Brain-Immune interactions: Implication for Cognitive impairments in Alzheimer's Disease and autoimmune disorders.

Progressive memory loss and cognitive dysfunction, encompassing deficits in learning, memory, problem-solving, spatial reasoning, verbal expression are characteristics of Alzheimer's disease and related dementia (ADRD). A wealth of studies has described multiple roles of the immune system in the development or exacerbation of dementia. Individuals with autoimmune disorders can also develop cognitive dysfunction, a phenomenon termed autoimmune dementia. Together, these findings underscore the pivotal role of the neuroimmune axis in both ADRD and autoimmune dementia. The dynamic interplay between adaptive and innate immunity, both in and outside the brain, significantly affects the etiology and progression of these conditions. Multidisciplinary research shows that cognitive dysfunction arises from a bidirectional relationship between the nervous and immune systems, though the specific mechanisms that drive cognitive impairments are not fully understood. Intriguingly, this reciprocal regulation occurs at multiple levels, where neuronal signals can modulate immune responses, and immune system-related processes can influence neuronal viability and function. In this review, we consider the implications of autoimmune responses in various autoimmune disorders and Alzheimer's disease and explore their effects on brain function. We also discuss the diverse cellular and molecular cross talk between the brain and the immune system as they may shed light on potential triggers of peripheral inflammation, their effect on the integrity of the blood-brain barrier (BBB) and brain function. Additionally, we assess challenges and possibilities associated with developing immune-based therapies for the treatment of cognitive decline.

A roadmap to human hippocampal neurogenesis in adulthood, aging and AD.

In the rodent, hippocampal neurogenesis plays critical roles in learning and memory1,2, is tightly regulated by inhibitory neurons3-7 and contributes to memory dysfunction in Alzheimer's disease (AD) mouse models8-10. In contrast, the mechanisms regulating neurogenesis in the adult human hippocampus, the dynamic shifts in the transcriptomic and epigenomic profiles in aging and AD and putative niche interactions within the cellular environment, remain largely unknown. Using single nuclei multi-omics of postmortem human hippocampi we map the molecular mechanisms of hippocampal neurogenesis across aging, cognitive decline, and AD neuropathology. Transcriptomic and epigenetic profiling of neural stem cells (NSCs), neuroblasts and immature neurons suggests that the earliest shift in the characteristics of neurogenesis takes place in NSCs in aging. Cognitive impairment was associated with changes in neuroblast profile. In AD, there was a widespread cessation of the transcription machinery in immature neurons, with robust downregulation of genes regulating ribosomal and mitochondrial function. Further, there was substantial loss of parvalbumin+ inhibitory neurons in the hippocampus in aging. The number of the rest of inhibitory neurons were reduced as a function of age and diagnosis. Notably, a similar system-level effect was observed between immature and inhibitory neurons in the transition from aging to AD, manifested by common molecular pathways that were ultimately lost in AD. The numbers of neuroblasts, immature and GABAergic neurons inversely correlated with extent of neuropathology. Using CellChat and NeuronChat, we inferred the ligands and receptors by which neurogenic cells communicate with their cellular environment. Loss of synaptic adhesion molecules and neurotransmitters, either sent or received by neurogenic cells, was observed in AD. Together, this study delineates the molecular mechanisms and dynamics of human neurogenesis, functional association with inhibitory neurons and a mechanism of hippocampal hyperexcitability in AD.

Memory circuits in dementia: The engram, hippocampal neurogenesis and Alzheimer's disease.

Here, we provide an in-depth consideration of our current understanding of engrams, spanning from molecular to network levels, and hippocampal neurogenesis, in health and Alzheimer's disease (AD). This review highlights novel findings in these emerging research fields and future research directions for novel therapeutic avenues for memory failure in dementia. Engrams, memory in AD, and hippocampal neurogenesis have each been extensively studied. The integration of these topics, however, has been relatively less deliberated, and is the focus of this review. We primarily focus on the dentate gyrus (DG) of the hippocampus, which is a key area of episodic memory formation. Episodic memory is significantly impaired in AD, and is also the site of adult hippocampal neurogenesis. Advancements in technology, especially opto- and chemogenetics, have made sophisticated manipulations of engram cells possible. Furthermore, innovative methods have emerged for monitoring neurons, even specific neuronal populations, in vivo while animals engage in tasks, such as calcium imaging. In vivo calcium imaging contributes to a more comprehensive understanding of engram cells. Critically, studies of the engram in the DG using these technologies have shown the important contribution of hippocampal neurogenesis for memory in both health and AD. Together, the discussion of these topics provides a holistic perspective that motivates questions for future research.

Temporal Alterations in White Matter in An App Knock-In Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common form of dementia and results in neurodegeneration and cognitive impairment. White matter (WM) is affected in AD and has implications for neural circuitry and cognitive function. The trajectory of these changes across age, however, is still not well understood, especially at earlier stages in life. To address this, we used the AppNL-G-F/NL-G-F knock-in (APPKI) mouse model that harbors a single copy knock-in of the human amyloid precursor protein (APP) gene with three familial AD mutations. We performed in vivo diffusion tensor imaging (DTI) to study how the structural properties of the brain change across age in the context of AD. In late age APPKI mice, we observed reduced fractional anisotropy (FA), a proxy of WM integrity, in multiple brain regions, including the hippocampus, anterior commissure (AC), neocortex, and hypothalamus. At the cellular level, we observed greater numbers of oligodendrocytes in middle age (prior to observations in DTI) in both the AC, a major interhemispheric WM tract, and the hippocampus, which is involved in memory and heavily affected in AD, prior to observations in DTI. Proteomics analysis of the hippocampus also revealed altered expression of oligodendrocyte-related proteins with age and in APPKI mice. Together, these results help to improve our understanding of the development of AD pathology with age, and imply that middle age may be an important temporal window for potential therapeutic intervention.

Caveolin-1 Autonomously Regulates Hippocampal Neurogenesis Via Mitochondrial Dynamics.

The mechanisms underlying adult hippocampal neurogenesis (AHN) are not fully understood. AHN plays instrumental roles in learning and memory. Understanding the signals that regulate AHN has implications for brain function and therapy. Here we show that Caveolin-1 (Cav-1), a protein that is highly enriched in endothelial cells and the principal component of caveolae, autonomously regulates AHN. Conditional deletion of Cav-1 in adult neural progenitor cells (nestin +) led to increased neurogenesis and enhanced performance of mice in contextual discrimination. Proteomic analysis revealed that Cav-1 plays a role in mitochondrial pathways in neural progenitor cells. Importantly, Cav-1 was localized to the mitochondria in neural progenitor cells and modulated mitochondrial fission-fusion, a critical process in neurogenesis. These results suggest that Cav-1 is a novel regulator of AHN and underscore the impact of AHN on cognition.

Reduced endothelial caveolin-1 underlies deficits in brain insulin signalling in type 2 diabetes.

Patients with type 2 diabetes exhibit severe impairments in insulin signalling in the brain and are five times more likely to develop Alzheimer's disease. However, what leads to these impairments is not fully understood. Here, we show reduced expression of endothelial cell caveolin-1 (Cav-1) in the db/db (Leprdb) mouse model of type 2 diabetes. This reduction correlated with alterations in insulin receptor expression and signalling in brain microvessels as well as brain parenchyma. These findings were recapitulated in the brains of endothelial cell-specific Cav-1 knock-out (Tie2Cre; Cav-1fl/fl) mice. Lack of Cav-1 in endothelial cells led to reduced response to insulin as well as reduced insulin uptake. Furthermore, we observed that Cav-1 was necessary for the stabilization of insulin receptors in lipid rafts. Interactome analysis revealed that insulin receptor interacts with Cav-1 and caveolae-associated proteins, insulin-degrading enzyme and the tight junction protein Zonula Occludence-1 in brain endothelial cells. Restoration of Cav-1 in Cav-1 knock-out brain endothelial cells rescued insulin receptor expression and localization. Overall, these results suggest that Cav-1 regulates insulin signalling and uptake by brain endothelial cells by modulating IR-α and IR-ß localization and function in lipid rafts. Furthermore, depletion of endothelial cell-specific Cav-1 and the resulting impairment in insulin transport leads to alteration in insulin signalling in the brain parenchyma of type 2 diabetics.

Adult hippocampal neurogenesis in Alzheimer's disease: A roadmap to clinical relevance.

Adult hippocampal neurogenesis (AHN) drops sharply during early stages of Alzheimer's disease (AD), via unknown mechanisms, and correlates with cognitive status in AD patients. Understanding AHN regulation in AD could provide a framework for innovative pharmacological interventions. We here combine molecular, behavioral, and clinical data and critically discuss the multicellular complexity of the AHN niche in relation to AD pathophysiology. We further present a roadmap toward a better understanding of the role of AHN in AD by probing the promises and caveats of the latest technological advancements in the field and addressing the conceptual and methodological challenges ahead.

Augmenting neurogenesis rescues memory impairments in Alzheimer's disease by restoring the memory-storing neurons.

Hippocampal neurogenesis is impaired in Alzheimer's disease (AD) patients and familial Alzheimer's disease (FAD) mouse models. However, it is unknown whether new neurons play a causative role in memory deficits. Here, we show that immature neurons were actively recruited into the engram following a hippocampus-dependent task. However, their recruitment is severely deficient in FAD. Recruited immature neurons exhibited compromised spine density and altered transcript profile. Targeted augmentation of neurogenesis in FAD mice restored the number of new neurons in the engram, the dendritic spine density, and the transcription signature of both immature and mature neurons, ultimately leading to the rescue of memory. Chemogenetic inactivation of immature neurons following enhanced neurogenesis in AD, reversed mouse performance, and diminished memory. Notably, AD-linked App, ApoE, and Adam10 were of the top differentially expressed genes in the engram. Collectively, these observations suggest that defective neurogenesis contributes to memory failure in AD.

Hippocampal functional connectivity across age in an App knock-in mouse model of Alzheimer's disease.

Introduction: Alzheimer's disease (AD) is a progressive neurodegenerative disease. The early processes of AD, however, are not fully understood and likely begin years before symptoms manifest. Importantly, disruption of the default mode network, including the hippocampus, has been implicated in AD. Methods: To examine the role of functional network connectivity changes in the early stages of AD, we performed resting-state functional magnetic resonance imaging (rs-fMRI) using a mouse model harboring three familial AD mutations (App NL-G-F/NL-G-F knock-in, APPKI) in female mice in early, middle, and late age groups. The interhemispheric and intrahemispheric functional connectivity (FC) of the hippocampus was modeled across age. Results: We observed higher interhemispheric functional connectivity (FC) in the hippocampus across age. This was reduced, however, in APPKI mice in later age. Further, we observed loss of hemispheric asymmetry in FC in APPKI mice. Discussion: Together, this suggests that there are early changes in hippocampal FC prior to heavy onset of amyloid ß plaques, and which may be clinically relevant as an early biomarker of AD.

Adult hippocampal neurogenesis in Alzheimer's disease.

New neurons are generated in the dentate gyrus of the adult brain throughout life. They incorporate in the granular cell layer of the dentate gyrus and integrate in the hippocampal circuitry. Increasing evidence suggests that new neurons play a role in learning and memory. In turn, a large body of evidence suggests that neurogenesis is impaired in Alzheimer's disease, contributing to memory deficits characterizing the disease. We outline here current knowledge about the biology of adult hippocampal neurogenesis and its function in learning and memory. In addition, we discuss evidence that neurogenesis is dysfunctional in Alzheimer's disease, address the controversy in the literature concerning the persistence of hippocampal neurogenesis in the adult and aging human brain, and evaluate the therapeutic potential of neurogenesis-based drug development for the treatment of cognitive deficits in Alzheimer's disease.

Deficits in hippocampal neurogenesis in obesity-dependent and -independent type-2 diabetes mellitus mouse models.

Hippocampal neurogenesis plays an important role in learning and memory function throughout life. Declines in this process have been observed in both aging and Alzheimer's disease (AD). Type 2 Diabetes mellitus (T2DM) is a disorder characterized by insulin resistance and impaired glucose metabolism. T2DM often results in cognitive decline in adults, and significantly increases the risk of AD development. The pathways underlying T2DM-induced cognitive deficits are not known. Some studies suggest that alterations in hippocampal neurogenesis may contribute to cognitive deterioration, however, the fate of neurogenesis in these studies is highly controversial. To address this problem, we utilized two models of T2DM: (1) obesity-independent MKR transgenic mice expressing a mutated form of the human insulin-like growth factor 1 receptor (IGF-1R) in skeletal muscle, and (2) Obesity-dependent db/db mice harboring a mutation in the leptin receptor. Our results show that both models of T2DM display compromised hippocampal neurogenesis. We show that the number of new neurons in the hippocampus of these mice is reduced. Clone formation capacity of neural progenitor cells isolated from the db/db mice is deficient. Expression of insulin receptor and epidermal growth factor receptor was reduced in hippocampal neurospheres isolated from db/db mice. Results from this study warrant further investigation into the mechanisms underlying decreased neurogenesis in T2DM and its link to the cognitive decline observed in this disorder.

Harnessing neurogenesis in the adult brain-A role in type 2 diabetes mellitus and Alzheimer's disease.

Some metabolic disorders, such as type 2 diabetes mellitus (T2DM) are risk factors for the development of cognitive deficits and Alzheimer's disease (AD). Epidemiological studies suggest that in people with T2DM, the risk of developing dementia is 2.5 times higher than that in the non-diabetic population. The signaling pathways that underlie the increased risk and facilitate cognitive deficits are not fully understood. In fact, the cause of memory deficits in AD is not fully elucidated. The dentate gyrus of the hippocampus plays an important role in memory formation. Hippocampal neurogenesis is the generation of new neurons and glia in the adult brain throughout life. New neurons incorporate in the granular cell layer of the dentate gyrus and play a role in learning and memory and hippocampal plasticity. A large body of studies suggests that hippocampal neurogenesis is impaired in mouse models of AD and T2DM. Recent evidence shows that hippocampal neurogenesis is also impaired in human patients exhibiting mild cognitive impairment or AD. This review discusses the role of hippocampal neurogenesis in the development of cognitive deficits and AD, and considers inflammatory and endothelial signaling pathways in T2DM that may compromise hippocampal neurogenesis and cognitive function, leading to AD.

Activated Mesenchymal Stem Cells Induce Recovery Following Stroke Via Regulation of Inflammation and Oligodendrogenesis.

Background Brain repair mechanisms fail to promote recovery after stroke, and approaches to induce brain regeneration are scarce. Mesenchymal stem cells (MSC) are thought to be a promising therapeutic option. However, their efficacy is not fully elucidated, and the mechanism underlying their effect is not known. Methods and Results The middle cerebral artery occlusion model was utilized to determine the efficacy of interferon-γ-activated mesenchymal stem cells (aMSCγ) as an acute therapy for stroke. Here we show that treatment with aMSCγ is a more potent therapy for stroke than naive MSC. aMSCγ treatment results in significant functional recovery assessed by the modified neurological severity score and open-field analysis compared with vehicle-treated animals. aMSCγ-treated animals showed significant reductions in infarct size and inhibition of microglial activation. The aMSCγ treatment suppressed the hypoxia-induced microglial proinflammatory phenotype more effectively than treatment with naive MSC. Importantly, treatment with aMSCγ induced recruitment and differentiation of oligodendrocyte progenitor cells to myelin-producing oligodendrocytes in vivo. To elucidate the mechanism underlying high efficacy of aMSCγ therapy, we examined the secretome of aMSCγ and compared it to that of naive MSC. Intriguingly, we found that aMSCγ but not nMSC upregulated neuron-glia antigen 2, an important extracellular signal and a hallmark protein of oligodendrocyte progenitor cells. Conclusions These results suggest that activation of MSC with interferon-γ induces a potent proregenerative, promyelinating, and anti-inflammatory phenotype of these cells, which increases the potency of aMSCγ as an effective therapy for ischemic stroke.

Depletion of Caveolin-1 in Type 2 Diabetes Model Induces Alzheimer's Disease Pathology Precursors.

Type 2 diabetes mellitus (T2DM) is a risk factor for the development of late-onset Alzheimer's disease (AD). However, the mechanism underlying the development of late-onset AD is largely unknown. Here we show that levels of the endothelial-enriched protein caveolin-1 (Cav-1) are reduced in the brains of T2DM patients compared with healthy aging, and inversely correlated with levels of ß-amyloid (Aß). Depletion of Cav-1 is recapitulated in the brains of db/db (Leprdb ) diabetic mice and corresponds with recognition memory deficits as well as the upregulation of amyloid precursor protein (APP), BACE-1, a trending increase in ß-amyloid Aß42/40 ratio and hyperphosphorylated tau (p-tau) species. Importantly, we show that restoration of Cav-1 levels in the brains of male db/db mice using adenovirus overexpressing Cav-1 (AAV-Cav-1) rescues learning and memory deficits and reduces pathology (i.e., APP, BACE-1 and p-tau levels). Knocking down Cav-1 using shRNA in HEK cells expressing the familial AD-linked APPswe mutant variant upregulates APP, APP carboxyl terminal fragments, and Aß levels. In turn, rescue of Cav-1 levels restores APP metabolism. Together, these results suggest that Cav-1 regulates APP metabolism, and that depletion of Cav-1 in T2DM promotes the amyloidogenic processing of APP and hyperphosphorylation of tau. This may suggest that depletion of Cav-1 in T2DM underlies, at least in part, the development of AD and imply that restoration of Cav-1 may be a therapeutic target for diabetic-associated sporadic AD.SIGNIFICANCE STATEMENT More than 95% of the Alzheimer's patients have the sporadic late-onset form (LOAD). The cause for late-onset Alzheimer's disease is unknown. Patients with Type 2 diabetes mellitus have considerably higher incidence of cognitive decline and AD compared with the general population, suggesting a common mechanism. Here we show that the expression of caveolin-1 (Cav-1) is reduced in the brain in Type 2 diabetes mellitus. In turn, reduced Cav-1 levels induce AD-associated neuropathology and learning and memory deficits. Restoration of Cav-1 levels rescues these deficits. This study unravels signals underlying LOAD and suggests that restoration of Cav-1 may be an effective therapeutic target.

Human Hippocampal Neurogenesis Persists in Aged Adults and Alzheimer's Disease Patients.

Whether hippocampal neurogenesis persists throughout life in the human brain is not fully resolved. Here, we demonstrate that hippocampal neurogenesis is persistent through the tenth decade of life and is detectable in patients with mild cognitive impairments and Alzheimer's disease. In a cohort of 18 participants with a mean age of 90.6 years, Nestin+Sox2+ neural progenitor cells (NPCs) and DCX+ neuroblasts and immature neurons were detected, but their numbers greatly varied between participants. Nestin+ cells localize in the anterior hippocampus, and NPCs, neuroblasts, and immature neurons are evenly distributed along the anterior to posterior axis. The number of DCX+PCNA+ cells is reduced in mild cognitive impairments, and higher numbers of neuroblasts are associated with better cognitive status. The number of DCX+PCNA+ cells correlates with functional interactions between presynaptic SNARE proteins. Our results suggest that hippocampal neurogenesis persists in the aged and diseased human brain and that it is possibly associated with cognition.

Vascular dysfunction-The disregarded partner of Alzheimer's disease.

Increasing evidence recognizes Alzheimer's disease (AD) as a multifactorial and heterogeneous disease with multiple contributors to its pathophysiology, including vascular dysfunction. The recently updated AD Research Framework put forth by the National Institute on Aging-Alzheimer's Association describes a biomarker-based pathologic definition of AD focused on amyloid, tau, and neuronal injury. In response to this article, here we first discussed evidence that vascular dysfunction is an important early event in AD pathophysiology. Next, we examined various imaging sequences that could be easily implemented to evaluate different types of vascular dysfunction associated with, and/or contributing to, AD pathophysiology, including changes in blood-brain barrier integrity and cerebral blood flow. Vascular imaging biomarkers of small vessel disease of the brain, which is responsible for >50% of dementia worldwide, including AD, are already established, well characterized, and easy to recognize. We suggest that these vascular biomarkers should be incorporated into the AD Research Framework to gain a better understanding of AD pathophysiology and aid in treatment efforts.

CREB signals as PBMC-based biomarkers of cognitive dysfunction: A novel perspective of the brain-immune axis.

To date, there is no reliable biomarker for the assessment or determination of cognitive dysfunction in Alzheimer's disease and related dementia. Such a biomarker would not only aid in diagnostics, but could also serve as a measure of therapeutic efficacy. It is widely acknowledged that the hallmarks of Alzheimer's disease, namely, amyloid deposits and neurofibrillary tangles, as well as their precursors and metabolites, are poorly correlated with cognitive function and disease stage and thus have low diagnostic or prognostic value. A lack of biomarkers is one of the major roadblocks in diagnosing the disease and in assessing the efficacy of potential therapies. The phosphorylation of cAMP Response Element Binding protein (pCREB) plays a major role in memory acquisition and consolidation. In the brain, CREB activation by phosphorylation at Ser133 and the recruitment of transcription cofactors such as CREB binding protein (CBP) is a critical step for the formation of memory. This set of processes is a prerequisite for the transcription of genes thought to be important for synaptic plasticity, such as Egr-1. Interestingly, recent work suggests that the expression of pCREB in peripheral blood mononuclear cells (PBMC) positively correlates with pCREB expression in the postmortem brain of Alzheimer's patients, suggesting not only that pCREB expression in PBMC might serve as a biomarker of cognitive dysfunction, but also that the dysfunction of CREB signaling may not be limited to the brain in AD, and that a link may exist between the regulation of CREB in the blood and in the brain. In this review we consider the evidence suggesting a correlation between the level of CREB signals in the brain and blood, the current knowledge about CREB in PBMC and its association with CREB in the brain, and the implications and mechanisms for a neuro-immune cross talk that may underlie this communication. This Review will discuss the possibility that peripheral dysregulation of CREB is an early event in AD pathogenesis, perhaps as a facet of immune system dysfunction, and that this impairment in peripheral CREB signaling modifies CREB signaling in the brain, thus exacerbating cognitive decline in AD. A more thorough understanding of systemic dysregulation of CREB in AD will facilitate the search for a biomarker of cognitive function in AD, and also aid in the understanding of the mechanisms underlying cognitive decline in AD.

Phytochemicals from Achillea fragrantissima are Modulators of AßPP Metabolism.

Plant derivatives offer a novel and natural source of therapeutics. The desert plant Achillea fragrantissima (Forssk) Sch. Bip (Af) is characterized by protective antioxidative and anti-inflammatory properties. Here, we examined the effect of two Af-derived phytochemicals on learning and memory, amyloid-ß protein precursor (AßPP) metabolism, and tau phosphorylation in the familial Alzheimer's disease-linked APPswe/PS1ΔE9 mouse model. We observed that mice that were injected with the phytochemicals showed a trend of improvement, albeit statistically insignificant, in the Novel Object Recognition task. However, we did not observe improvement in contextual fear conditioning, suggesting that the benefits of treatment may be either indirect or task-specific. In addition, we observed an increase in the full-length form of AßPP in the brains of mice treated with Af-derived phytochemicals. Interestingly, both in vivo and in vitro, there was no change in levels of soluble Aß, oligomeric Aß, or the carboxyl terminus fragments of AßPP (APP-CTFs), suggesting that the increase in full length AßPP does not exacerbate AßPP pathology, but may stabilize the full-length form of the molecule. Together, our data suggest that phytochemicals present in Af may have a modest positive impact on the progression of Alzheimer's disease.

Reciprocal regulation of eNOS and caveolin-1 functions in endothelial cells.

We hypothesized that the maintenance of vascular homeostasis is critically dependent on the expression and reciprocal regulation of caveolin-1 (Cav-1) and endothelial nitric oxide synthase (eNOS) in endothelial cells (ECs). Skeletal muscle biopsies from subjects with type 2 diabetes showed 50% less Cav-1 and eNOS than those from lean healthy controls. The Cav-1:eNOS expression ratio was 200:1 in primary culture human ECs. Cav-1 small interfering RNA (siRNA) reduced eNOS protein and gene expression in association with a twofold increase in eNOS phosphorylation and nitrate production per molecule of eNOS, which was reversed in cells overexpressing Adv-Cav-1-GFP. Upon addition of the Ca2+ ionophore A23187 to activate eNOS, we observed eNOS Ser1177 phosphorylation, its translocation to ß-catenin-positive cell-cell junctions, and increased colocalization of eNOS and Cav-1 within 5 min. We also observed Cav-1 S-nitrosylation and destabilization of Cav-1 oligomers in cells treated with A23187 as well as insulin or albumin, and this could be blocked by L-NAME, PP2, or eNOS siRNA. Finally, caveola-mediated endocytosis of albumin or insulin was reduced by Cav-1 or eNOS siRNA, and the effect of Cav-1 siRNA was rescued by Adv-Cav-1-GFP. Thus, Cav-1 stabilizes eNOS expression and regulates its activity, whereas eNOS-derived NO promotes caveola-mediated endocytosis.