Vascular endothelial growth factor associated dissimilar cerebrovascular phenotypes in two different mouse models of Alzheimer's Disease.

Vascular perturbations and cerebral hypometabolism are emerging as important components of Alzheimer's disease (AD). While various in vivo imaging modalities have been designed to detect changes of cerebral perfusion and metabolism in AD patients and animal models, study results were often heterogenous with respect to imaging techniques and animal models. We therefore evaluated cerebral perfusion and glucose metabolism of two popular transgenic AD mouse strains, TgCRND8 and 5xFAD, at 7 and 12 months-of-age under identical conditions and analyzed possible molecular mechanisms underlying heterogeneous cerebrovascular phenotypes. Results revealed disparate findings in these two strains, displaying important aspects of AD progression. TgCRND8 mice showed significantly decreased cerebral blood flow and glucose metabolism with unchanged cerebral blood volume (CBV) at 12 months-of-age whereas 5xFAD mice showed unaltered glucose metabolism with significant increase in CBV at 12 months-of-age and a biphasic pattern of early hypoperfusion followed by a rebound to normal cerebral blood flow in late disease. Finally, immunoblotting assays suggested that VEGF dependent vascular tone change may restore normoperfusion and increase CBV in 5xFAD.

Cerebral amyloid angiopathy-linked ß-amyloid mutations promote cerebral fibrin deposits via increased binding affinity for fibrinogen.

Cerebral amyloid angiopathy (CAA), where beta-amyloid (Aß) deposits around cerebral blood vessels, is a major contributor of vascular dysfunction in Alzheimer's disease (AD) patients. However, the molecular mechanism underlying CAA formation and CAA-induced cerebrovascular pathology is unclear. Hereditary cerebral amyloid angiopathy (HCAA) is a rare familial form of CAA in which mutations within the (Aß) peptide cause an increase in vascular deposits. Since the interaction between Aß and fibrinogen increases CAA and plays an important role in cerebrovascular damage in AD, we investigated the role of the Aß-fibrinogen interaction in HCAA pathology. Our work revealed the most common forms of HCAA-linked mutations, Dutch (E22Q) and Iowa (D23N), resulted in up to a 50-fold stronger binding affinity of Aß for fibrinogen. In addition, the stronger interaction between fibrinogen and mutant Aßs led to a dramatic perturbation of clot structure and delayed fibrinolysis. Immunofluorescence analysis of the occipital cortex showed an increase of fibrin(ogen)/Aß codeposition, as well as fibrin deposits in HCAA patients, compared to early-onset AD patients and nondemented individuals. Our results suggest the HCAA-type Dutch and Iowa mutations increase the interaction between fibrinogen and Aß, which might be central to cerebrovascular pathologies observed in HCAA.

Astrocytic laminin regulates pericyte differentiation and maintains blood brain barrier integrity.

Blood brain barrier (BBB) breakdown is not only a consequence of but also contributes to many neurological disorders, including stroke and Alzheimer's disease. How the basement membrane (BM) contributes to the normal functioning of the BBB remains elusive. Here we use conditional knockout mice and an acute adenovirus-mediated knockdown model to show that lack of astrocytic laminin, a brain-specific BM component, induces BBB breakdown. Using functional blocking antibody and RNAi, we further demonstrate that astrocytic laminin, by binding to integrin α2 receptor, prevents pericyte differentiation from the BBB-stabilizing resting stage to the BBB-disrupting contractile stage, and thus maintains the integrity of BBB. Additionally, loss of astrocytic laminin decreases aquaporin-4 (AQP4) and tight junction protein expression. Altogether, we report a critical role for astrocytic laminin in BBB regulation and pericyte differentiation. These results indicate that astrocytic laminin maintains the integrity of BBB through, at least in part, regulation of pericyte differentiation.

Nox2-derived radicals contribute to neurovascular and behavioral dysfunction in mice overexpressing the amyloid precursor protein.

Alterations in cerebrovascular regulation related to vascular oxidative stress have been implicated in the mechanisms of Alzheimer's disease (AD), but their role in the amyloid deposition and cognitive impairment associated with AD remains unclear. We used mice overexpressing the Swedish mutation of the amyloid precursor protein (Tg2576) as a model of AD to examine the role of reactive oxygen species produced by NADPH oxidase in the cerebrovascular alterations, amyloid deposition, and behavioral deficits observed in these mice. We found that 12- to 15-month-old Tg2576 mice lacking the catalytic subunit Nox2 of NADPH oxidase do not develop oxidative stress, cerebrovascular dysfunction, or behavioral deficits. These improvements occurred without reductions in brain amyloid-beta peptide (Abeta) levels or amyloid plaques. The findings unveil a previously unrecognized role of Nox2-derived radicals in the behavioral deficits of Tg2576 mice and provide a link between the neurovascular dysfunction and cognitive decline associated with amyloid pathology.

Convergence of heat shock protein 90 with ubiquitin in filamentous alpha-synuclein inclusions of alpha-synucleinopathies.

Heat shock proteins (Hsps) facilitate refolding of denatured polypeptides, but there is limited understanding about their roles in neurodegenerative diseases characterized by misfolded proteins. Because Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy are alpha-synucleinopathies characterized by filamentous alpha-synuclein (alpha-syn) inclusions, we assessed which Hsps might be implicated in these disorders by examining human brain samples, transgenic mouse models, and cell culture systems. Light and electron microscopic multiple-label immunohistochemistry showed Hsp90 was the predominant Hsp examined that co-localized with alpha-syn in Lewy bodies, Lewy neurites, and glial cell inclusions and that Hsp90 co-localized with alpha-syn filaments of Lewy bodies in PD. Hsp90 levels were most predominantly increased in PD brains, which correlated with increased levels of insoluble alpha-syn. These alterations in Hsp90 were recapitulated in a transgenic mouse model of PD-like alpha-syn pathologies. Cell culture studies also revealed that alpha-syn co-immunoprecipitated preferentially with Hsp90 and Hsc70 relative to other Hsps, and exposure of cells to proteasome inhibitors resulted in increased levels of Hsp90. These data implicate predominantly Hsp90 in the formation of alpha-syn inclusions in PD and related alpha-synucleinopathies.

Reversible inhibition of alpha-synuclein fibrillization by dopaminochrome-mediated conformational alterations.

Previous studies demonstrated that alpha-synuclein (alpha-syn) fibrillization is inhibited by dopamine, and studies to understand the molecular basis of this process were conducted (Conway, K. A., Rochet, J. C., Bieganski, R. M., and Lansbury, P. T., Jr. (2001) Science 294, 1346-1349). Dopamine inhibition of alpha-syn fibrillization generated exclusively spherical oligomers that depended on dopamine autoxidation but not alpha-syn oxidation, because mutagenesis of Met, His, and Tyr residues in alpha-syn did not abrogate this inhibition. However, truncation of alpha-syn at residue 125 restored the ability of alpha-syn to fibrillize in the presence of dopamine. Mutagenesis and competition studies with specific synthetic peptides identified alpha-syn residues 125-129 (i.e. YEMPS) as an important region in the dopamine-induced inhibition of alpha-syn fibrillization. Significantly, the dopamine oxidation product dopaminochrome was identified as a specific inhibitor of alpha-syn fibrillization. Dopaminochrome promotes the formation of spherical oligomers by inducing conformational changes, as these oligomers regained the ability to fibrillize by simple denaturation/renaturation. Taken together, these data indicate that dopamine inhibits alpha-syn fibrillization by inducing structural changes in alpha-syn that can occur through the interaction of dopaminochrome with the 125YEMPS129 motif of alpha-syn. These results suggest that the dopamine autoxidation can prevent alpha-syn fibrillization in dopaminergic neurons through a novel mechanism. Thus, decreased dopamine levels in substantia nigra neurons might promote alpha-syn aggregation in Parkinson's disease.