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

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

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

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

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

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

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

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

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

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

Dependence of glucose transport on autophagy and GAPDH activity.

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

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

[Figure: see text].

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

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

BRD4 modulates vulnerability of triple-negative breast cancer to targeting of integrin-dependent signaling pathways.

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 < 0.0109 or p < 0.0402, respectively). Furthermore, we found that 14 TNBC cell lines exhibited high vulnerabilities to the combination of JQ1 and VS-6063, potent pharmacological antagonists of the BRD4/c-Myc and integrin/FAK-dependent pathways, respectively. We also observed a cooperative inhibitory effect of JQ1 and VS-6063 on tumor growth and infiltration of Ly6G+ 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.

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

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

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

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

Elevating Insulin Signaling Using a Constitutively Active Insulin Receptor Increases Glucose Metabolism and Expression of GLUT3 in Hippocampal Neurons.

Insulin signaling is an integral component of healthy brain function, with evidence of positive insulin-mediated alterations in synaptic integrity, cerebral blood flow, inflammation, and memory. However, the specific pathways targeted by this peptide remain unclear. Previously, our lab used a molecular approach to characterize the impact of insulin signaling on voltage-gated calcium channels and has also shown that acute insulin administration reduces calcium-induced calcium release in hippocampal neurons. Here, we explore the relationship between insulin receptor signaling and glucose metabolism using similar methods. Mixed, primary hippocampal cultures were infected with either a control lentivirus or one containing a constitutively active human insulin receptor (IRß). 2-NBDG imaging was used to obtain indirect measures of glucose uptake and utilization. Other outcome measures include Western immunoblots of GLUT3 and GLUT4 on total membrane and cytosolic subcellular fractions. Glucose imaging data indicate that neurons expressing IRß show significant elevations in uptake and rates of utilization compared to controls. As expected, astrocytes did not respond to the IRß treatment. Quantification of Western immunoblots show that IRß is associated with significant elevations in GLUT3 expression, particularly in the total membrane subcellular fraction, but did not alter GLUT4 expression in either fraction. Our work suggests that insulin plays a significant role in mediating neuronal glucose metabolism, potentially through an upregulation in the expression of GLUT3. This provides further evidence for a potential therapeutic mechanism underlying the beneficial impact of intranasal insulin in the clinic.

Long-Term Intranasal Insulin Aspart: A Profile of Gene Expression, Memory, and Insulin Receptors in Aged F344 Rats.

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.

Deletion of tetraspanin CD151 alters the Wnt oncogene-induced mammary tumorigenesis: A cell type-linked function and signaling.

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 ß-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.

Broadening the definition of brain insulin resistance in aging and Alzheimer's disease.

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.

Aging-Related Calcium Dysregulation in Rat Entorhinal Neurons Homologous with the Human Entorhinal Neurons in which Alzheimer's Disease Neurofibrillary Tangles First Appear.

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.

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

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

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

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

Novel calcium-related targets of insulin in hippocampal neurons.

Both insulin signaling disruption and Ca2+ 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 Ca2+-dependent afterhyperpolarization (AHP), which is associated with cognitive decline. Recent studies suggest that insulin reduces the Ca2+-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 Ca2+. 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 Ca2+ transients. Results indicate that insulin reduced Ca2+ transients, which appears to have involved a reduction in ryanodine receptor function. Together, these results suggest insulin regulates pathways that control intracellular Ca2+ 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.

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

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