The Role of Basement Membranes in Cerebral Amyloid Angiopathy.

Dementia is a neuropsychiatric syndrome characterized by cognitive decline in multiple domains, often leading to functional impairment in activities of daily living, disability, and death. The most common causes of age-related progressive dementia include Alzheimer's disease (AD) and vascular cognitive impairment (VCI), however, mixed disease pathologies commonly occur, as epitomized by a type of small vessel pathology called cerebral amyloid angiopathy (CAA). In CAA patients, the small vessels of the brain become hardened and vulnerable to rupture, leading to impaired neurovascular coupling, multiple microhemorrhage, microinfarction, neurological emergencies, and cognitive decline across multiple functional domains. While the pathogenesis of CAA is not well understood, it has long been thought to be initiated in thickened basement membrane (BM) segments, which contain abnormal protein deposits and amyloid-ß (Aß). Recent advances in our understanding of CAA pathogenesis link BM remodeling to functional impairment of perivascular transport pathways that are key to removing Aß from the brain. Dysregulation of this process may drive CAA pathogenesis and provides an important link between vascular risk factors and disease phenotype. The present review summarizes how the structure and composition of the BM allows for perivascular transport pathways to operate in the healthy brain, and then outlines multiple mechanisms by which specific dementia risk factors may promote dysfunction of perivascular transport pathways and increase Aß deposition during CAA pathogenesis. A better understanding of how BM remodeling alters perivascular transport could lead to novel diagnostic and therapeutic strategies for CAA patients.

The ubiquitin ligase UBE4B regulates amyloid precursor protein ubiquitination, endosomal trafficking, and amyloid ß42 generation and secretion.

The extracellular accumulation of amyloid ß (Aß) fragments of amyloid precursor protein (APP) in brain parenchyma is a pathological hallmark of Alzheimer's disease (AD). APP can be cleaved into Aß on late endosomes/multivesicular bodies (MVBs). E3 ubiquitin ligases have been linked to Aß production, but specific E3 ligases associated with APP ubiquitination that may affect targeting of APP to endosomes have not yet been described. Using cultured cortical neurons isolated from rat pups, we reconstituted APP movement into the internal vesicles (ILVs) of MVBs. Loss of endosomal sorting complexes required for transport (ESCRT) components inhibited APP movement into ILVs and increased endosomal Aß42 generation, implying a requirement for APP ubiquitination. We identified an ESCRT-binding and APP-interacting endosomal E3 ubiquitin ligase, ubiquitination factor E4B (UBE4B) that regulates APP ubiquitination. Depleting UBE4B in neurons inhibited APP ubiquitination and internalization into MVBs, resulting in increased endosomal Aß42 levels and increased neuronal secretion of Aß42. When we examined AD brains, we found levels of the UBE4B-interacting ESCRT component, hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), were significantly decreased in AD brains. These data suggest that ESCRT components critical for membrane protein sorting in the endocytic pathway are altered in AD. These results indicate that the molecular machinery underlying endosomal trafficking of APP, including the ubiquitin ligase UBE4B, regulates Aß levels and may play an essential role in AD progression.

Transforming growth factor-ß promotes basement membrane fibrosis, alters perivascular cerebrospinal fluid distribution, and worsens neurological recovery in the aged brain after stroke.

Aging and stroke alter the composition of the basement membrane and reduce the perivascular distribution of cerebrospinal fluid and solutes, which may contribute to poor functional recovery in elderly patients. Following stroke, TGF-ß induces astrocyte activation and subsequent glial scar development. This is dysregulated with aging and could lead to chronic, detrimental changes within the basement membrane. We hypothesized that TGF-ß induces basement membrane fibrosis after stroke, leading to impaired perivascular CSF distribution and poor functional recovery in aged animals. We found that CSF entered the aged brain along perivascular tracts; this process was reduced by experimental stroke and was rescued by TGF-ß receptor inhibition. Brain fibronectin levels increased with experimental stroke, which was reversed with inhibitor treatment. Exogenous TGF-ß stimulation increased fibronectin expression, both in vivo and in primary cultured astrocytes. Oxygen-glucose deprivation of cultured astrocytes induced multiple changes in genes related to astrocyte activation and extracellular matrix production. Finally, in stroke patients, we found that serum TGF-ß levels correlated with poorer functional outcomes, suggesting that serum levels may act as a biomarker for functional recovery. These results support a potential new treatment strategy to enhance recovery in elderly stroke patients.

Sex-specific Association of Matrix Metalloproteinases with Secondary Injury and Outcomes after Intracerebral Hemorrhage.

OBJECTIVE: Intracerebral hemorrhage affects approximately 2 million individuals per year. While the incidence is roughly equal in men and women, few studies have examined the influence of sex on secondary injury and associated long-term functional outcomes. Matrix metalloproteinases (MMPs) promote vessel rupture and worsen outcomes by potentiating blood-brain barrier breakdown after injury. We hypothesized that different MMP isoform levels would be predictive of injury severity, secondary injury, and long-term functional outcomes in males and females, respectively. METHODS: We examined the levels of MMP isoforms in serum samples from a prospective patient biobank (n = 55). Baseline clinical, radiographic, and laboratory data were also analyzed. RESULTS: We found that MMP-1 (P = .036), MMP-2 (P = .014), MMP-3 (P < .001), and MMP-9 (P = .02) levels gradually increased over time in male patients until 10 DPI. In female patients, we found a different pattern of activation: MMP-8 (P = .02) was the only isoform that significantly changed with time, which reached a peak at 3-5 days postinjury. Several MMP isoforms correlated with markers of secondary injury in female patients (all P < .05). Additionally, serum levels of MMP-3 (P = .011) in males and MMP-10 (P = .044) in females were significantly associated with long-term functional outcomes in a sex-specific manner. CONCLUSIONS: This is the first sex-specific study to examine serum MMP levels and their correlation with clinicoradiologic measures after intracerebral hemorrhage, and identifies potential biomarkers of secondary injury and long-term outcomes in both sexes.

Inhibition of calcium/calmodulin-dependent protein kinase kinase (CaMKK) exacerbates impairment of endothelial cell and blood-brain barrier after stroke.

Brain microvascular endothelial cells play an essential role in maintaining blood-brain barrier (BBB) integrity, and disruption of the BBB aggravates the ischemic injury. CaMKK (α and ß) is a major kinase activated by elevated intracellular calcium. Previously, we demonstrated that inhibition of CaMKK exacerbated outcomes, conversely, overexpression reduced brain injury after stroke in mice. Interestingly, CaMKK has been shown to activate a key endothelial protector, sirtuin 1 (SIRT1). We hypothesized that CaMKK protects brain endothelial cells via SIRT1 activation after stroke. In this study, Oxygen-Glucose Deprivation (OGD) was performed in human brain microvascular endothelial cells. Stroke was induced by middle cerebral artery occlusion (MCAO) in male mice. Knockdown of CaMKK ß using siRNA increased cell death following OGD. Inhibition of CaMKK ß by STO-609 significantly and selectively down-regulated levels of phosphorylated SIRT1 after OGD. Changes in the downstream targets of SIRT1 were observed following STO-609 treatment. The effect of STO-609 on cell viability after OGD was absent, when SIRT1 was concurrently inhibited. We also demonstrated that STO-609 increased endothelial expression of the pro-inflammatory proteins ICAM-1 and VCAM-1 and inhibition of CaMKK exacerbated OGD-induced leukocyte-endothelial adhesion. Finally, intracerebroventricular injection of STO-609 exacerbated endothelial apoptosis and reduced BBB integrity after 24-hr reperfusion following MCAO in vivo. Collectively, these results demonstrated that CaMKK inhibition reduced endothelial cell viability, exacerbated inflammatory responses and aggravated BBB impairment after ischemia. CaMKK activation may attenuate ischemic brain injury via protection of the microvascular system and a reduction in the infiltration of pro-inflammatory factors.

Serum Markers of Blood-Brain Barrier Remodeling and Fibrosis as Predictors of Etiology and Clinicoradiologic Outcome in Intracerebral Hemorrhage.

Background: Intracerebral hemorrhage (ICH) is a stroke subtype associated with high disability and mortality. There is a clinical need for blood-based biomarkers that can aid in diagnosis, risk stratification, and prognostication. Given their role in the pathophysiology of ICH, we hypothesized markers of blood-brain barrier disruption and fibrosis would associate with neurologic deterioration and/or long-term functional outcomes. We also hypothesized these markers may be unique in patients with ICH due to cerebral amyloid angiopathy (CAA) vs. other etiologies. Methods: Seventy-nine patients enrolled in prospective ICH registries at two separate hospitals (the University of Texas Health Science Center at Houston and Hartford Hospital) were included in this study. We assessed initial injury severity and admission variables along with measures of inpatient deterioration (hematoma expansion, perihematomal edema (PHE), and early and delayed neurologic deterioration) and functional outcome [modified Rankin Scale (mRS) score at discharge and 90 days]. Serial biospecimens were obtained at 5 pre-specified timepoints (within 24 h, 1-2, 3-5, 6-8, and 10 days); serum samples were analyzed for fibronectin, all three TGF-ß isoforms, and 7 matrix metalloproteinases (MMPs). Results: In our initial correlation analysis, MMP 10 and 3 were associated with hematoma expansion and early neurologic deterioration, whereas MMP 8 and MMP 1 were associated with PHE and delayed neurologic deterioration (respectively). Subacute levels of MMP 8 (sampled from day 6-10) positively correlated with PHE even after adjusting for multiple comparisons (p = 0.02). Acute levels of MMP 1, TGF-ß1, and TGF-ß3 were predictive of functional outcome, with TGF-ß1 and TGF-ß3 associating with 90 day mRS independent of age, hematoma volume, hemorrhage location, GCS, and IVH [p = 0.02; OR 1.03 (95% CI 1.0-1.05); p = 0.03; OR 3.1 (95% CI 1.1-8.8)]. When evaluated together as a panel, the cytokines distinguished patients with ICH due to CAA vs. ICH due to hypertension (AUC 0.81). Conclusions: Serum levels of fibronectin, TGF-ß, and MMPs may be useful in refining ICH etiology and prognosis. Further large-scale studies are needed to confirm these findings, particularly regarding patients with CAA.

Fibronectin induces the perivascular deposition of cerebrospinal fluid-derived amyloid-ß in aging and after stroke.

Cerebral amyloid angiopathy occurs after stroke, but the mechanism underlying the initial amyloid-ß deposition is not fully understood. This study investigates whether overexpression of fibronectin and its receptor, integrin-α5, induces the perivascular deposition of cerebrospinal fluid-derived amyloid-ß after stroke in young and aged animals. We found that stroke impaired the bulk flow of cerebrospinal fluid into the brain parenchyma and further showed that perivascular amyloid-ß deposition was enhanced in aged animals with stroke, which colocalized with integrin-α5 in the basement membrane. Furthermore, we found that stroke dramatically increased the cortical levels of fibronectin and integrin-α5, with further increases in integrin-α5 in aged animals with stroke, fibronectin bound amyloid-ß in vitro, and fibronectin administration increased amyloid-ß deposition in vivo. Finally, aging and stroke impaired performance on the Barnes maze. These results indicate that fibronectin induces the perivascular deposition of amyloid-ß and that increased integrin-α5 further "primes" the aged brain for amyloid-ß binding. This provides a novel molecular and physiological mechanism for perivascular amyloid-ß deposition after stroke, particularly in aged individuals.

Prevention and management of stroke in women.

Stroke is the leading cause of acquired disability and the third leading cause of death in women worldwide. Sex differences in risk factors, treatment response and quality of life after stroke complicate stroke management in women. Women have an increased lifetime incidence of stroke compared to men, largely due to a sharp increase in stroke risk in older postmenopausal women. Women also have an increased lifetime prevalence of stroke risk factors, including hypertension and atrial fibrillation in postmenopausal women, as well as abdominal obesity and metabolic syndrome in middle-aged women. Controversy continues over the risks of oral contraceptives, hormone therapy and surgical intervention for carotid stenosis in women. Pregnancy and the postpartum period represent a time of increased risk, presenting challenges to stroke management. Recognition of these issues is critical to improving acute care and functional recovery after stroke in women.

Hippocampal theta, gamma, and theta-gamma coupling: effects of aging, environmental change, and cholinergic activation.

Hippocampal theta and gamma oscillations coordinate the timing of multiple inputs to hippocampal neurons and have been linked to information processing and the dynamics of encoding and retrieval. One major influence on hippocampal rhythmicity is from cholinergic afferents. In both humans and rodents, aging is linked to impairments in hippocampus-dependent function along with degradation of cholinergic function. Cholinomimetics can reverse some age-related memory impairments and modulate oscillations in the hippocampus. Therefore, one would expect corresponding changes in these oscillations and possible rescue with the cholinomimetic physostigmine. Hippocampal activity was recorded while animals explored a familiar or a novel maze configuration. Reexposure to a familiar situation resulted in minimal aging effects or changes in theta or gamma oscillations. In contrast, exploration of a novel maze configuration increased theta power; this was greater in adult than old animals, although the deficit was reversed with physostigmine. In contrast to the theta results, the effects of novelty, age, and/or physostigmine on gamma were relatively weak. Unrelated to the behavioral situation were an age-related decrease in the degree of theta-gamma coupling and the fact that physostigmine lowered the frequency of theta in both adult and old animals. The results indicate that age-related changes in gamma and theta modulation of gamma, while reflecting aging changes in hippocampal circuitry, seem less related to aging changes in information processing. In contrast, the data support a role for theta and the cholinergic system in encoding and that hippocampal aging is related to impaired encoding of new information.