-related changes in neuronal networks in the C56BL/6J mouse. Following imaging, gait behavior was characterized to test for changes in locomotor stability. During ambulation, in both young adult and aged mice, an increase in network connectivity and synchronicity was noted. An age-dependent increase in synchronicity was seen in ambulating aged males only. Additionally, females displayed increases in the number of active neurons, Ca2+ transients, and neuronal activity compared to males, particularly during ambulation. These results suggest S1 Ca2+ dynamics and network synchronicity are likely contributors of locomotor stability. We believe this work raises awareness of age- and sex-dependent alterations in S1 neuronal networks, perhaps underlying the increase in falls with age.

RIT1 regulation of CNS lipids RIT1 deficiency Alters cerebral lipid metabolism and reduces white matter tract oligodendrocytes and conduction velocities.

Oligodendrocytes (OLs) generate lipid-rich myelin membranes that wrap axons to enable efficient transmission of electrical impulses. Using a RIT1 knockout mouse model and in situ high-resolution matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) coupled with MS-based lipidomic analysis to determine the contribution of RIT1 to lipid homeostasis. Here, we report that RIT1 loss is associated with altered lipid levels in the central nervous system (CNS), including myelin-associated lipids within the corpus callosum (CC). Perturbed lipid metabolism was correlated with reduced numbers of OLs, but increased numbers of GFAP+ glia, in the CC, but not in grey matter. This was accompanied by reduced myelin protein expression and axonal conduction deficits. Behavioral analyses revealed significant changes in voluntary locomotor activity and anxiety-like behavior in RIT1KO mice. Together, these data reveal an unexpected role for RIT1 in the regulation of cerebral lipid metabolism, which coincide with altered white matter tract oligodendrocyte levels, reduced axonal conduction velocity, and behavioral abnormalities in the CNS.

Disrupting function of FK506-binding protein 1b/12.6 induces the Ca²+-dysregulation aging phenotype in hippocampal neurons.

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.

Dependence of glucose transport on autophagy and GAPDH activity.

Glucose uptake in the brain is critically important to brain health. Using two widely used cell line model systems, we have found that siramesine, a lysosomotropic agent and ligand for the sigma-2 receptor, inhibits glucose uptake and decreases pools of the GLUT1 glucose transporter at the plasma membrane. Siramesine induces autophagy but also disrupts degradation of autophagy substrates, providing a potential mechanism for its action on glucose uptake. In other cell systems, many of the effects of siramesine can be suppressed by α -tocopherol, a type of vitamin E and potent antioxidant, and α-tocopherol also suppressed the effect of siramesine on glucose uptake, suggesting a role for reactive oxygen species and membrane maintenance. We have also identified a novel mechanism for siramesine in which it inhibited plasma membrane levels of GAPDH, a key protein in glycolysis which localizes to the plasma membrane in some cell types. Indeed, GAPDH inhibitors decreased glucose uptake, like siramesine, likely through an overlapping pathway with siramesine. GAPDH inhibitors induced autophagy but inhibited degradation of autophagy targets. Thus, we have identified novel mechanisms required for glucose uptake which may have important implications in disease.

Falling Short: The Contribution of Central Insulin Receptors to Gait Dysregulation in Brain Aging.

Insulin resistance, which manifests as a reduction of insulin receptor signaling, is known to correlate with pathological changes in peripheral tissues as well as in the brain. Central insulin resistance has been associated with impaired cognitive performance, decreased neuronal health, and reduced brain metabolism; however, the mechanisms underlying central insulin resistance and its impact on brain regions outside of those associated with cognition remain unclear. Falls are a leading cause of both fatal and non-fatal injuries in the older population. Despite this, there is a paucity of work focused on age-dependent alterations in brain regions associated with ambulatory control or potential therapeutic approaches to target these processes. Here, we discuss age-dependent alterations in central modalities that may contribute to gait dysregulation, summarize current data supporting the role of insulin signaling in the brain, and highlight key findings that suggest insulin receptor sensitivity may be preserved in the aged brain. Finally, we present novel results showing that administration of insulin to the somatosensory cortex of aged animals can alter neuronal communication, cerebral blood flow, and the motivation to ambulate, emphasizing the need for further investigations of intranasal insulin as a clinical management strategy in the older population.

Sensitivity of the S1 neuronal calcium network to insulin and Bay-K 8644 in vivo: Relationship to gait, motivation, and aging processes.

Neuronal hippocampal Ca2+ dysregulation is a critical component of cognitive decline in brain aging and Alzheimer's disease and is suggested to impact communication and excitability through the activation of a larger after hyperpolarization. However, few studies have tested for the presence of Ca2+ dysregulation in vivo, how it manifests, and whether it impacts network function across hundreds of neurons. Here, we tested for neuronal Ca2+ network dysregulation in vivo in the primary somatosensory cortex (S1) of anesthetized young and aged male Fisher 344 rats using single-cell resolution techniques. Because S1 is involved in sensory discrimination and proprioception, we tested for alterations in ambulatory performance in the aged animal and investigated two potential pathways underlying these central aging- and Ca2+ -dependent changes. Compared to young, aged animals displayed increased overall activity and connectivity of the network as well as decreased ambulatory speed. In aged animals, intranasal insulin (INI) increased network synchronicity and ambulatory speed. Importantly, in young animals, delivery of the L-type voltage-gated Ca2+ channel modifier Bay-K 8644 altered network properties, replicating some of the changes seen in the older animal. These results suggest that hippocampal Ca2+ dysregulation may be generalizable to other areas, such as S1, and might engage modalities that are associated with locomotor stability and motivation to ambulate. Further, given the safety profile of INI in the clinic and the evidence presented here showing that this central dysregulation is sensitive to insulin, we suggest that these processes can be targeted to potentially increase motivation and coordination while also reducing fall frequency with age.

Aging-related gene expression in hippocampus proper compared with dentate gyrus is selectively associated with metabolic syndrome variables in rhesus monkeys.

Age-dependent metabolic syndrome (MetS) is a well established risk factor for cardiovascular disease, but it also confers major risk for impaired cognition in normal aging or Alzheimer's disease (AD). However, little is known about the specific pathways mediating MetS-brain interactions. Here, we performed the first studies quantitatively linking MetS variables to aging changes in brain genome-wide expression and mitochondrial function. In six young adult and six aging female rhesus monkeys, we analyzed gene expression in two major hippocampal subdivisions critical for memory/cognitive function [hippocampus proper, or cornu ammonis (CA), and dentate gyrus (DG)]. Genes that changed with aging [aging-related genes (ARGs)] were identified in each region. Serum variables reflecting insulin resistance and dyslipidemia were used to construct a quantitative MetS index (MSI). This MSI increased with age and correlated negatively with hippocampal mitochondrial function (state III oxidation). More than 2000 ARGs were identified in CA and/or DG, in approximately equal numbers, but substantially more ARGs in CA than in DG were correlated selectively with the MSI. Pathways represented by MSI-correlated ARGs were determined from the Gene Ontology Database and literature. In particular, upregulated CA ARGs representing glucocorticoid receptor (GR), chromatin assembly/histone acetyltransferase, and inflammatory/immune pathways were closely associated with the MSI. These results suggest a novel model in which MetS is associated with upregulation of hippocampal GR-dependent transcription and epigenetic coactivators, contributing to decreased mitochondrial function and brain energetic dysregulation. In turn, these MSI-associated neuroenergetic changes may promote inflammation, neuronal vulnerability, and risk of cognitive impairment/AD.

Epididymal Fat-Derived Sympathoexcitatory Signals Exacerbate Neurogenic Hypertension in Obese Male Mice Exposed to Early Life Stress.

[Figure: see text].

Hippocampal and cognitive aging across the lifespan: a bioenergetic shift precedes and increased cholesterol trafficking parallels memory impairment.

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.

Neuronal Calcium Imaging, Excitability, and Plasticity Changes in the Aldh2-/- Mouse Model of Sporadic Alzheimer's Disease.

BACKGROUND: Dysregulated signaling in neurons and astrocytes participates in pathophysiological alterations seen in the Alzheimer's disease brain, including increases in amyloid-ß, hyperphosphorylated tau, inflammation, calcium dysregulation, and oxidative stress. These are often noted prior to the development of behavioral, cognitive, and non-cognitive deficits. However, the extent to which these pathological changes function together or independently is unclear. OBJECTIVE: Little is known about the temporal relationship between calcium dysregulation and oxidative stress, as some reports suggest that dysregulated calcium promotes increased formation of reactive oxygen species, while others support the opposite. Prior work has quantified several key outcome measures associated with oxidative stress in aldehyde dehydrogenase 2 knockout (Aldh2-/-) mice, a non-transgenic model of sporadic Alzheimer's disease. METHODS: Here, we tested the hypothesis that early oxidative stress can promote calcium dysregulation across aging by measuring calcium-dependent processes using electrophysiological and imaging methods and focusing on the afterhyperpolarization (AHP), synaptic activation, somatic calcium, and long-term potentiation in the Aldh2-/- mouse. RESULTS: Our results show a significant age-related decrease in the AHP along with an increase in the slow AHP amplitude in Aldh2-/- animals. Measures of synaptic excitability were unaltered, although significant reductions in long-term potentiation maintenance were noted in the Aldh2-/- animals compared to wild-type. CONCLUSION: With so few changes in calcium and calcium-dependent processes in an animal model that shows significant increases in HNE adducts, Aß, p-tau, and activated caspases across age, the current findings do not support a direct link between neuronal calcium dysregulation and uncontrolled oxidative stress.

Electrophysiological and Imaging Calcium Biomarkers of Aging in Male and Female 5×FAD Mice.

BACKGROUND: In animal models and tissue preparations, calcium dyshomeostasis is a biomarker of aging and Alzheimer's disease that is associated with synaptic dysfunction, neuritic pruning, and dysregulated cellular processes. It is unclear, however, whether the onset of calcium dysregulation precedes, is concurrent with, or is the product of pathological cellular events (e.g., oxidation, amyloid-ß production, and neuroinflammation). Further, neuronal calcium dysregulation is not always present in animal models of amyloidogenesis, questioning its reliability as a disease biomarker. OBJECTIVE: Here, we directly tested for the presence of calcium dysregulation in dorsal hippocampal neurons in male and female 5×FAD mice on a C57BL/6 genetic background using sharp electrodes coupled with Oregon-green Bapta-1 imaging. We focused on three ages that coincide with the course of amyloid deposition: 1.5, 4, and 10 months old. METHODS: Outcome variables included measures of the afterhyperpolarization, short-term synaptic plasticity, and calcium kinetics during synaptic activation. Quantitative analyses of spatial learning and memory were also conducted using the Morris water maze. Main effects of sex, age, and genotype were identified on measures of electrophysiology and calcium imaging. RESULTS: Measures of resting Oregon-green Bapta-1 fluorescence showed significant reductions in the 5×FAD group compared to controls. Deficits in spatial memory, along with increases in Aß load, were detectable at older ages, allowing us to test for temporal associations with the onset of calcium dysregulation. CONCLUSION: Our results provide evidence that reduced, rather than elevated, neuronal calcium is identified in this 5×FAD model and suggests that this surprising result may be a novel biomarker of AD.

Molecular elevation of insulin receptor signaling improves memory recall in aged Fischer 344 rats.

As demonstrated by increased hippocampal insulin receptor density following learning in animal models and decreased insulin signaling, receptor density, and memory decline in aging and Alzheimer's diseases, numerous studies have emphasized the importance of insulin in learning and memory processes. This has been further supported by work showing that intranasal delivery of insulin can enhance insulin receptor signaling, alter cerebral blood flow, and improve memory recall. Additionally, inhibition of insulin receptor function or expression using molecular techniques has been associated with reduced learning. Here, we sought a different approach to increase insulin receptor activity without the need for administering the ligand. A constitutively active, modified human insulin receptor (IRß) was delivered to the hippocampus of young (2 months) and aged (18 months) male Fischer 344 rats in vivo. The impact of increasing hippocampal insulin receptor expression was investigated using several outcome measures, including Morris water maze and ambulatory gait performance, immunofluorescence, immunohistochemistry, and Western immunoblotting. In aged animals, the IRß construct was associated with enhanced performance on the Morris water maze task, suggesting that this receptor was able to improve memory recall. Additionally, in both age-groups, a reduced stride length was noted in IRß-treated animals along with elevated hippocampal insulin receptor levels. These results provide new insights into the potential impact of increasing neuronal insulin signaling in the hippocampus of aged animals and support the efficacy of molecularly elevating insulin receptor activity in vivo in the absence of the ligand to directly study this process.