Novel Targets and Interventions for Cognitive Complications of Diabetes

Diabetes and cognitive dysfunction, ranging from mild cognitive impairment to dementia, often coexist in individuals over 65 years of age. Vascular contributions to cognitive impairment/dementia (VCID) are the second leading cause of dementias under the umbrella of Alzheimer’s disease and related dementias (ADRD). Over half of dementia patients have VCID either as a single pathology or a mixed dementia with AD. While the prevalence of type 2 diabetes in individuals with dementia can be as high as 39% and diabetes increases the risk of cerebrovascular disease and stroke, VCID remains to be one of the less understood and less studied complications of diabetes. We have identified cerebrovascular dysfunction and compromised endothelial integrity leading to decreased cerebral blood flow and iron deposition into the brain, respectively, as targets for intervention for the prevention of VCID in diabetes. This review will focus on targeted therapies that improve endothelial function or remove iron without systemic effects, such as agents delivered intranasally, that may result in actionable and disease-modifying novel treatments in the high-risk diabetic population.

Luseogliflozin, a sodium-glucose co-transporter 2 inhibitor, reverses cerebrovascular dysfunction and cognitive impairments in 18-month-old diabetic animals

Diabetes mellitus (DM) is a leading risk factor for age-related dementia, but the mechanisms involved remain to be elucidated. We previously discovered that hyperglycemia-induced impaired myogenic response (MR) and cerebral blood flow (CBF) autoregulation in 18-month-old DM rats associated with blood-brain barrier (BBB) leakage, impaired neurovascular coupling, and cognitive impairment. In the present study, we examined whether reduction of plasma glucose with a sodium-glucose co-transporter 2 inhibitor (SGLT2i) luseogliflozin can ameliorate cerebral vascular and cognitive function in diabetic rats. Plasma glucose and HbA1c levels of 18-month-old DM rats were reduced, and blood pressure was not altered after treatment with luseogliflozin. SGLT2i treatment restored the impaired MR of middle cerebral arteries (MCAs) and parenchymal arterioles, and surface and deep cortical CBF autoregulation in DM rats. Luseogliflozin treatment also rescued neurovascular uncoupling, reduced BBB leakage and cognitive deficits in DM rats. However, SGLT2i did not have direct constrictive effects on vascular smooth muscle cells and MCAs isolated from normal rats, although it decreased reactive oxygen species production in cerebral vessels of DM rats. These results provide evidence that normalization of hyperglycemia with an SGLT2i can reverse cerebrovascular dysfunction and cognitive impairments in rats with long-standing hyperglycemia, possibly by ameliorating oxidative stress-caused vascular damage.

Sex Differences in Large Artery Stiffness: Implications for Cerebrovascular Dysfunction and Alzheimer’s Disease

Two in every three Alzheimer’s disease diagnoses are females, calling attention to the need to understand sexual dimorphisms with aging and neurodegenerative disease progression. Dysfunction and damage to the vasculature with aging are strongly linked to Alzheimer’s disease. With aging there is an increase in stiffness of the large elastic arteries, and this stiffening is associated with cerebrovascular dysfunction and cognitive impairment. However, it is unclear how the deleterious effects of arterial stiffness may differ between females and males. While environmental, chromosomal, and sex hormone factors influence aging, there is evidence that the deficiency of estrogen post-menopause in females is a contributor to vascular aging and Alzheimer’s disease progression. The purpose of this mini review is to describe the recent developments in our understanding of sex differences in large artery stiffness, cerebrovascular dysfunction, and cognitive impairment, and their intricate relations. Furthermore, we will focus on the impact of the loss of estrogen post-menopause as a potential driving factor for these outcomes. Overall, a better understanding of how sex differences influence aging physiology is crucial to the prevention and treatment of neurodegenerative diseases.

Endothelial-Derived Extracellular Vesicles Induce Cerebrovascular Dysfunction in Inflammation

Blood–brain barrier (BBB) dysfunction is a key hallmark in the pathology of many neuroinflammatory disorders. Extracellular vesicles (EVs) are lipid membrane-enclosed carriers of molecular cargo that are involved in cell-to-cell communication. Circulating endothelial EVs are increased in the plasma of patients with neurological disorders, and immune cell-derived EVs are known to modulate cerebrovascular functions. However, little is known about whether brain endothelial cell (BEC)-derived EVs themselves contribute to BBB dysfunction. Human cerebral microvascular cells (hCMEC/D3) were treated with TNFα and IFNy, and the EVs were isolated and characterised. The effect of EVs on BBB transendothelial resistance (TEER) and leukocyte adhesion in hCMEC/D3 cells was measured by electric substrate cell-substrate impedance sensing and the flow-based T-cell adhesion assay. EV-induced molecular changes in recipient hCMEC/D3 cells were analysed by RT-qPCR and Western blotting. A stimulation of naïve hCMEC/D3 cells with small EVs (sEVs) reduced the TEER and increased the shear-resistant T-cell adhesion. The levels of microRNA-155, VCAM1 and ICAM1 were increased in sEV-treated hCMEC/D3 cells. Blocking the expression of VCAM1, but not of ICAM1, prevented sEV-mediated T-cell adhesion to brain endothelia. These results suggest that sEVs derived from inflamed BECs promote cerebrovascular dysfunction. These findings may provide new insights into the mechanisms involving neuroinflammatory disorders.

Impaired Hippocampal Neurovascular Coupling in a Mouse Model of Alzheimer’s Disease

Alzheimer’s disease (AD), the most common form of dementia, is characterized by neuronal degeneration and cerebrovascular dysfunction. Increasing evidence indicates that cerebrovascular dysfunction may be a key or an aggravating pathogenic factor in AD. This emphasizes the importance to investigate the tight coupling between neuronal activity and cerebral blood flow (CBF) termed neurovascular coupling (NVC). NVC depends on all cell types of the neurovascular unit within which astrocytes are important players in the progression of AD. Hence, the objective of this study was to characterize the hippocampal NVC in a mouse model of AD. Hippocampal NVC was studied in 6-month-old amyloid-beta precursor protein (APP) transgenic mice and their corresponding wild-type littermates using in vivo laser Doppler flowmetry to measure CBF in area CA1 of the hippocampus in response to Schaffer collaterals stimulation. Ex vivo two-photon microscopy experiments were performed to determine astrocytic Ca2+ and vascular responses to electrical field stimulation (EFS) or caged Ca2+ photolysis in hippocampal slices. Neuronal synaptic transmission, astrocytic endfeet Ca2+ in correlation with reactive oxygen species (ROS), and vascular reactivity in the presence or absence of Tempol, a mimetic of superoxide dismutase, were further investigated using electrophysiological, caged Ca2+ photolysis or pharmacological approaches. Whisker stimulation evoked-CBF increases and ex vivo vascular responses to EFS were impaired in APP mice compared with their age-matched controls. APP mice were also characterized by decreased basal synaptic transmission, a shorter astrocytic Ca2+ increase, and altered vascular response to elevated perivascular K+. However, long-term potentiation, astrocytic Ca2+ amplitude in response to EFS, together with vascular responses to nitric oxide remained unchanged. Importantly, we found a significantly increased Ca2+ uncaging-induced ROS production in APP mice. Tempol prevented the vascular response impairment while normalizing astrocytic Ca2+ in APP mice. These findings suggest that NVC is altered at many levels in APP mice, at least in part through oxidative stress. This points out that therapies against AD should include an antioxidative component to protect the neurovascular unit.

Depletion of microglia mitigates cerebrovascular dysfunction in diet-induced obesity mice

Obesity is frequently associated with cerebrovascular dysfunction, however, the underlying mechanism remains less well understood. In this study, by using pharmacological approaches, we show that neuroinflammation involving microglia plays an important role in obesity-related cerebrovascular dysfunction. PLX3397 treatment, which leads to depletion of microglia, reduced the wall thickness and collagen deposition in the basilar artery of diet-induced obesity (DIO) mice. Besides, the phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser1177 was enhanced, suggesting improved endothelial function of the basilar artery. The wire myography data show that acetylcholine-elicited relaxation of basilar artery isolated from DIO mice was improved after the treatment with PLX3397. Moreover, our data demonstrate that brain administration of IL-18 impaired cerebrovascular function in mice with normal body weight. Together, these data suggest that neuroinflammation involving microglia is important in obesity-related vascular dysfunction in the brain.

APOE genotype dependent molecular abnormalities in the cerebrovasculature of Alzheimer’s disease and age-matched non-demented brains

Cerebrovascular dysfunction is a hallmark feature of Alzheimer's disease (AD). One of the greatest risk factors for AD is the apolipoprotein E4 (E4) allele. The APOE4 genotype has been shown to negatively impact vascular amyloid clearance, however, its direct influence on the molecular integrity of the cerebrovasculature compared to other APOE variants (APOE2 and APOE3) has been largely unexplored. To address this, we employed a 10-plex tandem isobaric mass tag approach in combination with an ultra-high pressure liquid chromatography MS/MS (Q-Exactive) method, to interrogate unbiased proteomic changes in cerebrovessels from AD and healthy control brains with different APOE genotypes. We first interrogated changes between healthy control cases to identify underlying genotype specific effects in cerebrovessels. EIF2 signaling, regulation of eIF4 and 70S6K signaling and mTOR signaling were the top significantly altered pathways in E4/E4 compared to E3/E3 cases. Oxidative phosphorylation, EIF2 signaling and mitochondrial dysfunction were the top significant pathways in E2E2 vs E3/E3cases. We also identified AD-dependent changes and their interactions with APOE genotype and found the highest number of significant proteins from this interaction was observed in the E3/E4 (192) and E4/E4 (189) cases. As above, EIF2, mTOR signaling and eIF4 and 70S6K signaling were the top three significantly altered pathways in E4 allele carriers (i.e. E3/E4 and E4/E4 genotypes). Of all the cerebrovascular cell-type specific markers identified in our proteomic analyses, endothelial cell, astrocyte, and smooth muscle cell specific protein markers were significantly altered in E3/E4 cases, while endothelial cells and astrocyte specific protein markers were altered in E4/E4 cases. These proteomic changes provide novel insights into the longstanding link between APOE4 and cerebrovascular dysfunction, implicating a role for impaired autophagy, ER stress, and mitochondrial bioenergetics. These APOE4 dependent changes we identified could provide novel cerebrovascular targets for developing disease modifying strategies to mitigate the effects of APOE4 genotype on AD pathogenesis.

Long Term Cerebrovascular Dysfunction in the Offspring from Maternal Electronic Cigarette Use during Pregnancy

Electronic cigarettes (E-cigs) have been promoted as harm-free or less-risky than smoking, even for women during pregnancy. These claims are made largely on E-cig aerosol having fewer number of toxic chemicals compared to cigarette smoke. Given that even low levels of smoking are found to produce adverse birth outcomes, we sought to test the hypothesis that vaping during pregnancy (with or without nicotine) would not be harm-free, and would result in vascular dysfunction that would be evident in offspring during adolescent and/or adult life. Pregnant female Sprague Dawley rats were exposed to E-cig aerosol (1-hour/day, 5 days/week, starting on gestational day 2 until pups were weaned) using e-liquid with 0 mg/ml (E-cig0) or 18 mg/ml nicotine (E-cig18) and compared to ambient air exposed controls. Body mass at birth and at weaning were not different between groups. Assessment of middle cerebral artery (MCA) reactivity revealed a 51-56% reduction in endothelial-dependent dilation response to acetylcholine (ACh) for both E-cig0 and E-cig18 in 1-month, 3-month (adolescent), and 7-month old (adult) offspring (p<0.05 compared to air, all time points). MCA response to sodium nitroprusside (SNP) and myogenic tone were not different across groups suggesting that endothelial-independent responses were not altered. The MCA vasoconstrictor response (5-hydroxytryptamine, 5-HT) was also not different across treatment and age groups. These data demonstrate that maternal vaping during pregnancy is not harm-free and confers significant cerebrovascular health risk/dysfunction to offspring that persists into adult life.