Sex difference in brain functional connectivity of hippocampus in Alzheimer's disease.

Alzheimer's disease (AD), affecting nearly 6.5 million people, is the fifth leading cause of death in individuals 65 years or older in the USA. Prior research has shown that AD disproportionality affects females; females have a greater incidence rate, perform worse on a variety of neuropsychological tasks, and have greater total brain atrophy. Recent research has linked these sex differences to neuroimaging markers of brain pathology, such as hippocampal volumes. Specifically, research from our lab found that functional connectivity from the hippocampus to the precuneus cortex and brain stem was significantly stronger in males than in females with mild cognitive impairment. The aim of this study was to extend our understanding to individuals with AD and to determine if these potential sex-specific functional connectivity biomarkers extend through different disease stages. The resting state fMRI and T2 MRI of cognitively normal individuals (n = 32, female = 16) and individuals with AD (n = 32, female = 16) from the Alzheimer's Disease Neuroimaging Initiative (ADNI) were analyzed using the Functional Connectivity Toolbox (CONN). Our results demonstrate that males had a significantly stronger interhemispheric functional connectivity between the left and right hippocampus compared to females. These results improve our current understanding of the role of the hippocampus in sex differences in AD. Understanding the contribution of impaired functional connectivity sex differences may aid in the development of sex-specific precision medicine for improved AD treatment.

Impaired Neurovascular Coupling and Increased Functional Connectivity in the Frontal Cortex Predict Age-Related Cognitive Dysfunction.

Impaired cerebrovascular function contributes to the genesis of age-related cognitive decline. In this study, the hypothesis is tested that impairments in neurovascular coupling (NVC) responses and brain network function predict cognitive dysfunction in older adults. Cerebromicrovascular and working memory function of healthy young (n = 21, 33.2±7.0 years) and aged (n = 30, 75.9±6.9 years) participants are assessed. To determine NVC responses and functional connectivity (FC) during a working memory (n-back) paradigm, oxy- and deoxyhemoglobin concentration changes from the frontal cortex using functional near-infrared spectroscopy are recorded. NVC responses are significantly impaired during the 2-back task in aged participants, while the frontal networks are characterized by higher local and global connection strength, and dynamic FC (p < 0.05). Both impaired NVC and increased FC correlate with age-related decline in accuracy during the 2-back task. These findings suggest that task-related brain states in older adults require stronger functional connections to compensate for the attenuated NVC responses associated with working memory load.

Elimination of senescent cells by treatment with Navitoclax/ABT263 reverses whole brain irradiation-induced blood-brain barrier disruption in the mouse brain.

Whole brain irradiation (WBI), a commonly employed therapy for multiple brain metastases and as a prophylactic measure after cerebral metastasis resection, is associated with a progressive decline in neurocognitive function, significantly impacting the quality of life for approximately half of the surviving patients. Recent preclinical investigations have shed light on the multifaceted cerebrovascular injury mechanisms underlying this side effect of WBI. In this study, we aimed to test the hypothesis that WBI induces endothelial senescence, contributing to chronic disruption of the blood-brain barrier (BBB) and microvascular rarefaction. To accomplish this, we utilized transgenic p16-3MR mice, which enable the identification and selective elimination of senescent cells. These mice were subjected to a clinically relevant fractionated WBI protocol (5 Gy twice weekly for 4 weeks), and cranial windows were applied to both WBI-treated and control mice. Quantitative assessment of BBB permeability and capillary density was performed using two-photon microscopy at the 6-month post-irradiation time point. The presence of senescent microvascular endothelial cells was assessed by imaging flow cytometry, immunolabeling, and single-cell RNA-sequencing (scRNA-seq). WBI induced endothelial senescence, which associated with chronic BBB disruption and a trend for decreased microvascular density in the mouse cortex. In order to investigate the cause-and-effect relationship between WBI-induced senescence and microvascular injury, senescent cells were selectively removed from animals subjected to WBI treatment using Navitoclax/ABT263, a well-known senolytic drug. This intervention was carried out at the 3-month post-WBI time point. In WBI-treated mice, Navitoclax/ABT263 effectively eliminated senescent endothelial cells, which was associated with decreased BBB permeability and a trend for increased cortical capillarization. Our findings provide additional preclinical evidence that senolytic treatment approaches may be developed for prevention of the side effects of WBI.

Progressive cognitive impairment after recovery from neuroinvasive and non-neuroinvasive Listeria monocytogenes infection.

Background: Neuro-cognitive impairment is a deleterious complication of bacterial infections that is difficult to treat or prevent. Listeria monocytogenes (Lm) is a neuroinvasive bacterial pathogen and commonly used model organism for studying immune responses to infection. Antibiotic-treated mice that survive systemic Lm infection have increased numbers of CD8+ and CD4+ T-lymphocytes in the brain that include tissue resident memory (TRM) T cells, but post-infectious cognitive decline has not been demonstrated. We hypothesized that Lm infection would trigger cognitive decline in accord with increased numbers of recruited leukocytes. Methods: Male C57BL/6J mice (age 8 wks) were injected with neuroinvasive Lm 10403s, non-neuroinvasive Δhly mutants, or sterile saline. All mice received antibiotics 2-16d post-injection (p.i.) and underwent cognitive testing 1 month (mo) or 4 mo p.i. using the Noldus PhenoTyper with Cognition Wall, a food reward-based discrimination procedure using automated home cage based observation and monitoring. After cognitive testing, brain leukocytes were quantified by flow cytometry. Results: Changes suggesting cognitive decline were observed 1 mo p.i. in both groups of infected mice compared with uninfected controls, but were more widespread and significantly worse 4 mo p.i. and most notably after Lm 10403s. Impairments were observed in learning, extinction of prior learning and distance moved. Infection with Lm 10403s, but not Δhly Lm, significantly increased numbers of CD8+ and CD4+ T-lymphocytes, including populations expressing CD69 and TRM cells, 1 mo p.i. Numbers of CD8+, CD69+CD8+ T-lymphocytes and CD8+ TRM remained elevated at 4 mo p.i. but numbers of CD4+ cells returned to homeostatic levels. Higher numbers of brain CD8+ T-lymphocytes showed the strongest correlations with reduced cognitive performance. Conclusions: Systemic infection by neuroinvasive as well as non-neuroinvasive Lm triggers a progressive decline in cognitive impairment. Notably, the deficits are more profound after neuroinvasive infection that triggers long-term retention of CD8+ T-lymphocytes in the brain, than after non-neuroinvasive infection, which does not lead to retained cells in the brain. These results support the conclusion that systemic infections, particularly those that lead to brain leukocytosis trigger a progressive decline in cognitive function and implicate CD8+ T-lymphocytes, including CD8+TRM in the etiology of this impairment.

Cognitive heterogeneity reveals molecular signatures of age-related impairment.

The greatest risk factor for cognitive decline is aging. The biological mechanisms for this decline remain enigmatic due, in part, to the confounding of normal aging mechanisms and those that contribute to cognitive impairment. Importantly, many individuals exhibit impaired cognition in age, while some retain functionality despite their age. Here, we establish a behavioral testing paradigm to characterize age-related cognitive heterogeneity in inbred aged C57BL/6 mice and reliably separate animals into cognitively "intact" (resilient) and "impaired" subgroups using a high-resolution home-cage testing paradigm for spatial discrimination. RNA sequencing and subsequent pathway analyses of cognitively stratified mice revealed molecular signatures unique to cognitively impaired animals, including transcriptional down-regulation of genes involved in mitochondrial oxidative phosphorylation (OXPHOS) and sirtuin (Sirt1 and Sirt3) expression in the hippocampus. Mitochondrial function assessed using high-resolution respirometry indicated a reduced OXPHOS coupling efficiency in cognitively impaired animals with subsequent hippocampal analyses revealing an increase in the oxidative damage marker (3-nitrotyrosine) and an up-regulation of antioxidant enzymes (Sod2, Sod1, Prdx6, etc.). Aged-impaired animals also showed increased levels of IL-6 and TNF-α gene expression in the hippocampus and increased serum levels of proinflammatory cytokines, including IL-6. These results provide critical insight into the diversity of brain aging in inbred animals and reveal the unique mechanisms that separate cognitive resilience from cognitive impairment. Our data indicate the importance of cognitive stratification of aging animals to delineate the mechanisms underlying cognitive impairment and test the efficacy of therapeutic interventions.

Measurements of cerebral microvascular blood flow, oxygenation, and morphology in a mouse model of whole-brain irradiation-induced cognitive impairment by two-photon microscopy and optical coherence tomography: evidence for microvascular injury in the cerebral white matter.

Whole-brain irradiation (WBI, also known as whole-brain radiation therapy) is a mainstay treatment modality for patients with multiple brain metastases. It is also used as a prophylactic treatment for microscopic tumors that cannot be detected by magnetic resonance imaging. WBI induces a progressive cognitive decline in ~ 50% of the patients surviving over 6 months, significantly compromising the quality of life. There is increasing preclinical evidence that radiation-induced injury to the cerebral microvasculature and accelerated neurovascular senescence plays a central role in this side effect of WBI. To better understand this side effect, male C57BL/6 mice were first subjected to a clinically relevant protocol of fractionated WBI (5 Gy, two doses per week, for 4 weeks). Nine months post the WBI treatment, we applied two-photon microscopy and Doppler optical coherence tomography to measure capillary red-blood-cell (RBC) flux, capillary morphology, and microvascular oxygen partial pressure (PO2) in the cerebral somatosensory cortex in the awake, head-restrained, WPI-treated mice and their age-matched controls, through a cover-glass-sealed chronic cranial window. Thanks to the extended penetration depth with the fluorophore - Alexa680, measurements of capillary blood flow properties (e.g., RBC flux, speed, and linear density) in the cerebral subcortical white matter were enabled. We found that the WBI-treated mice exhibited a significantly decreased capillary RBC flux in the white matter. WBI also caused a significant reduction in capillary diameter, as well as a large (although insignificant) reduction in segment density at the deeper cortical layers (e.g., 600-700 µm), while the other morphological properties (e.g., segment length and tortuosity) were not obviously affected. In addition, we found that PO2 measured in the arterioles and venules, as well as the calculated oxygen saturation and oxygen extraction fraction, were not obviously affected by WBI. Lastly, WBI was associated with a significant increase in the erythrocyte-associated transients of PO2, while the changes of other cerebral capillary PO2 properties (e.g., capillary mean-PO2, RBC-PO2, and InterRBC-PO2) were not significant. Collectively, our findings support the notion that WBI results in persistent cerebral white matter microvascular impairment, which likely contributes to the WBI-induced brain injury and cognitive decline. Further studies are warranted to assess the WBI-induced changes in brain tissue oxygenation and malfunction of the white matter microvasculature as well.

Decreased lifespan in female "Munchkin" actors from the cast of the 1939 film version of The Wizard of Oz does not support the hypothesis linking hypopituitary dwarfism to longevity.

In laboratory mice, pituitary dwarfism caused by genetic reduction or elimination of the activity of growth hormone (GH) significantly extends lifespan. The effects of congenital pituitary dwarfism on human longevity are not well documented. To analyse the effects of untreated pituitary dwarfism on human lifespan, the longevity of a diverse group of widely known little people, the 124 adults who played "Munchkins" in the 1939 movie The Wizard of Oz was investigated. Survival of "Munchkin" actors with those of controls defined as cast members of The Wizard of Oz and those of other contemporary Academy Award winning Hollywood movies was compared. According to the Kaplan-Meier survival curves, survival of female and male "Munchkin" actors was shorter than cast controls and Hollywood controls of respective sexes. Cox regression analyses showed that female "Munchkin" actors had significantly higher risk ratios compared to both female cast controls (RR, 1.70; 95% CI, 1.05 to 2.77) and female Hollywood controls (RR, 1.52; 95% CI, 1.03 to 2.24). Similar trends were also discernible for men, albeit point estimates were not significant. The lack of lifespan extension in "Munchkin" actors does not support the hypothesis that hereditary GH deficiency regulates longevity in humans.

Sex differences in brain functional connectivity of hippocampus in mild cognitive impairment.

Mild cognitive impairment (MCI) is the prodromal stage of Alzheimer's Disease (AD). Prior research shows that females are more impacted by MCI than males. On average females have a greater incidence rate of any dementia and current evidence suggests that they suffer greater cognitive deterioration than males in the same disease stage. Recent research has linked these sex differences to neuroimaging markers of brain pathology, such as hippocampal volumes. Specifically, the rate of hippocampal atrophy affects the progression of AD in females more than males. This study was designed to extend our understanding of the sex-related differences in the brain of participants with MCI. Specifically, we investigated the difference in the hippocampal connectivity to different areas of the brain. The Resting State fMRI and T2 MRI of cognitively normal individuals (n = 40, female = 20) and individuals with MCI (n = 40, female = 20) from the Alzheimer's Disease Neuroimaging Initiative (ADNI) were analyzed using the Functional Connectivity Toolbox (CONN). Our results demonstrate that connectivity of hippocampus to the precuneus cortex and brain stem was significantly stronger in males than in females. These results improve our current understanding of the role of hippocampus-precuneus cortex and hippocampus-brainstem connectivity in sex differences in MCI. Understanding the contribution of impaired functional connectivity sex differences may aid in the development of sex specific precision medicine to manipulate hippocampal-precuneus cortex and hippocampal-brainstem connectivity to decrease the progression of MCI to AD.

Systemic Listeria monocytogenes infection in aged mice induces long-term neuroinflammation: the role of miR-155.

BACKGROUND: Understanding mechanisms of pathologic neuroinflammation is essential for improving outcomes after central nervous system infections. Brain tissue-resident memory T cells (bTRM) are recruited during central nervous system infection and promote pathogen control as well as noxious inflammation. Our prior studies in young mice showed optimal recruitment of CD8+ bTRM during neuroinvasive Listeria monocytogenes (Lm) infection required miR-155, and was significantly inhibited by anti-miR-155 oligonucleotides. Since Lm is an important pathogen in the elderly, we hypothesized anti-miR-155 would also inhibit accumulation of CD8+ bTRM in aged mice infected with Lm. METHODS: Young (2 mo) and aged (> 18 mo) male C57BL/6 mice were infected intra-peritoneally with wild-type Lm, or avirulent Lm mutants lacking the genes required for intracellular motility (ΔactA) or phagosomal escape (Δhly), then were given antibiotics. Brain leukocytes and their intracellular cytokine production were quantified by flow cytometry >28d post-infection (p.i.). The role of miR-155 was tested by injecting mice with anti-miR-155 or control oligonucleotides along with antibiotics. RESULTS: Aged mice had significantly more homeostatic CD8+ bTRM than did young mice, which did not increase after infection with wild-type Lm despite 50% mortality, whereas young mice suffered no mortality after a larger inoculum. For direct comparison of post-infectious neuroinflammation after the same inoculum, young and aged mice were infected with 107 CFU ΔactA Lm. This mutant caused no mortality and significantly increased CD8+ bTRM 28d p.i. in both groups, whereas bone marrow-derived myeloid cells, particularly neutrophils, increased only in aged mice. Notably, anti-miR-155 reduced accumulation of brain myeloid cells in aged mice after infection, whereas CD8+ bTRM were unaffected. CONCLUSIONS: Systemic infection with Lm ΔactA is a novel model for studying infection-induced brain inflammation in aged mice without excessive mortality. CD8+ bTRM increase in both young and aged mice after infection, whereas only in aged mice bone marrow-derived myeloid cells increase long-term. In aged mice, anti-miR-155 inhibits brain accumulation of myeloid cells, but not CD8+ bTRM. These results suggest young and aged mice differ in manifestations and mechanisms of infection-induced neuroinflammation and give insight for developing therapies to ameliorate brain inflammation following severe infection in the elderly.

Increased Susceptibility to Cerebral Microhemorrhages Is Associated With Imaging Signs of Microvascular Degeneration in the Retina in an Insulin-Like Growth Factor 1 Deficient Mouse Model of Accelerated Aging.

Age-related cerebrovascular defects contribute to vascular cognitive impairment and dementia (VCID) as well as other forms of dementia. There has been great interest in developing biomarkers and other tools for studying cerebrovascular disease using more easily accessible tissues outside the brain such as the retina. Decreased circulating insulin-like growth factor 1 (IGF-1) levels in aging are thought to contribute to the development of cerebrovascular impairment, a hypothesis that has been supported by the use of IGF-1 deficient animal models. Here we evaluate vascular and other retinal phenotypes in animals with circulating IGF-1 deficiency and ask whether the retina mimics common age-related vascular changes in the brain such as the development of microhemorrhages. Using a hypertension-induced model, we confirm that IGF-1 deficient mice exhibited worsened microhemorrhages than controls. The retinas of IGF-1 deficient animals do not exhibit microhemorrhages but do exhibit signs of vascular damage and retinal stress such as patterns of vascular constriction and Müller cell activation. These signs of retinal stress are not accompanied by retinal degeneration or impaired neuronal function. These data suggest that the role of IGF-1 in the retina is complex, and while IGF-1 deficiency leads to vascular defects in both the brain and the retina, not all brain pathologies are evident in the retina.

Oklahoma Nathan Shock Aging Center - assessing the basic biology of aging from genetics to protein and function.

The Oklahoma Shock Nathan Shock Center is designed to deliver unique, innovative services that are not currently available at most institutions. The focus of the Center is on geroscience and the development of careers of young investigators. Pilot grants are provided through the Research Development Core to junior investigators studying aging/geroscience throughout the USA. However, the services of our Center are available to the entire research community studying aging and geroscience. The Oklahoma Nathan Shock Center provides researchers with unique services through four research cores. The Multiplexing Protein Analysis Core uses the latest mass spectrometry technology to simultaneously measure the levels, synthesis, and turnover of hundreds of proteins associated with pathways of importance to aging, e.g., metabolism, antioxidant defense system, proteostasis, and mitochondria function. The Genomic Sciences Core uses novel next-generation sequencing that allows investigators to study the effect of age, or anti-aging manipulations, on DNA methylation, mitochondrial genome heteroplasmy, and the transcriptome of single cells. The Geroscience Redox Biology Core provides investigators with a comprehensive state-of-the-art assessment of the oxidative stress status of a cell, e.g., measures of oxidative damage and redox couples, which are important in aging as well as many major age-related diseases as well as assays of mitochondrial function. The GeroInformatics Core provides investigators assistance with data analysis, which includes both statistical support as well as analysis of large datasets. The Core also has developed number of unique software packages to help with interpretation of results and discovery of new leads relevant to aging. In addition, the Geropathology Research Resource in the Program Enhancement Core provides investigators with pathological assessments of mice using the recently developed Geropathology Grading Platform.

Measuring Behavior in the Home Cage: Study Design, Applications, Challenges, and Perspectives.

The reproducibility crisis (or replication crisis) in biomedical research is a particularly existential and under-addressed issue in the field of behavioral neuroscience, where, in spite of efforts to standardize testing and assay protocols, several known and unknown sources of confounding environmental factors add to variance. Human interference is a major contributor to variability both within and across laboratories, as well as novelty-induced anxiety. Attempts to reduce human interference and to measure more "natural" behaviors in subjects has led to the development of automated home-cage monitoring systems. These systems enable prolonged and longitudinal recordings, and provide large continuous measures of spontaneous behavior that can be analyzed across multiple time scales. In this review, a diverse team of neuroscientists and product developers share their experiences using such an automated monitoring system that combines Noldus PhenoTyper® home-cages and the video-based tracking software, EthoVision® XT, to extract digital biomarkers of motor, emotional, social and cognitive behavior. After presenting our working definition of a "home-cage", we compare home-cage testing with more conventional out-of-cage tests (e.g., the open field) and outline the various advantages of the former, including opportunities for within-subject analyses and assessments of circadian and ultradian activity. Next, we address technical issues pertaining to the acquisition of behavioral data, such as the fine-tuning of the tracking software and the potential for integration with biotelemetry and optogenetics. Finally, we provide guidance on which behavioral measures to emphasize, how to filter, segment, and analyze behavior, and how to use analysis scripts. We summarize how the PhenoTyper has applications to study neuropharmacology as well as animal models of neurodegenerative and neuropsychiatric illness. Looking forward, we examine current challenges and the impact of new developments. Examples include the automated recognition of specific behaviors, unambiguous tracking of individuals in a social context, the development of more animal-centered measures of behavior and ways of dealing with large datasets. Together, we advocate that by embracing standardized home-cage monitoring platforms like the PhenoTyper, we are poised to directly assess issues pertaining to reproducibility, and more importantly, measure features of rodent behavior under more ethologically relevant scenarios.

Sleep deprivation impairs cognitive performance, alters task-associated cerebral blood flow and decreases cortical neurovascular coupling-related hemodynamic responses.

Sleep deprivation (SD) is a common condition and an important health concern. In addition to metabolic and cardiovascular risks, SD associates with decreases in cognitive performance. Neurovascular coupling (NVC, "functional hyperemia") is a critical homeostatic mechanism, which maintains adequate blood supply to the brain during periods of intensive neuronal activity. To determine whether SD alters NVC responses and cognitive performance, cognitive and hemodynamic NVC assessments were conducted prior to and 24 h post-SD in healthy young male individuals (n = 10, 27 ± 3 years old). Cognition was evaluated with a battery of tests from the Cambridge Neuropsychological Test Automated Battery (CANTAB). Hemodynamic components of NVC were measured by transcranial Doppler sonography (TCD) during cognitive stimulation, dynamic retinal vessel analysis (DVA) during flicker light stimulation, and functional near infrared spectroscopy (fNIRS) during finger tapping motor task. Cognitive assessments revealed impairments in reaction time and sustained attention after 24 h of SD. Functional NIRS analysis revealed that SD significantly altered hemodynamic responses in the prefrontal cortex and somatosensory cortex during a motor task. NVC-related vascular responses measured by DVA and TCD did not change significantly. Interestingly, TCD detected decreased task-associated cerebral blood flow (CBF) in the right middle cerebral artery in sleep deprived participants. Our results demonstrate that 24 h of SD lead to impairments in cognitive performance together with altered CBF and hemodynamic components of cortical NVC responses.

Endothelial deficiency of insulin-like growth factor-1 receptor (IGF1R) impairs neurovascular coupling responses in mice, mimicking aspects of the brain aging phenotype.

Age-related impairment of neurovascular coupling (NVC; or "functional hyperemia") compromises moment-to-moment adjustment of regional cerebral blood flow to increased neuronal activity and thereby contributes to the pathogenesis of vascular cognitive impairment (VCI). Previous studies established a causal link among age-related decline in circulating levels of insulin-like growth factor-1 (IGF-1), neurovascular dysfunction and cognitive impairment. Endothelium-mediated microvascular dilation plays a central role in NVC responses. To determine the functional consequences of impaired IGF-1 input to cerebromicrovascular endothelial cells, endothelium-mediated NVC responses were studied in a novel mouse model of accelerated neurovascular aging: mice with endothelium-specific knockout of IGF1R (VE-Cadherin-CreERT2/Igf1rf/f). Increases in cerebral blood flow in the somatosensory whisker barrel cortex (assessed using laser speckle contrast imaging through a cranial window) in response to contralateral whisker stimulation were significantly attenuated in VE-Cadherin-CreERT2/Igf1rf/f mice as compared to control mice. In VE-Cadherin-CreERT2/Igf1rf/f mice, the effects of the NO synthase inhibitor L-NAME were significantly decreased, suggesting that endothelium-specific disruption of IGF1R signaling impairs the endothelial NO-dependent component of NVC responses. Collectively, these findings provide additional evidence that IGF-1 is critical for cerebromicrovascular endothelial health and maintenance of normal NVC responses.

Sleep deprivation alters task-related changes in functional connectivity of the frontal cortex: A near-infrared spectroscopy study.

Sleep deprivation (SD) is known to be associated with decreased cognitive performance; however, the underlying mechanisms are poorly understood. As interactions between distinct brain regions depend on mental state, functional brain networks established by these connections typically show a reorganization during task. Hence, analysis of functional connectivity (FC) could reveal the task-related change in the examined frontal brain networks. Our objective was to assess the impact of SD on static FC in the prefrontal and motor cortices and find whether changes in FC correlate with changes in neuropsychological scores. Healthy young male individuals (n = 10, 27.6 ± 3.7 years of age) participated in the study. A battery of tests from the Cambridge Neuropsychological Test Automated Battery (CANTAB) and 48 channel functional near-infrared spectroscopy (fNIRS) measurements were performed before and after 24 hr of SD. Network metrics were obtained by graph theoretical analysis using the fNIRS records in resting state and during finger-tapping sessions. During task, SD resulted in a significantly smaller decrease in the number and strength of functional connections (characterizing FC) in the frontal cortex. Changes in the global connection strengths correlated with decreased performance in the paired association learning test. These results indicate a global impact of SD on functional brain networks in the frontal lobes.

IGF1R signaling regulates astrocyte-mediated neurovascular coupling in mice: implications for brain aging.

Aging is associated with a significant deficiency in circulating insulin-like growth factor-1 (IGF-1), which has an important role in the pathogenesis of age-related vascular cognitive impairment (VCI). Impairment of moment-to-moment adjustment of regional cerebral blood flow via neurovascular coupling (NVC) importantly contributes to VCI. Previous studies established a causal link between circulating IGF-1 deficiency and neurovascular dysfunction. Release of vasodilator mediators from activated astrocytes plays a key role in NVC. To determine the impact of impaired IGF-1 signaling on astrocytic function, astrocyte-mediated NVC responses were studied in a novel mouse model of astrocyte-specific knockout of IGF1R (GFAP-CreERT2/Igf1rf/f) and accelerated neurovascular aging. We found that mice with disrupted astrocytic IGF1R signaling exhibit impaired NVC responses, decreased stimulated release of the vasodilator gliotransmitter epoxy-eicosatrienoic acids (EETs), and upregulation of soluble epoxy hydrolase (sEH), which metabolizes and inactivates EETs. Collectively, our findings provide additional evidence that IGF-1 promotes astrocyte health and maintains normal NVC, protecting cognitive health.

Neuroinvasive Listeria monocytogenes infection triggers accumulation of brain CD8+ tissue-resident memory T cells in a miR-155-dependent fashion.

BACKGROUND: Brain inflammation is a key cause of cognitive decline after central nervous system (CNS) infections. A thorough understanding of immune responses to CNS infection is essential for developing anti-inflammatory interventions that improve outcomes. Tissue-resident memory T cells (TRM) are non-recirculating memory T cells that provide surveillance of previously infected tissues. However, in addition to protecting the brain against reinfection, brain TRM can contribute to post-infectious neuroinflammation. We hypothesized that accumulation of CD8+ TRM in the brain could be reduced by inhibiting microRNA (miR)-155, a microRNA that influences development of cytotoxic CD8+ T lymphocytes during infection. METHODS: C57BL/6J mice were infected by intraperitoneal injection with a lethal inoculum of Listeria monocytogenes (Lm) then treated with antibiotics. Flow cytometry was used to quantify specific populations of brain leukocytes 28-29 days (d) post-infection (p.i.). To test the degree to which miR-155 altered leukocyte influxes into the brain, infected mice were injected with a miR-155 inhibitor or locked nucleic acid (LNA) scramble control 2d, 4d, 6d, and 8d p.i. along with antibiotic treatment. Bacterial loads in spleen and liver and body weights were measured up to 7d p.i. Brain leukocytes were analyzed 14d and 28d p.i. Confirmatory studies were performed in mutated mice lacking miR-155 (miR-155-/-) RESULTS: Lm infection significantly increased the numbers of brain CD3+CD8+ lymphocytes at 28d p.i. These cells were extravascular, and displayed markers characteristic of TRM, with the predominant phenotype of CD44+CD62L-CD69+CX3CR1-. Further analysis showed that > 75% of brain TRM also expressed CD49a, PD-1, Ly6C, CD103, and CD127. Mice injected with miR-155 inhibitor lost less weight through 7d p.i. than did control mice, whereas bacterial loads in brain, liver, and spleen were not different from controls. By 28d p.i., the numbers of brain CD8+ TRM cells were significantly decreased in mice treated with the inhibitor compared with controls. Similarly, miR-155-/- mice showed significantly reduced numbers of brain CD8+ TRM cells by 28d p.i. CONCLUSIONS: Brain CD8+ TRM populations are established during neuroinvasive Lm infection. Accumulation of brain CD8+ TRM cells is reduced by blocking miR-155 and in miR-155-/- mice, indicating that this molecule has a critical role in development of these specialized cells. Administering anti-miR-155 during infection could provide a novel avenue for reducing post-infectious neuroinflammation.

Interleukin 6 reduces allopregnanolone synthesis in the brain and contributes to age-related cognitive decline in mice.

Cognitive decline with age is a harmful process that can reduce quality of life. Multiple factors have been established to contribute to cognitive decline, but the overall etiology remains unknown. Here, we hypothesized that cognitive dysfunction is mediated, in part, by increased levels of inflammatory cytokines that alter allopregnanolone (AlloP) levels, an important neurosteroid in the brain. We assessed the levels and regulation of AlloP and the effects of AlloP supplementation on cognitive function in 4-month-old and 24-month-old male C57BL/6 mice. With age, the expression of enzymes involved in the AlloP synthetic pathway was decreased and corticosterone (CORT) synthesis increased. Supplementation of AlloP improved cognitive function. Interestingly, interleukin 6 (IL-6) infusion in young animals significantly reduced the production of AlloP compared with controls. It is notable that inhibition of IL-6 with its natural inhibitor, soluble membrane glycoprotein 130, significantly improved spatial memory in aged mice. These findings were supported by in vitro experiments in primary murine astrocyte cultures, indicating that IL-6 decreases production of AlloP and increases CORT levels. Our results indicate that age-related increases in IL-6 levels reduce progesterone substrate availability, resulting in a decline in AlloP levels and an increase in CORT. Furthermore, our results indicate that AlloP is a critical link between inflammatory cytokines and the age-related decline in cognitive function.

Pharmacological or genetic depletion of senescent astrocytes prevents whole brain irradiation-induced impairment of neurovascular coupling responses protecting cognitive function in mice.

Whole brain irradiation (WBI, also known as whole brain radiation therapy or WBRT) is a mainstream therapy for patients with identifiable brain metastases and as a prophylaxis for microscopic malignancies. WBI accelerates brain aging, causing progressive cognitive dysfunction in ~ 50% of surviving patients, thus compromising quality of life. The mechanisms responsible for this WBI side effect remain obscure, and there are no effective treatments or prevention strategies. Here, we test the hypothesis that WBI induces astrocyte senescence, which contributes to impaired astrocytic neurovascular coupling (NVC) responses and the genesis of cognitive decline. To achieve this goal, we used transgenic p16-3MR mice, which allows the detection and selective elimination of senescent cells. We subjected these mice to a clinically relevant protocol of fractionated WBI (5 Gy twice weekly for 4 weeks). WBI-treated and control mice were tested for spatial memory performance (radial arm water maze), astrocyte-dependent NVC responses (whisker-stimulation-induced increases in cerebral blood flow, assessed by laser speckle contrast imaging), NVC-related gene expression, astrocytic release of eicosanoid gliotransmitters and the presence of senescent astrocytes (by flow cytometry, immunohistochemistry and gene expression profiling) at 6 months post-irradiation. WBI induced senescence in astrocytes, which associated with NVC dysfunction and impaired performance on cognitive tasks. To establish a causal relationship between WBI-induced senescence and NVC dysfunction, senescent cells were depleted from WBI-treated animals (at 3 months post-WBI) by genetic (ganciclovir treatment) or pharmacological (treatment with the BCL-2/BCL-xL inhibitor ABT263/Navitoclax, a known senolytic drug) means. In WBI-treated mice, both treatments effectively eliminated senescent astrocytes, rescued NVC responses, and improved cognitive performance. Our findings suggest that the use of senolytic drugs can be a promising strategy for preventing the cognitive impairment associated with WBI.

Accelerated decline in cognition in a mouse model of increased oxidative stress.

Mice deficient in the antioxidant enzyme Cu/Zn-superoxide dismutase (Sod1KO mice) have a significant reduction in lifespan, exhibit many phenotypes of accelerated aging, and have high levels of oxidative stress in various tissues. Age-associated cognitive decline is a hallmark of aging and the increase in oxidative stress/damage with age is one of the mechanisms proposed for cognitive decline with age. Therefore, the goal of this study was to determine if Sod1KO mice exhibit an accelerated loss in cognitive function similar to that observed in aged animals. Cognition was assessed in Sod1KO and wild type (WT) mice using an automated home-cage testing apparatus (Noldus PhenoTyper) that included an initial discrimination and reversal task. Comparison of the total distance moved by the mice during light and dark phases of the study demonstrated that the Sod1KO mice do not show a deficit in movement. Assessment of cognitive function showed no significant difference between Sod1KO and WT mice during the initial discrimination phase of learning. However, during the reversal task, Sod1KO mice showed a significantly greater number of incorrect entries compared to WT mice indicating a decline in cognition similar to that observed in aged animals. Markers of oxidative stress (4-Hydroxynonenal, 4-HNE) and neuroinflammation [proinflammatory cytokines (IL6 and IL-1ß) and neuroinflammatory markers (CD68, TLR4, and MCP1)] were significantly elevated in the hippocampus of male and female Sod1KO compared to WT mice. This study provides important evidence that increases in oxidative stress alone are sufficient to induce neuroinflammation and cognitive dysfunction that parallels the memory deficits seen in advanced aging and neurodegenerative diseases.