A transient brain endothelial translatome response to endotoxin is associated with mild cognitive changes post-shock in young mice.

Sepsis-associated encephalopathy (SAE) is a common manifestation in septic patients that is associated with increased risk of long-term cognitive impairment. SAE is driven, at least in part, by brain endothelial dysfunction in response to systemic cytokine signaling. However, the mechanisms driving SAE and its consequences remain largely unknown. Here, we performed translating ribosome affinity purification and RNA-sequencing (TRAP-seq) from the brain endothelium to determine the transcriptional changes after an acute endotoxemic (LPS) challenge. LPS induced a strong acute transcriptional response in the brain endothelium that partially correlates with the whole brain transcriptional response and suggested an endothelial-specific hypoxia response. Consistent with a crucial role for IL-6, loss of the main regulator of this pathway, SOCS3, leads to a broadening of the population of genes responsive to LPS, suggesting that an overactivation of the IL-6/JAK/STAT3 pathway leads to an increased transcriptional response that could explain our prior findings of severe brain injury in these mice. To identify any potential sequelae of this acute response, we performed brain TRAP-seq following a battery of behavioral tests in mice after apparent recovery. We found that the transcriptional response returns to baseline within days post-challenge. Despite the transient nature of the response, we observed that mice that recovered from the endotoxemic shock showed mild, sex-dependent cognitive impairment, suggesting that the acute brain injury led to sustained, non-transcriptional effects. A better understanding of the transcriptional and non-transcriptional changes in response to shock is needed in order to prevent and/or revert the devastating consequences of septic shock.

Effects of High Fat Diet on Metabolic Health Vary by Age of Menopause Onset.

Menopause accelerates metabolic dysfunction, including (pre-)diabetes, obesity and visceral adiposity. However, the effects of endocrine vs. chronological aging in this progression are poorly understood. We hypothesize that menopause, especially in the context of middle-age, will exacerbate the metabolic effects of a high fat diet. Using young-adult and middle-aged C57BL/6J female mice, we modeled diet-induce obesity via chronic administration of high fat (HF) diet vs. control diet. We modeled peri-menopause/menopause via injections of 4-vinylcyclohexene diepoxide, which accelerates ovarian failure vs. vehicle. We performed glucose tolerance tests 2.5 and 7 months after diet onset, during the peri-menopausal and menopausal phases, respectively. Peri-menopause increased the severity of glucose intolerance and weight gain in middle-aged, HF-fed mice. Menopause increased weight gain in all mice regardless of age and diet, while chronological aging drove changes in adipose tissue distribution towards more visceral vs. subcutaneous adiposity. These data are in line with clinical data showing that post-menopausal women are more susceptible to metabolic dysfunction and suggest that greater chorological age exacerbates the effects of endocrine aging (menopause). This work highlights the importance of considering both chronological and endocrine aging in studies of metabolic health.

Brain Specific Estrogen Ameliorates Cognitive Effects of Surgical Menopause in Mice.

Menopause is a major endocrinological shift that leads to an increased vulnerability to the risk factors for cognitive impairment and dementia. This is thought to be due to the loss of circulating estrogens, which exert many potent neuroprotective effects in the brain. Systemic replacement of estrogen post-menopause has many limitations, including increased risk for estrogen-sensitive cancers. A more promising therapeutic approach therefore might be to deliver estrogen only to the brain thus limiting adverse peripheral side effects. We examined whether we could enhance cognitive performance by delivering estrogen exclusively to the brain in post-menopausal mice. We modeled surgical menopause via bilateral ovariectomy (OVX). We treated mice with the pro-drug 10ß,17ß-dihydroxyestra-1,4-dien-3-one (DHED), which can be administered systemically but is converted to 17ß-estradiol only in the brain. Young (2.5-month) and middle-aged (11-month-old) female C57BL/6J mice received ovariectomy and a subcutaneous implant containing vehicle (cholesterol) or DHED. At 3.5 months old (young group) and 14.5 months old (middle-aged group), mice underwent behavior testing to assess memory. DHED did not significantly alter metabolic status in middle-aged, post-menopausal mice. In both young and middle-aged mice, the brain-specific estrogen DHED improved spatial memory. Additional testing in middle-aged mice also showed that DHED improved working and recognition memory. These promising results lay the foundation for future studies aimed at determining if this intervention is as efficacious in models of dementia that have comorbid risk factors.

Sex differences in metabolic phenotype and hypothalamic inflammation in the 3xTg-AD mouse model of Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is notably associated with cognitive decline resulting from impaired function of hippocampal and cortical areas; however, several other domains and corresponding brain regions are affected. One such brain region is the hypothalamus, shown to atrophy and develop amyloid and tau pathology in AD patients. The hypothalamus controls several functions necessary for survival, including energy and glucose homeostasis. Changes in appetite and body weight are common in AD, often seen several years prior to the onset of cognitive symptoms. Therefore, altered metabolic processes may serve as a biomarker for AD, as well as a target for treatment, considering they are likely both a result of pathological changes and contributor to disease progression. Previously, we reported sexually dimorphic metabolic disturbances in ~ 7-month-old 3xTg-AD mice, accompanied by differences in systemic and hypothalamic inflammation. METHODS: In the current study, we investigated metabolic outcomes and hypothalamic inflammation in 3xTg-AD males and females at 3, 6, 9, and 12 months of age to determine when these sex differences emerge. RESULTS: In agreement with our previous study, AD males displayed less weight gain and adiposity, as well as reduced blood glucose levels following a glucose challenge, compared to females. These trends were apparent by 6-9 months of age, coinciding with increased expression of inflammatory markers (Iba1, GFAP, TNF-α, and IL-1ß) in the hypothalamus of AD males. CONCLUSIONS: These findings provide additional evidence for sex-dependent effects of AD pathology on energy and glucose homeostasis, which may be linked to hypothalamic inflammation.

Alzheimer's disease (AD), often associated with memory loss, can also affect other parts of the brain and body, resulting in several other symptoms. Changes in appetite and body weight are commonly seen in people with AD, often before they start showing signs of memory loss. These metabolism-related changes are likely due in part to AD affecting a part of the brain called the hypothalamus, which controls important functions like energy balance (calories in vs. calories out) and blood sugar levels. This study aimed to examine whether changes in metabolism and the hypothalamus could serve as early signs of AD, and even help in treating the disease. We also wanted to see if these changes were influenced by biological sex, as two-thirds of AD patients are women, and our previous studies showed many differences between males and females. In this study, we observed male and female mice at different ages to see when these changes began to appear. We found that male AD mice gained less weight, had less body fat, and had better blood sugar control, compared to female AD mice. These differences became noticeable at the same age that we noticed signs of increased inflammation in the hypothalamus of male mice. These findings suggest that AD affects males and females differently, particularly in terms of energy balance and blood sugar control, and this might be related to inflammation in the hypothalamus. This research could provide valuable insights into understanding, diagnosing, and treating Alzheimer's disease.

Menopause causes metabolic and cognitive impairments in a chronic cerebral hypoperfusion model of vascular contributions to cognitive impairment and dementia.

BACKGROUND: The vast majority of women with dementia are post-menopausal. Despite clinical relevance, menopause is underrepresented in rodent models of dementia. Before menopause, women are less likely than men to experience strokes, obesity, and diabetes-known risk factors for vascular contributions to cognitive impairment and dementia (VCID). During menopause, ovarian estrogen production stops and the risk of developing these dementia risk factors spikes. Here, we aimed to determine if menopause worsens cognitive impairment in VCID. We hypothesized that menopause would cause metabolic dysfunction and increase cognitive impairment in a mouse model of VCID. METHODS: We performed a unilateral common carotid artery occlusion surgery to produce chronic cerebral hypoperfusion and model VCID in mice. We used 4-vinylcyclohexene diepoxide to induce accelerated ovarian failure and model menopause. We evaluated cognitive impairment using behavioral tests including novel object recognition, Barnes maze, and nest building. To assess metabolic changes, we measured weight, adiposity, and glucose tolerance. We explored multiple aspects of brain pathology including cerebral hypoperfusion and white matter changes (commonly observed in VCID) as well as changes to estrogen receptor expression (which may mediate altered sensitivity to VCID pathology post-menopause). RESULTS: Menopause increased weight gain, glucose intolerance, and visceral adiposity. VCID caused deficits in spatial memory regardless of menopausal status. Post-menopausal VCID specifically led to additional deficits in episodic-like memory and activities of daily living. Menopause did not alter resting cerebral blood flow on the cortical surface (assessed by laser speckle contrast imaging). In the white matter, menopause decreased myelin basic protein gene expression in the corpus callosum but did not lead to overt white matter damage (assessed by Luxol fast blue). Menopause did not significantly alter estrogen receptor expression (ERα, ERß, or GPER1) in the cortex or hippocampus. CONCLUSIONS: Overall, we have found that the accelerated ovarian failure model of menopause caused metabolic impairment and cognitive deficits in a mouse model of VCID. Further studies are needed to identify the underlying mechanism. Importantly, the post-menopausal brain still expressed estrogen receptors at normal (pre-menopausal) levels. This is encouraging for any future studies attempting to reverse the effects of estrogen loss by activating brain estrogen receptors.

Nearly all women with dementia are menopausal. Reduced blood flow to the brain, resulting from damaged blood vessels, can lead to vascular dementia. Vascular dementia is the second most common cause of dementia. Before menopause, women are less likely than men to experience strokes, obesity, and diabetes­known risk factors for vascular dementia. During menopause, estrogen levels drop and the risk of developing these dementia risk factors increases. The goal of this study was to determine how menopause impacts risk factors (obesity, diabetes), memory and brain pathology in vascular dementia. This study used mouse models of vascular dementia and menopause. Menopause increased weight gain and other indicators of poor metabolic health. In mice with vascular dementia, menopausal mice had worse memory than pre-menopausal mice. After menopause, the brain still expressed estrogen receptors at normal (pre-menopausal) levels. This is encouraging for any future studies attempting to reverse the effects of estrogen loss by activating brain estrogen receptors.

Sex differences in the effects of high fat diet on underlying neuropathology in a mouse model of VCID.

BACKGROUND: Damage to the cerebral vasculature can lead to vascular contributions to cognitive impairment and dementia (VCID). A reduction in blood flow to the brain leads to neuropathology, including neuroinflammation and white matter lesions that are a hallmark of VCID. Mid-life metabolic disease (obesity, prediabetes, or diabetes) is a risk factor for VCID which may be sex-dependent (female bias). METHODS: We compared the effects of mid-life metabolic disease between males and females in a chronic cerebral hypoperfusion mouse model of VCID. C57BL/6J mice were fed a control or high fat (HF) diet starting at ~ 8.5 months of age. Three months after diet initiation, sham or unilateral carotid artery occlusion surgery (VCID model) was performed. Three months later, mice underwent behavior testing and brains were collected to assess pathology. RESULTS: We have previously shown that in this VCID model, HF diet causes greater metabolic impairment and a wider array of cognitive deficits in females compared to males. Here, we report on sex differences in the underlying neuropathology, specifically white matter changes and neuroinflammation in several areas of the brain. White matter was negatively impacted by VCID in males and HF diet in females, with greater metabolic impairment correlating with less myelin markers in females only. High fat diet led to an increase in microglia activation in males but not in females. Further, HF diet led to a decrease in proinflammatory cytokines and pro-resolving mediator mRNA expression in females but not males. CONCLUSIONS: The current study adds to our understanding of sex differences in underlying neuropathology of VCID in the presence of a common risk factor (obesity/prediabetes). This information is crucial for the development of effective, sex-specific therapeutic interventions for VCID.

Reduced blood flow to the brain resulting from damaged blood vessels can lead to vascular dementia. Neuroinflammation and white matter damage are characteristics of vascular dementia. Middle-age is a time when obesity and prediabetes can increase risk for vascular dementia. This increase in risk is greater for women. A high fat diet causes obesity and prediabetes in mice. We compared the effects of diet-induced obesity in middle-age between males and females in a mouse model of vascular dementia. We have previously shown that a high fat diet causes greater obesity and prediabetes and a wider array of learning and memory problems in females compared to males. Here, we report on sex differences in the damage to the brain. White matter was negatively impacted by vascular dementia in males and high fat diet in females, with more severe prediabetes correlating with less white matter markers in females only. High fat diet led to an increase in activation of microglia (immune cells in the brain) in males but not in females. High fat diet also led to a decrease in pro-inflammatory and pro-resolving mediators expression in females but not males. The current study adds to our understanding of sex differences in underlying damage to the brain caused by vascular dementia in the presence of common risk factors (obesity and prediabetes). This information is needed for the development of effective, sex-specific treatments for vascular dementia.

High-fat diet exacerbates cognitive decline in mouse models of Alzheimer's disease and mixed dementia in a sex-dependent manner.

BACKGROUND: Approximately 70% of Alzheimer's disease (AD) patients have co-morbid vascular contributions to cognitive impairment and dementia (VCID); this highly prevalent overlap of dementia subtypes is known as mixed dementia (MxD). AD is more prevalent in women, while VCID is slightly more prevalent in men. Sex differences in risk factors may contribute to sex differences in dementia subtypes. Unlike metabolically healthy women, diabetic women are more likely to develop VCID than diabetic men. Prediabetes is 3× more prevalent than diabetes and is linked to earlier onset of dementia in women, but not men. How prediabetes influences underlying pathology and cognitive outcomes across different dementia subtypes is unknown. To fill this gap in knowledge, we investigated the impact of diet-induced prediabetes and biological sex on cognitive function and neuropathology in mouse models of AD and MxD. METHODS: Male and female 3xTg-AD mice received a sham (AD model) or unilateral common carotid artery occlusion surgery to induce chronic cerebral hypoperfusion (MxD model). Mice were fed a control or high fat (HF; 60% fat) diet from 3 to 7 months of age. In both sexes, HF diet elicited a prediabetic phenotype (impaired glucose tolerance) and weight gain. RESULTS: In females, but not males, metabolic consequences of a HF diet were more severe in AD or MxD mice compared to WT. In both sexes, HF-fed AD or MxD mice displayed deficits in spatial memory in the Morris water maze (MWM). In females, but not males, HF-fed AD and MxD mice also displayed impaired spatial learning in the MWM. In females, but not males, AD or MxD caused deficits in activities of daily living, regardless of diet. Astrogliosis was more severe in AD and MxD females compared to males. Further, AD/MxD females had more amyloid beta plaques and hippocampal levels of insoluble amyloid beta 40 and 42 than AD/MxD males. In females, but not males, more severe glucose intolerance (prediabetes) was correlated with increased hippocampal microgliosis. CONCLUSIONS: High-fat diet had a wider array of metabolic, cognitive, and neuropathological consequences in AD and MxD females compared to males. These findings shed light on potential underlying mechanisms by which prediabetes may lead to earlier dementia onset in women.

Role of sex and high-fat diet in metabolic and hypothalamic disturbances in the 3xTg-AD mouse model of Alzheimer's disease.

BACKGROUND: Hypothalamic dysfunction occurs early in the clinical course of Alzheimer's disease (AD), likely contributing to disturbances in feeding behavior and metabolic function that are often observed years prior to the onset of cognitive symptoms. Late-life weight loss and low BMI are associated with increased risk of dementia and faster progression of disease. However, high-fat diet and metabolic disease (e.g., obesity, type 2 diabetes), particularly in mid-life, are associated with increased risk of AD, as well as exacerbated AD pathology and behavioral deficits in animal models. In the current study, we explored possible relationships between hypothalamic function, diet/metabolic status, and AD. Considering the sex bias in AD, with women representing two-thirds of AD patients, we sought to determine whether these relationships vary by sex. METHODS: WT and 3xTg-AD male and female mice were fed a control (10% fat) or high-fat (HF 60% fat) diet from ~ 3-7 months of age, then tested for metabolic and hypothalamic disturbances. RESULTS: On control diet, male 3xTg-AD mice displayed decreased body weight, reduced fat mass, hypoleptinemia, and mild systemic inflammation, as well as increased expression of gliosis- and inflammation-related genes in the hypothalamus (Iba1, GFAP, TNF-α, IL-1ß). In contrast, female 3xTg-AD mice on control diet displayed metabolic disturbances opposite that of 3xTg-AD males (increased body and fat mass, impaired glucose tolerance). HF diet resulted in expected metabolic alterations across groups (increased body and fat mass; glucose intolerance; increased plasma insulin and leptin, decreased ghrelin; nonalcoholic fatty liver disease-related pathology). HF diet resulted in the greatest weight gain, adiposity, and glucose intolerance in 3xTg-AD females, which were associated with markedly increased hypothalamic expression of GFAP and IL-1ß, as well as GFAP labeling in several hypothalamic nuclei that regulate energy balance. In contrast, HF diet increased diabetes markers and systemic inflammation preferentially in AD males but did not exacerbate hypothalamic inflammation in this group. CONCLUSIONS: These findings provide further evidence for the roles of hypothalamic and metabolic dysfunction in AD, which in the 3xTg-AD mouse model appears to be dependent on both sex and diet.

Sex-specific effects of high-fat diet on cognitive impairment in a mouse model of VCID.

Mid-life metabolic disease (ie, obesity, diabetes, and prediabetes) causes vascular dysfunction and is a risk factor for vascular contributions to cognitive impairment and dementia (VCID), particularly in women. Using middle-aged mice, we modeled metabolic disease (obesity/prediabetes) via chronic high-fat (HF) diet and modeled VCID via unilateral common carotid artery occlusion. VCID impaired spatial memory in both sexes, but episodic-like memory in females only. HF diet caused greater weight gain and glucose intolerance in middle-aged females than males. HF diet alone impaired episodic-like memory in both sexes, but spatial memory in females only. Finally, the combination of HF diet and VCID elicited cognitive impairments in all tests, in both sexes. Sex-specific correlations were found between metabolic outcomes and memory. Notably, both visceral fat and the pro-inflammatory cytokine tumor necrosis factor alpha correlated with spatial memory deficits in middle-aged females, but not males. Overall, our data show that HF diet causes greater metabolic impairment and a wider array of cognitive deficits in middle-aged females than males. The combination of HF diet with VCID elicits deficits across multiple cognitive domains in both sexes. Our data are in line with clinical data, which shows that mid-life metabolic disease increases VCID risk, particularly in females.

Depletion of NK Cells Improves Cognitive Function in the Alzheimer Disease Mouse Model.

Despite mounting evidence suggesting the involvement of the immune system in regulating brain function, the specific role of immune and inflammatory cells in neurodegenerative diseases remain poorly understood. In this study, we report that depletion of NK cells, a type of innate lymphocytes, alleviates neuroinflammation, stimulates neurogenesis, and improves cognitive function in a triple-transgenic Alzheimer disease (AD) mouse model. NK cells in the brains of triple-transgenic AD mouse model (3xTg-AD) mice exhibited an enhanced proinflammatory profile. Depletion of NK cells by anti-NK1.1 Abs drastically improved cognitive function of 3xTg-AD mice. NK cell depletion did not affect amyloid ß concentrations but enhanced neurogenesis and reduced neuroinflammation. Notably, in 3xTg-AD mice depleted of NK cells, microglia demonstrated a homeostatic-like morphology, decreased proliferative response and reduced expression of neurodestructive proinflammatory cytokines. Together, our results suggest a proinflammatory role for NK cells in 3xTg-AD mice and indicate that targeting NK cells might unlock novel strategies to combat AD.

Activation of group 2 innate lymphoid cells alleviates aging-associated cognitive decline.

Increasing evidence has challenged the traditional view about the immune privilege of the brain, but the precise roles of immune cells in regulating brain physiology and function remain poorly understood. Here, we report that tissue-resident group 2 innate lymphoid cells (ILC2) accumulate in the choroid plexus of aged brains. ILC2 in the aged brain are long-lived, are relatively resistant to cellular senescence and exhaustion, and are capable of switching between cell cycle dormancy and proliferation. They are functionally quiescent at homeostasis but can be activated by IL-33 to produce large amounts of type 2 cytokines and other effector molecules in vitro and in vivo. Intracerebroventricular transfer of activated ILC2 revitalized the aged brain and enhanced the cognitive function of aged mice. Administration of IL-5, a major ILC2 product, was sufficient to repress aging-associated neuroinflammation and alleviate aging-associated cognitive decline. Targeting ILC2 in the aged brain may provide new avenues to combat aging-associated neurodegenerative disorders.

High-Fat Diet-Induced Obesity Causes Sex-Specific Deficits in Adult Hippocampal Neurogenesis in Mice.

Adult hippocampal neurogenesis (AHN) is suppressed by high-fat (HF) diet and metabolic disease, including obesity and type 2 diabetes. Deficits in AHN may contribute to cognitive decline and increased risk of dementia and mood disorders, which have higher prevalence in women. However, sex differences in the effects of HF diet/metabolic disease on AHN have yet to be thoroughly investigated. Herein, male and female C57BL/6J mice were fed an HF or control (CON) diet from ∼2 to 6 months of age. After 3 months on the diet, mice were injected with 5-ethynyl-2'-deoxyuridine (EdU) then killed 4 weeks later. Cell proliferation, differentiation/maturation, and survival of new neurons in the dentate gyrus were assessed with immunofluorescence for EdU, Ki67, doublecortin (DCX), and NeuN. CON females had more proliferating cells (Ki67+) and neuroblasts/immature neurons (DCX+) compared with CON males; however, HF diet reduced these cells in females to the levels of males. Diet did not affect neurogenesis in males. Further, the numbers of proliferating cells and immature neurons were inversely correlated with both weight gain and glucose intolerance in females only. These effects were robust in the dorsal hippocampus, which supports cognitive processes. Assessment of microglia in the dentate gyrus using immunofluorescence for Iba1 and CD68 uncovered sex-specific effects of diet, which may contribute to observed differences in neurogenesis. These findings demonstrate sex-specific effects of HF diet/metabolic disease on AHN, and highlight the potential for targeting neurogenic deficits to treat cognitive decline and reduce the risk of dementia associated with these conditions, particularly in females.

Contributions of sex to cerebrovascular function and pathology.

Sex differences exist in how cerebral blood vessels function under both physiological and pathological conditions, contributing to observed sex differences in risk and outcomes of cerebrovascular diseases (CBVDs), such as vascular contributions to cognitive impairment and dementia (VCID) and stroke. Throughout most of the lifespan, women are protected from CBVDs; however, risk increases following menopause, suggesting sex hormones may play a significant role in this protection. The cerebrovasculature is a target for sex hormones, including estrogens, progestins, and androgens, where they can influence numerous vascular functions and pathologies. While there is a plethora of information on estrogen, the effects of progestins and androgens on the cerebrovasculature are less well-defined. Estrogen decreases cerebral tone and increases cerebral blood flow, while androgens increase tone. Both estrogens and androgens enhance angiogenesis/cerebrovascular remodeling. While both estrogens and androgens attenuate cerebrovascular inflammation, pro-inflammatory effects of androgens under physiological conditions have also been demonstrated. Sex hormones exert additional neuroprotective effects by attenuating oxidative stress and maintaining integrity and function of the blood brain barrier. Most animal studies utilize young, healthy, gonadectomized animals, which do not mimic the clinical conditions of aging individuals likely to get CBVDs. This is also concerning, as sex hormones appear to mediate cerebrovascular function differently based on age and disease state (e.g. metabolic syndrome). Through this review, we hope to inspire others to consider sex as a key biological variable in cerebrovascular research, as greater understanding of sex differences in cerebrovascular function will assist in developing personalized approaches to prevent and treat CBVDs.

Sex differences in the metabolic effects of diet-induced obesity vary by age of onset.

Sex differences in the effect of diet-induced obesity (DIO) have been reported in juvenile mice. However, thorough side by side comparisons of the effects of DIO in males and females at different ages of onset have yet to be examined. We hypothesized that aged females would lose their protection, relative to males, from the effects of DIO. We examined the effect of DIO on body weight and glucose tolerance in juvenile, young adult, and middle-aged male and female mice. Our data show DIO in juvenile mice causes a greater increase in body weight and greater impairment in glucose tolerance in males than females. However, if the diet is initiated in young adult mice, these sex differences are absent. Further, if the diet is initiated in middle-aged mice, the sex difference is reversed, and females gain more weight and have greater impairment in glucose tolerance than males. Our data show that sex differences in the effect of DIO vary by age of onset; thus highlighting the importance of both age and sex as biological variables in DIO research.