Astrocytes are a glial cell type responsible for many protective functions in the brain. While they are primarily recognized for regulating synaptic activity, they’re also essential for maintaining the neurovascular unit, and cerebral hyperperfusion during metabolic demand. Calcium signaling has been identified as a regulatory process for these functions. Amyloid Beta deposits (Aβ) are a primary diagnostic marker of Alzheimer’s disease, and are linked to synaptic degeneration, astrocyte reactivity, and cognitive decline. Alzheimer’s disease also frequently presents with vascular damage and hypoperfusion, suggesting impaired astrocyte function. We investigated the impact of amyloid burden on stimulation-evoked vasoreactivity and astrocyte calcium signaling to determine potential patterns of physiological impairment that may contribute to Alzheimer’s disease pathology. Six-month-old 5XFAD and littermate control mice were injected with AAV2/5-Gfa104-jGCaMP8f into barrel cortex and imaged three weeks later, while awake, using two-photon microscopy. Neurovascular coupling experiments were conducted using timed air puff stimulation of whiskers, and calcium signals were recorded from activated astrocytes. Calcium transient properties were analyzed over different cellular compartments by custom developed Matlab applications. Vascular tone in response to stimulation was also measured, and correlations calculated with endfeet signaling. Astrocytes from 5XFAD mice showed a significant reduction in calcium signaling amplitudes, (F(1,53) = 8.735, p = 0.0047, n = 25,32) compared to wild type controls with a significant deficit in female 5XFAD mice. Correlations between other signaling properties such as rise/decay kinetics, and network connectivity were also characterized between the 5XFAD and control mice. Astrocyte endfoot compartments also showed reduced transient amplitudes. (F(1,30) = 3.226, p = 0.033, n = 17,15) Neurovascular coupling in the 5XFAD mice was reduced (F(1,39) = 2.511, p = 0.015, n = 20,19) despite no changes in arteriole elasticity. A correlation between astrocyte calcium signaling and the magnitude of stimulation-induced vasodilation was observed in wild-type mice but not in the 5XFAD group. Amyloid induced pathology impairs the brain’s adaptivity to neuronal stimuli at the neurovascular unit. The uncoupling between vasoreactivity and astrocyte signaling processes implies that amyloid accumulation may render the brain vulnerable to conditions of neuronal hyperexcitability and metabolic dysregulation observed in Alzheimer’s disease.

Amyloid deposits (Aβ) in the brain are a primary diagnostic marker of Alzheimer’s disease, and are associated with the degeneration of synapses and cognitive decline. However, recent advances in cell specific approaches have revealed that glial processes may also contribute to the progression of disease pathology. Astrocytes are a glial cell in the brain responsible for a plethora of essential functions in the brain, such as the removal of synaptic glutamate, and control over cerebrovascular function. Astrocyte Ca2+ signaling is closely coupled to these functions. These pathways are dysregulated with disease onset, and contribute to astrocyte reactivity. Our group showed previously in a diet model of VCID, that changes in astrocyte calcium signaling and network synchronicity were associated with deficits in cerebrovascular function. Here we examined the spatial and temporal relationship of astrocyte Ca2+ signals to neurovascular coupling in 5XFAD mice to determine the relationship between reactive astrocytes and cerebrovascular function. 5XFAD and littermate controls aged to 6 months were injected with AAV2/5-Gfa104-jGCaMP8f into barrel cortex before cranial window installation. At 7 months, mice were briefly anaesthetized before retroorbital injection of 500kb rhodamine dextran. Mice were then imaged awake under a two-photon microscope for functional hyperemia measures in response to timed air puff whisker stimulation. Spontaneous and evoked Ca2+ transients were measured by ∆F/F calculation of Ca2+ peaks extracted by custom MATLAB algorithms. Analysis of astrocyte endfoot Ca2+ was measured along vessels stimulated for neurovascular coupling to relate vasoactivity to Ca2+ signaling kinetics and intensity. 5XFAD mice showed a significant reduction is astrocyte Ca2+ rise time kinetics compared to wild type controls, as well as parameters of functional hyperemia. Timing of neurovascular coupling in response to stimulation and the latency of astrocyte calcium kinetics was characterized for the 5XFAD model and shown to differ between soma and endfoot processes. Amyloid induced pathology induces changes in astrocyte Ca2+ signaling. Dysregulation of astrocyte signaling may have a mechanistic role in the kinetics of neurovascular coupling and/or cerebrovascular dysfunction with disease progression. However, further analysis of astrocyte endfoot coverage and manipulation of astrocyte signaling during functional hyperemia is needed to further clarify mechanism.

Mice exposed to a diet deficient in B6/B12 vitamins and enriched in methionine exhibit hyperhomocysteinemia (HHcy) concomitant with many pathologic features of vascular cognitive impairment and dementia: a very common Alzheimer’s disease related dementia. Here, we evaluated the impact of astrocyte Ca2+ dysregulation and astrocytic Ca2+ signaling pathways in the context of functional hyperemia in fully awake mice exposed to HHcy diet. Adult C57/BL6 mice were fed for 15 weeks with control chow, or chow deficient in vitamins B6/B12 and enriched in methionine (HHcy diet). Spontaneous and evoked (via air puff whisker stimulation) Ca2+ transients in astrocytes of fully awake mice were investigated in barrel cortex using the Ca2+ sensor GCaMP6f (expressed specifically in astrocytes using AAV) and two-photon imaging. In other experiments, mice were treated with AAV-Gfa2-VIVIT to inhibit the Ca2+-sensitive calcineurin/NFAT pathway in astrocytes, which regulates reactive astrocyte phenotypes. Two-photon imaging was then used to assess diet and VIVIT effects on functional hyperemia in barrel cortex of fully awake mice. Ca2+ transients in individual barrel cortex astrocytes were significantly augmented (greater in amplitude, with faster rise/decay kinetics) in mice treated with HHcy diet. However, the functional connectivity in astrocyte networks of HHcy mice was significantly impaired, particularly during engagement of brain activity with whisker stimulation. Compared to control diet mice, HHcy mice also showed impaired functional hyperemia during whisker stimulation characterized by impaired arteriole dilations and reduced red blood cell velocity in nearby capillaries. Inhibition of reactive astrocyte signaling in HHcy diet mice with VIVIT significantly ameliorated these neurovascular coupling deficits. The results show that astrocyte Ca2+ dysregulation is a major feature of HHcy and contributes to cerebrovascular dysfunction through hyperactivation of the astrocytic CN/NFAT pathway.

The p38alpha MAPK (p38) signaling pathway is ubiquitously expressed in the brain where it plays central roles in neuroinflammation, metabolism, synaptic development and communication, and modulation of blood brain barrier integrity. Its activity in astrocytes has largely been described as immunomodulatory; however, it has also recently been reported to contribute to long term depression and associated behaviors. Astrocytes are also known to be important to healthy brain aging and neuroprotection, but the specific involvement of astrocytic p38 signaling is unclear. We therefore used conditional KO mice to determine how the loss of p38 in astrocytes early in adulthood (3 months) affects synaptic function and cytokine expression phenotypes in later life (20+ months). Mice were bred homozygous for floxed p38, heterozygous for a Rosa reporter, and with or without tamoxifen-inducible Cre recombinase under control of the astrocyte aldh1l1 promoter. When animals were about 3 months of age they were placed on tamoxifen diet (400 ppm) for 4 weeks, after which they were returned to standard chow and allowed to age until 20-24 months prior to undergoing electrophysiological endpoints or collection of brain tissue for biochemical analyses. Electrically evoked field excitatory post synaptic potentials (fEPSPs) were recorded in hippocampal CA1 stratum radiatum in acutely prepared brain slices and cytokines from the whole hippocampus were measured from a subset of mice that did not undergo electrophysiological recordings. Males and females showed baseline differences in measures of I/O, fiber volley amplitude, early vs. late potentiation, and paired pulse facilitation. Additionally, knockout of astrocyte p38 generally had opposite effects on these parameters in males and females. Interestingly, the pattern of hippocampal IL-1β cytokine levels mirrored these differences. These data indicate that there may be qualitative differences in male versus female brain aging that are differentially impacted by astrocyte p38 signaling. The implications of these findings are currently being explored to determine the underlying mechanisms for these sexually dimorphic effects and further determine what functional differences may exist for hippocampal-dependent behaviors.

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.

Cerebrovascular pathology, often found in autopsy confirmed Alzheimer’s disease (AD), is widely believed to exacerbate AD pathophysiology and complicate anti-AD treatment strategies. Perivascular cells such as astrocytes maintain the microenvironment for proper brain cell function and vascular integrity. Early occurrence of cerebral small vessel disease and astrocyte reactivity could lower the threshold of neuronal disorders and AD. Studies of astrocyte and microvascular pathology and function in the brain have been limited and reaction of astrocytes to vascular damage has yet to be fully understood. AAV-Gfa-EGFP or AAV-Gfa-GCaMP6 was injected into the barrel cortex of wild type mice followed by glass window installation. After 3 weeks recovery, rhodamine dextran was IV -injected and intravital brain imaging through two-photon microscopy was performed at pre- and post-photoinduction. Photoactivation of IV-injected Rose Bengal dye (RB) technique was used to create vascular pathology. ImageJ was used for data analysis and GraphPad prism was used for statistical analysis. After photoinduction, green fluorescence intensity was increased in blood stream of both EGFP and GCaMP expressing brains. This result indicates a leakage of brain parenchyma component to blood stream. Astrocytic EGFP fluorescence intensity and astrocyte processes complexity were reduced after photoinduction in a time-dependent manner. Interestingly, astrocyte calcium signaling was strikingly increased after photoinduction and returned back to the baseline at ∼1,000s post-induction. The highest peak of astrocyte calcium signaling was located near a site of vascular damage, indicated by an increased in rhodamine fluorescence intensity in the brain parenchyma area. Astrocytic GCaMP intensity at the astrocyte endfeet surrounding small vasculature was increased, on the other hand, GCaMP intensity at the astrocyte processes are decreased in a time-dependent manner. Consistently, we observed a reduction of vascular diameter after photoinduction in both EGFP and GCaMP expressing brains. Acute vascular injury induced by rose bengal photoinduction altered astrocyte integrity and cellular signaling. This injury model resembles vascular constriction and hemorrhages found in human brain that could accelerate neurodegeneration and AD progression.

Background: Dysregulated signaling in neurons and astrocytes participates in pathophysiological alterations seen in the Alzheimer’s disease brain, including increases in amyloid-β, 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, Aβ, p-tau, and activated caspases across age, the current findings do not support a direct link between neuronal calcium dysregulation and uncontrolled oxidative stress. Keywords Afterhyperpolarization , aging , calcium dysregulation , electrophysiology , hippocampus , HNE , intracellular , oxidative stress

Background: Dysregulated signaling in neurons and astrocytes participates in pathophysiological alterations seen in the Alzheimer’s disease brain, including increases in amyloid-β, 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, Aβ, p-tau, and activated caspases across age, the current findings do not support a direct link between neuronal calcium dysregulation and uncontrolled oxidative stress.

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-β 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 Aβ 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.

Increasing evidence suggests that vitamin D plays a role in maintaining cognitive function and that vitamin D deficiency may accelerate age-related cognitive decline. Here, we determined if a long-term enhanced vitamin D (VitD3, cholecalciferol) diet, higher than the standard dietary level, maintains or improves cognitive function in aging male and female rats. We also examined if the high VitD3 diet affected the gut microbiome. Beginning at 12 months of age 20 male and 20 female F344 rats were fed an AIN-93 diet containing either standard (1000 IU/kg diet) or higher (10,000 IU/kg) VitD3 for 6 months. The Morris water maze (MWM) was then used to assess learning and memory. Following the MWM, the gut microbiome from undigested chyme collected from the intestinal cecum was identified and taxonomically classified by Argonne National Laboratory using 16S rRNA sequences. ZRT Laboratory determined 25-(OH)VitD3 levels from cardiac blood. A two-way ANOVA and Tukey post-hoc was used to test for statistical significance. After 3 days of training the probe test on day 4 indicated that the higher VitD3 diet significantly reduced path length and latency (P = 0.01) to the digital platform in females but not males. On day 5 platform location was changed and animals received one day of reversal training. On day 8, three days after reversal training, the reversal probe indicated that higher dietary VitD3 improved performance in males but not females by significantly reducing path length and latency to the digital platform (P < 0.05). Analyses of the cecal microbiome content indicated that for numerous bacteria sex specific differences were present. Further, the Shannon Diversity Index of the gut microbiome indicated a significant treatment effect of higher dietary VitD3 in females (P = 0.01). The higher VitD3 diet significantly elevated 25-(OH)VitD3 blood levels. These results indicate that a high VitD3 diet may preserve cognitive acuity during aging. Further, VitD3 may have sexually dimorphic effects on memory formation. The present results replicate our previous study that a high VitD3 diet preserves cognition in aging male rats (Latimer et al. 2014). The microbiome showed sexually dimorphic differences and the high VitD3 diet appeared to affect the microbiome in females more than males. The significance of this is not clear. NIA, NIDDK.

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 in entorhinal cortex, implying that aging processes in entorhinal neurons promote NFT pathogenesis.Using electrophysiology and immunohistochemistry, we find pronounced aging-related Ca 2+ dysregulation in rat entorhinal neurons homologous with the human neurons in which NFTs originate.Considering that humans recapitulate many aspects of animal brain aging, these results support the hypothesis that aging-related Ca 2+ dysregulation occurs in human entorhinal neurons and promotes NFT pathogenesis.

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.

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.

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.

Peripheral insulin resistance is a key component of metabolic syndrome associated with obesity, dyslipidemia, hypertension, and type 2 diabetes. While the impact of insulin resistance is well recognized in the periphery, it is also becoming apparent in the brain. Recent studies suggest that insulin resistance may be a factor in brain aging and Alzheimer's disease (AD) whereby intranasal insulin therapy, which delivers insulin to the brain, improves cognition and memory in AD patients. Here, we tested a clinically relevant delivery method to determine the impact of two forms of insulin, short-acting insulin lispro (Humalog) or long-acting insulin detemir (Levemir), on cognitive functions in aged F344 rats. We also explored insulin effects on the Ca(2+)-dependent hippocampal afterhyperpolarization (AHP), a well-characterized neurophysiological marker of aging which is increased in the aged, memory impaired animal. Low-dose intranasal insulin improved memory recall in aged animals such that their performance was similar to that seen in younger animals. Further, because ex vivo insulin also reduced the AHP, our results suggest that the AHP may be a novel cellular target of insulin in the brain, and improved cognitive performance following intranasal insulin therapy may be the result of insulin actions on the AHP.

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

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.

Background Thiazolidinediones (TZDs) activate peroxisome proliferator-activated receptor gamma (PPARγ) and are used clinically to help restore peripheral insulin sensitivity in Type 2 diabetes (T2DM). Interestingly, long-term treatment of mouse models of Alzheimer's disease (AD) with TZDs also has been shown to reduce several well-established brain biomarkers of AD including inflammation, oxidative stress and Aβ accumulation. While TZD's actions in AD models help to elucidate the mechanisms underlying their potentially beneficial effects in AD patients, little is known about the functional consequences of TZDs in animal models of normal aging. Because aging is a common risk factor for both AD and T2DM, we investigated whether the TZD, pioglitazone could alter brain aging under non-pathological conditions. Methods and Findings We used the F344 rat model of aging, and monitored behavioral, electrophysiological, and molecular variables to assess the effects of pioglitazone (PIO-Actos® a TZD) on several peripheral (blood and liver) and central (hippocampal) biomarkers of aging. Starting at 3 months or 17 months of age, male rats were treated for 4–5 months with either a control or a PIO-containing diet (final dose approximately 2.3 mg/kg body weight/day). A significant reduction in the Ca2+-dependent afterhyperpolarization was seen in the aged animals, with no significant change in long-term potentiation maintenance or learning and memory performance. Blood insulin levels were unchanged with age, but significantly reduced by PIO. Finally, a combination of microarray analyses on hippocampal tissue and serum-based multiplex cytokine assays revealed that age-dependent inflammatory increases were not reversed by PIO. Conclusions While current research efforts continue to identify the underlying processes responsible for the progressive decline in cognitive function seen during normal aging, available medical treatments are still very limited. Because TZDs have been shown to have benefits in age-related conditions such as T2DM and AD, our study was aimed at elucidating PIO's potentially beneficial actions in normal aging. Using a clinically-relevant dose and delivery method, long-term PIO treatment was able to blunt several indices of aging but apparently affected neither age-related cognitive decline nor peripheral/central age-related increases in inflammatory signaling.