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Supplemental Digital Content is available in the text., Previously, we have shown that male mice exposed to maternal separation and early weaning (MSEW)—a mouse model of early life stress—display increased mean arterial pressure compared with controls when fed a high-fat diet. As the stimulation of sensory nerves from fat has been shown to trigger the adipose afferent reflex, we tested whether MSEW male mice show obesity-associated hypertension via the hyperactivation of this sympathoexcitatory mechanism. After 16 weeks on high-fat diet, MSEW mice displayed increased blood pressure, sympathetic activation, and greater depressor response to an α-adrenergic blocker when compared with controls (P

Neuronal hippocampal Ca

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.

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.

Tetraspanin CD151 is increasingly implicated as a multifaceted mediator of cancer development and progression. Here we investigated the role of CD151 in breast cancer in the context of the Wnt oncogenic activation. Our data showed that removal of one or both of CD151 alleles in the MMTV-Wnt1 model significantly decreased the tumor-free survival of mice from 34 weeks on average to 22 weeks and 18 weeks, respectively. This effect coincided with an accelerated tumor growth and an increased number of Ki-67+ proliferative cells. Mechanistically, the CD151-deficient tumors were largely ER+, and exhibited hyperactivation of the Wnt pathway as reflected by a marked upregulation in β-catenin and Cyclin D1, and their target genes. In addition, E-cadherin displayed a cytosolic distribution and transcription factor Snail was markedly upregulated. Collectively, this data implies that CD151 suppresses the Wnt1-driven tumorigenesis, at least in part, via counteracting the epithelial-mesenchymal transition (EMT)-like program in luminal epithelial cells. Meanwhile, the proportion of tumor cells expressing CK5 or p63, the biomarkers of myoepithelial/basal cells, markedly decreased in the absence of CD151. This change was accompanied by a decreased invasiveness of tumors and their incompetence to form a long-term cell culture. Consistent with this basal cell-linked role, the CD151 downregulation impairs mammosphere formation in MCF-10A cells and the defect was rescued by re-expression of intact CD151 ORF, but not its integrin binding-defective mutant. Overall, our study suggests that CD151 is a key player in the Wnt oncogene-driven tumorigenesis and impacts breast cancer malignancy in a cell type-dependent manner.

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.

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 (IRβ) 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 IRβ 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 IRβ‐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., To assess the impact of increasing insulin signaling on ambulatory performance, spatial memory, and receptor dynamics without the need for intranasal insulin delivery, we expressed a constitutively active insulin receptor (IRβ) in the hippocampus of Fischer 344 rats. In aged animals, IRβ was associated with improved memory performance, higher receptor levels, and increased downstream signaling. This work demonstrates the efficacy of molecularly enhancing receptor activity in vivo.

Purpose Stemming from a myriad of genetic and epigenetic alterations, triple-negative breast cancer (TNBC) is tied to poor clinical outcomes and aspires for individualized therapies. Here we investigated the therapeutic potential of co-inhibiting integrin-dependent signaling pathway and BRD4, a transcriptional and epigenetic mediator, for TNBC. Methods Two independent patient cohorts were subjected to bioinformatic and IHC examination for clinical association of candidate cancer drivers. The efficacy and biological bases for co-targeting these drivers were interrogated using cancer cell lines, a protein kinase array, chemical inhibitors, RNAi/CRISPR/Cas9 approaches, and a 4 T1-Balb/c xenograft model. Results We found that amplification of the chromosome 8q24 region occurred in nearly 20% of TNBC tumors, and that it coincided with co-upregulation or amplification of c-Myc and FAK, a key effector of integrin-dependent signaling. This co-upregulation at the mRNA or protein level correlated with a poor patient survival (p p + myeloid-derived suppressor cells in vivo. Finally, we found that JQ1 and VS-6063 cooperatively induced apoptotic cell death by altering XIAP, Bcl2/Bcl-xl and Bim levels, impairing c-Src/p130Cas-, PI3K/Akt- and RelA-associated signaling, and were linked to EMT-inducing transcription factor Snail- and Slug-dependent regulation. Conclusion Based on our results, we conclude that the BRD4/c-Myc- and integrin/FAK-dependent pathways act in concert to promote breast cancer cell survival and poor clinical outcomes. As such, they represent promising targets for a synthetic lethal-type of therapy against TNBC.

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.

Memory and cognitive decline are the product of numerous physiological changes within the aging brain. Multiple theories have focused on the oxidative, calcium, cholinergic, vascular, and inflammation hypotheses of brain aging, with recent evidence suggesting that reductions in insulin signaling may also contribute. Specifically, a reduction in insulin receptor density and mRNA levels has been implicated, however, overcoming these changes remains a challenge. While increasing insulin receptor occupation has been successful in offsetting cognitive decline, alternative molecular approaches should be considered as they could bypass the need for brain insulin delivery. Moreover, this approach may be favorable to test the impact of continued insulin receptor signaling on neuronal function. Here we used hippocampal cultures infected with lentivirus with or without IRβ, a constitutively active, truncated form of the human insulin receptor, to characterize the impact continued insulin receptor signaling on voltage-gated calcium channels. Infected cultures were harvested between DIV 13 and 17 (48 hrs after infection) for Western blot analysis on pAKT and AKT. These results were complemented with whole-cell patch-clamp recordings of individual pyramidal neurons starting 96 hrs post-infection. Results indicate that while a significant increase in neuronal pAKT/AKT ratio was seen at the time point tested, effects on voltage-gated calcium channels were not detected. These results suggest that there is a significant difference between constitutively active insulin receptors and the actions of insulin on an intact receptor, highlighting potential alternate mechanisms of neuronal insulin resistance and mode of 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.

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.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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

Intranasal insulin is a safe and effective method for ameliorating memory deficits associated with pathological brain aging. However, the impact of different formulations and the duration of treatment on insulin’s efficacy and the cellular processes targeted by the treatment remain unclear. Here, we tested whether intranasal insulin aspart, a short-acting insulin formulation, could alleviate memory decline associated with aging and whether long-term treatment affected regulation of insulin receptors and other potential targets. Outcome variables included measures of spatial learning and memory, autoradiography and immunohistochemistry of the insulin receptor, and hippocampal microarray analyses. Aged Fischer 344 rats receiving long-term (3 months) intranasal insulin did not show significant memory enhancement on the Morris water maze task. Autoradiography results showed that long-term treatment reduced insulin binding in the thalamus but not the hippocampus. Results from hippocampal immunofluorescence revealed age-related decreases in insulin immunoreactivity that were partially offset by intranasal administration. Microarray analyses highlighted numerous insulin-sensitive genes, suggesting insulin aspart was able to enter the brain and alter hippocampal RNA expression patterns including those associated with tumor suppression. Our work provides insights into potential mechanisms of intranasal insulin and insulin resistance, and highlights the importance of treatment duration and the brain regions targeted.

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 Ca2 + 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 Ca2 + dysregulation occurs in human entorhinal neurons and promotes NFT pathogenesis.

It has been >20 years since studies first revealed that the brain is insulin sensitive, highlighted by the expression of insulin receptors in neurons and glia, the presence of circulating brain insulin, and even localized insulin production. Following these discoveries, evidence of decreased brain insulin receptor number and function was reported in both clinical samples and animal models of aging and Alzheimer's disease, setting the stage for the hypothesis that neuronal insulin resistance may underlie memory loss in these conditions. The development of therapeutic insulin delivery to the brain using intranasal insulin administration has been shown to improve aspects of memory or learning in both humans and animal models. However, whether this approach functions by compensating for poorly signaling insulin receptors, for reduced insulin levels in the brain, or for reduced trafficking of insulin into the brain remains unclear. Direct measures of insulin's impact on cellular physiology and metabolism in the brain have been sparse in models of Alzheimer's disease, and even fewer studies have analyzed these processes in the aged brain. Nevertheless, recent evidence supports the role of brain insulin as a mediator of glucose metabolism through several means, including altering glucose transporters. Here, we provide a review of contemporary literature on brain insulin resistance, highlight the rationale for improving memory function using intranasal insulin, and describe initial results from experiments using a molecular approach to more directly measure the impact of insulin receptor activation and signaling on glucose uptake in neurons.

Novel therapies have turned to delivering compounds to the brain using nasal sprays, bypassing the blood brain barrier, and enriching treatment options for brain aging and/or Alzheimer's disease. We conducted a series of in vivo experiments to test the impact of intranasal Apidra, a zinc-free insulin formulation, on the brain of young and aged F344 rats. Both single acute and repeated daily doses were compared to test the hypothesis that insulin could improve memory recall in aged memory-deficient animals. We quantified insulin signaling in different brain regions and at different times following delivery. We measured cerebral blood flow (CBF) using MRI and also characterized several brain metabolite levels using MR spectroscopy. We show that neither acute nor chronic Apidra improved memory or recall in young or aged animals. Within 2 hours of a single dose, increased insulin signaling was seen in ventral areas of the aged brains only. Although chronic Apidra was able to offset reduced CBF with aging, it also caused significant reductions in markers of neuronal integrity. Our data suggest that this zinc-free insulin formulation may actually hasten cognitive decline with age when used chronically.

Thiazolidinedione drugs (TZDs) such as pioglitazone are approved by the U.S. Food and Drug Administration for the treatment of insulin resistance in type 2 diabetes. However, whether TZDs reduce painful diabetic neuropathy (PDN) remains unknown. Therefore, we tested the hypothesis that chronic administration of pioglitazone would reduce PDN in Zucker Diabetic Fatty (ZDF fa/fa [ZDF]) rats. Compared with Zucker Lean (ZL fa/+ ) controls, ZDF rats developed: 1) increased blood glucose, hemoglobin A1c, methylglyoxal, and insulin levels; 2) mechanical and thermal hyperalgesia in the hind paw; 3) increased avoidance of noxious mechanical probes in a mechanical conflict avoidance behavioral assay, to our knowledge, the first report of a measure of affective–motivational pain-like behavior in ZDF rats; and 4) exaggerated lumbar dorsal horn immunohistochemical expression of pressure-evoked phosphorylated extracellular signal-regulated kinase. Seven weeks of pioglitazone (30 mg/kg/d in food) reduced blood glucose, hemoglobin A1c, hyperalgesia, and phosphorylated extracellular signal-regulated kinase expression in ZDF. To our knowledge, this is the first report to reveal hyperalgesia and spinal sensitization in the same ZDF animals, both evoked by a noxious mechanical stimulus that reflects pressure pain frequently associated with clinical PDN. Because pioglitazone provides the combined benefit of reducing hyperglycemia, hyperalgesia, and central sensitization, we suggest that TZDs represent an attractive pharmacotherapy in patients with type 2 diabetes-associated pain. Perspective To our knowledge, this is the first preclinical report to show that: 1) ZDF rats exhibit hyperalgesia and affective–motivational pain concurrent with central sensitization; and 2) pioglitazone reduces hyperalgesia and spinal sensitization to noxious mechanical stimulation within the same subjects. Further studies are needed to determine the anti-PDN effect of TZDs in humans.

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.

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.

Both insulin signaling disruption and Ca 2+ dysregulation are closely related to memory loss during aging and increase the vulnerability to Alzheimer’s disease (AD). In hippocampal neurons, aging-related changes in calcium regulatory pathways have been shown to lead to higher intracellular calcium levels and an increase in the Ca 2+ -dependent afterhyperpolarization (AHP), which is associated with cognitive decline. Recent studies suggest that insulin reduces the Ca 2+ -dependent AHP. Given the sensitivity of neurons to insulin and evidence that brain insulin signaling is reduced with age, insulin-mediated alterations in calcium homeostasis may underlie the beneficial actions of insulin in the brain. Indeed, increasing insulin signaling in the brain via intranasal delivery has yielded promising results such as improving memory in both clinical and animal studies. However, while several mechanisms have been proposed, few have focused on regulation on intracellular Ca 2+ . In the present study, we further examined the effects of acute insulin on calcium pathways in primary hippocampal neurons in culture. Using the whole-cell patch-clamp technique, we found that acute insulin delivery reduced voltage-gated calcium currents. Fura-2 imaging was used to also address acute insulin effects on spontaneous and depolarization-mediated Ca 2+ transients. Results indicate that insulin reduced Ca 2+ transients, which appears to have involved a reduction in ryanodine receptor function. Together, these results suggest insulin regulates pathways that control intracellular Ca 2+ which may reduce the AHP and improve memory. This may be one mechanism contributing to improved memory recall in response to intranasal insulin therapy in the clinic.

Accumulating evidence is associating disorders of lipid and glucose metabolism, including the overlapping conditions of insulin resistance/metabolic syndrome, obesity and diabetes, with moderate cognitive impairment in normal aging and elevated risk of Alzheimer’s disease. It appears that a common feature of these conditions is deficient insulin signaling, likely affecting the brain as well as canonical peripheral target tissues. A number of studies have documented that insulin directly affects brain processes and that reduced insulin signaling results in impaired learning and memory. Several studies have also shown that deficient insulin signaling induces Ca2+ dysregulation in neurons. Because brain aging is associated with substantial Ca2+ dyshomeostasis, it has been proposed that deficient insulin signaling exacerbates or accelerates aging-related Ca2+ dyshomeostasis. However, there have been few studies examining insulin interactions with Ca2+ regulation in aging animals. We have been testing predictions of the Ca2+ dysregulation/diabetes/brain aging hypothesis and have found that insulin and insulin sensitizers (thiazolidinediones) target several hippocampal Ca2+-related processes affected by aging, including larger Ca2+ transients and Ca2+-dependent afterhyperpolarizations, and counteract the effects of aging on those processes. Thus, while additional testing is needed, the results to date are consistent with the view that effects of deficient insulin signaling on brain aging are mediated in part by neuronal Ca2+ dyshomeostasis.

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.

Thiazolidinediones (TZDs) are agonists at peroxisome proliferator-activated gamma-type (PPAR-γ) receptors and are used clinically for the treatment of type 2 diabetes where they have been shown to reestablish insulin sensitivity, improve lipid profiles, and reduce inflammation. Recent work also suggests that TZDs may be beneficial in Alzheimer’s disease (AD), ameliorating cognitive decline early in the disease process. However, there have been only a few studies identifying mechanisms through which cognitive benefits may be exerted. Starting at 10 months of age, the triple transgenic mouse model of AD (3×Tg-AD) with accelerated amyloid-β (Aβ) deposition and tau pathology was treated with the TZD pioglitazone (PIO-Actos®) at 18 mg/Kg body weight/day. After four months, PIO-treated animals showed multiple beneficial effects, including improved learning on the active avoidance task, reduced serum cholesterol, decreased hippocampal amyloid-β and tau deposits, and enhanced short- and long-term plasticity. Electrophysiological membrane properties and post-treatment blood glucose levels were unchanged by PIO. Gene microarray analyses of hippocampal tissue identified predicted transcriptional responses following TZD treatment as well as potentially novel targets of TZDs, including facilitation of estrogenic processes and decreases in glutamatergic and lipid metabolic/cholesterol dependent processes. Taken together, these results confirm prior animal studies showing that TZDs can ameliorate cognitive deficits associated with AD-related pathology, but also extend these findings by pointing to novel molecular targets in the brain.

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.

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.

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.

Type 2 diabetes mellitus is a metabolic disorder characterized by hyperglycemia and is especially prevalent in the elderly. Because aging is a risk factor for type 2 diabetes mellitus, and insulin resistance may contribute to the pathogenesis of Alzheimer's disease (AD), anti-diabetic agents (thiazolidinediones-TZDs) are being studied for the treatment of cognitive decline associated with AD. These agents normalize insulin sensitivity in the periphery and can improve cognition and verbal memory in AD patients. Based on evidence that Ca(2+) dysregulation is a pathogenic factor of brain aging/AD, we tested the hypothesis that TZDs could impact Ca(2+) signaling/homeostasis in neurons. We assessed the effects of pioglitazone and rosiglitazone (TZDs) on two major sources of Ca(2+) influx in primary hippocampal cultured neurons, voltage-gated Ca(2+) channel (VGCC) and the NMDA receptor (NMDAR). VGCC- and NMDAR-mediated Ca(2+) currents were recorded using patch-clamp techniques, and Ca(2+) intracellular levels were monitored with Ca(2+) imaging techniques. Rosiglitazone, but not pioglitazone reduced VGCC currents. In contrast, NMDAR-mediated currents were significantly reduced by pioglitazone but not rosiglitazone. These results show that TZDs modulate Ca(2+)-dependent pathways in the brain and have different inhibitory profiles on two major Ca(2+) sources, potentially conferring neuroprotection to an area of the brain that is particularly vulnerable to the effects of aging and/or AD.

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.

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.

// Pengcheng Zhou 1, * , Sonia Erfani 2, * , Zeyi Liu 2, 3, * , Changhe Jia 2, 4, * , Yecang Chen 5, * , Bingwei Xu 2 , Xinyu Deng 2 , Jose E. Alfaro 1 , Li Chen 2 , Dana Napier 6 , Michael Lu 8 , Jian-An Huang 3 , Chunming Liu 7 , Olivier Thibault 2 , Rosalind Segal 1 , Binhua P. Zhou 7 , Natasha Kyprianou 9 , Craig Horbinski 6, # , Xiuwei H. Yang 2, # 1 Department of Cancer Biology and Pediatric Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA 2 Department of Pharmacology and Nutritional Sciences, Markey Cancer Center and University of Kentucky, Lexington, KY, USA 3 Department of Respiratory Medicine, First Affiliated Hospital of Soochow University, Suzhou, Jiangsu Province, P. R. China 4 Department of Gastroenterology, Provincial People's Hospital, Zhengzhou, Henan Province, P. R. China 5 Department of Chemistry, University of Kentucky, Lexington, KY, USA 6 Department of Pathology and Laboratory Medicine, Markey Cancer Center and University of Kentucky, Lexington, KY, USA 7 Department of Molecular and Cellular Biochemistry, Markey Cancer Center and University of Kentucky, Lexington, KY, USA 8 Department of Biomedical Science, Florida Atlantic University, Boca Raton, FL, USA 9 Department of Urology, Markey Cancer Center and University of Kentucky, Lexington, KY, USA * These authors have contributed equally to this study # Co-equal contribution Correspondence to: Xiuwei H. Yang, email: xiuwei-yang@uky.edu Craig Horbinski, email: craig.horbinski@uky.edu Keywords: glioblastoma, CD151, α3 integrin, EGFR, cell invasion and motility Received: May 23, 2015 Accepted: August 03, 2015 Published: August 13, 2015 ABSTRACT Glioblastoma, one of the most aggressive forms of brain cancer, is featured by high tumor cell motility and invasiveness, which not only fuel tumor infiltration, but also enable escape from surgical or other clinical interventions. Thus, better understanding of how these malignant traits are controlled will be key to the discovery of novel biomarkers and therapies against this deadly disease. Tetraspanin CD151 and its associated α3β1 integrin have been implicated in facilitating tumor progression across multiple cancer types. How these adhesion molecules are involved in the progression of glioblastoma, however, remains largely unclear. Here, we examined an in-house tissue microarray-based cohort of 96 patient biopsies and TCGA dataset to evaluate the clinical significance of CD151 and α3β1 integrin. Functional and signaling analyses were also conducted to understand how these molecules promote the aggressiveness of glioblastoma at molecular and cellular levels. Results from our analyses showed that CD151 and α3 integrin were significantly elevated in glioblastomas at both protein and mRNA levels, and exhibited strong inverse correlation with patient survival ( p < 0.006). These adhesion molecules also formed tight protein complexes and synergized with EGF/EGFR to accelerate tumor cell motility and invasion. Furthermore, disruption of such complexes enhanced the survival of tumor-bearing mice in a xenograft model, and impaired activation of FAK and small GTPases. Also, knockdown- or pharmacological agent-based attenuation of EGFR, FAK or Graf (ARHGAP26)/small GTPase-mediated pathways markedly mitigated the aggressiveness of glioblastoma cells. Collectively, our findings provide clinical, molecular and cellular evidence of CD151-α3β1 integrin complexes as promising prognostic biomarkers and therapeutic targets for glioblastoma.

Delayed excitotoxic neuronal death after insult from exposure to high glutamate concentrations appears important in several CNS disorders. Although delayed excitotoxicity is known to depend on NMDA receptor (NMDAR) activity and Ca2+elevation, the electrophysiological mechanisms underlying postinsult persistence of NMDAR activation are not well understood. Membrane depolarization and nonspecific cationic current in the postinsult period were reported previously, but were not sensitive to NMDAR antagonists. Here, we analyzed mechanisms of the postinsult period using parallel current- and voltage-clamp recording and Ca2+imaging in primary hippocampal cultured neurons. We also compared more vulnerable older neurons [about 22 days in vitro (DIV)] to more resistant younger (about 15 DIV) neurons, to identify processes selectively associated with cell death in older neurons. During exposure to a modest glutamate insult (20 μM, 5 min), similar degrees of Ca2+elevation, membrane depolarization, action potential block, and increased inward current occurred in younger and older neurons. However, after glutamate withdrawal, these processes recovered rapidly in younger but not in older neurons. The latter also exhibited a concurrent postinsult increase in spontaneous miniature excitatory postsynaptic currents, reflecting glutamate release. Importantly, postinsult NMDAR antagonist administration reversed all of these persisting responses in older cells. Conversely, repolarization of the membrane by voltage clamp immediately after glutamate exposure reversed the NMDAR-dependent Ca2+elevation. Together, these data suggest that, in vulnerable neurons, excitotoxic insult induces a sustained positive feedback loop between NMDAR-dependent current and depolarization-mediated glutamate release, which persists after withdrawal of exogenous glutamate and drives Ca2+elevation and delayed excitotoxicity.

Basic principles of the neurobiology of aging were reviewed within selected topic areas chosen for their potential relevance to epileptogenesis in the aging brain. The availability of National Institute on Aging-supported aged mouse and rat strains and other biological resources for studies of aging and age-associated diseases was presented, and general principles of animal use in gerontological research were discussed. Neurobiological changes during normal brain aging were compared and contrasted with neuropathological events of Alzheimer's disease (AD) and age-associated memory impairment (AAMI). Major themes addressed were the loss of synaptic connections as vulnerable neurons die and circuits deteriorate in AD, the absence of significant neuron loss but potential synaptic alteration in the same circuits in AAMI, and the effects of decreased estrogen on normal aging. The "calcium hypothesis of brain aging" was examined by a review of calcium dyshomeostasis and synaptic communication in aged hippocampus, with particular emphasis on the role of L-type voltage-gated calcium channels during normal aging. Established and potential mechanisms of hippocampal plasticity during aging were discussed, including long-term potentiation, changes in functional connectivity, and increased gap junctions, the latter possibly being related to enhanced network excitability. Lastly, application of microarray gene chip technology to aging brain studies was presented and use of the hippocampal "zipper slice" preparation to study aged neurons was described.

It has been recognized for some years that a prolonged Ca 2+ elevation that is predictive of impending cell death develops in cultured neurons following excitotoxic insult. In addition, neurons exhibit enhanced sensitivity to excitotoxic insult with increasing age in culture. However, little is known about the processes that selectively regulate the post-insult Ca 2+ elevation and therefore, it remains unclear whether it is associated specifically with age-dependent toxicity. Here, we tested the hypothesis that a group I metabotropic glutamate receptor antagonist selectively modulates the prolonged Ca 2+ elevation in direct association with its protective effects against excitotoxicity. Rat hippocampal cultures of two ages (8–9 and 21–28 days in vitro ) were exposed to a 5-min glutamate insult (400 μM in younger and 10 μM in older cultures) sufficient to kill >50% of the neurons, and were treated with vehicle or the specific group I metabotropic glutamate receptor antagonist 1-aminoindan-1,5-dicarboxylic acid (AIDA; 1 mM), throughout and following the insult. Neuronal survival was quantified 24 h after insult. In parallel studies, neurons of similar age in culture were imaged ratiometrically with a confocal microscope during and for 60 min after the glutamate insult. A large post-insult Ca 2+ elevation was present in older but not most younger neurons. The N -methyl- D -aspartate receptor antagonist, MK-801, blocked the Ca 2+ elevation both during and following the insult. In contrast, AIDA blocked only the post-insult prolonged Ca 2+ elevation in older neurons. Moreover, AIDA was neuroprotective in older but not younger cultures. From these results we suggest that the post-insult Ca 2+ elevation is regulated differently from the Ca 2+ elevation during glutamate insult and is modulated by group I metabotropic glutamate receptors. Further, the prolonged Ca 2+ elevation appears to be directly linked to an age-dependent component of vulnerability.