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Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is the most common genetic small vessel disease caused by variants in the gene. Patients with CADASIL experience recurrent strokes, developing into cognitive defect and vascular dementia. CADASIL is a late-onset vascular condition, but migraine and brain MRI lesions appear in CADASIL patients as early as their teens and twenties, suggesting an abnormal neurovascular interaction at the neurovascular unit (NVU) where microvessels meet the brain parenchyma. To understand the molecular mechanisms of CADASIL, we established induced pluripotent stem cell (iPSC) models from CADASIL patients and differentiated the iPSCs into the major NVU cell types including brain microvascular endothelial-like cells (BMECs), vascular mural cells (MCs), astrocytes and cortical projection neurons. We then built an NVU model by co-culturing different neurovascular cell types in Transwells and evaluated the blood brain barrier (BBB) function by measuring transendothelial electrical resistance (TEER). Results showed that, while the wild-type MCs, astrocytes and neurons could all independently and significantly enhance TEER of the iPSC-BMECs, such capability of MCs from iPSCs of CADASIL patients was significantly impaired. Additionally, the barrier function of the BMECs from CADASIL iPSCs was significantly decreased, accompanied with disorganized tight junctions in iPSC-BMECs, which could not be rescued by the wild-type MCs or sufficiently rescued by the wild-type astrocytes and neurons. Our findings provide new insight into early disease pathologies on the neurovascular interaction and BBB function at the molecular and cellular levels for CADASIL, which helps inform future therapeutic development.

Several studies of Parkinson's disease (PD) have reported dysregulation of cerebral metals, particularly decreases in copper and increases in iron in substantia nigra (SN). However, few studies have investigated regions outside the SN, fewer have measured levels of multiple metals across different regions within the same brains, and there are no currently-available reports of metal levels in Parkinson's disease dementia (PDD). This study aimed to compare concentrations of nine essential metals across nine different brain regions in cases of PDD and controls. Investigated were: primary motor cortex (MCX); cingulate gyrus (CG); primary visual cortex (PVC); hippocampus (HP); cerebellar cortex (CB); SN; locus coeruleus (LC); medulla oblongata (MED); and middle temporal gyrus (MTG), thus covering regions with severe, moderate, or low levels of neuronal loss in PDD. Levels of eight essential metals and selenium were determined using an analytical methodology involving the use of inductively-coupled plasma mass spectrometry (ICP-MS), and compared between cases and controls, to better understand the extent and severity of metal perturbations. Findings were also compared with those from our previous study of sporadic Alzheimer's disease dementia (ADD), which employed equivalent methods, to identify differences and similarities between these conditions. Widespread copper decreases occurred in PDD in seven of nine regions (exceptions being LC and CB). Four PDD-affected regions showed similar decreases in ADD: CG, HP, MTG, and MCX. Decreases in potassium and manganese were present in HP, MTG and MCX; decreased manganese was also found in SN and MED. Decreased selenium and magnesium were present in MCX, and decreased zinc in HP. There was no evidence for increased iron in SN or any other region. These results identify alterations in levels of several metals across multiple regions of PDD brain, the commonest being widespread decreases in copper that closely resemble those in ADD, pointing to similar disease mechanisms in both dementias.

The cellular prion protein (PrPC) is a ubiquitously expressed protein of currently unresolved but potentially diverse function. Of putative relevance to normal biological activity, PrPC is recognized to undergo both α- and β-endoproteolysis, producing the cleavage fragment pairs N1/C1 and N2/C2, respectively. Experimental evidence suggests the likelihood that these processing events serve differing cellular needs. Through the engineering of a C-terminal c-myc tag onto murine PrPC, as well as the selective use of a far-C-terminal anti-PrP antibody, we have identified a new PrPC fragment, nominally ‘C3’, and elaborating existing nomenclature, ‘γ-cleavage’ as the responsible proteolysis. Our studies indicate that this novel γ-cleavage event can occur during transit through the secretory pathway after exiting the endoplasmic reticulum, and after PrPC has reached the cell surface, by a matrix metalloprotease. We found that C3 is GPI-anchored like other C-terminal and full length PrPC species, though it does not localize primarily at the cell surface, and is preferentially cleaved from an unglycosylated substrate. Importantly, we observed that C3 exists in diverse cell types as well as mouse and human brain tissue, and of possible pathogenic significance, γ-cleavage may increase in human prion diseases. Given the likely relevance of PrPC processing to both its normal function, and susceptibility to prion disease, the potential importance of this previously underappreciated and overlooked cleavage event warrants further consideration.

Widespread elevations in brain urea have, in recent years, been reported in certain types of age-related dementia, notably Alzheimer’s disease (AD) and Huntington’s disease (HD). Urea increases in these diseases are substantive, and approximate in magnitude to levels present in uraemic encephalopathy. In AD and HD, elevated urea levels are widespread, and not only in regions heavily affected by neurodegeneration. However, measurements of brain urea have not hitherto been reported in Parkinson’s disease dementia (PDD), a condition which shares neuropathological and symptomatic overlap with both AD and HD. Here we report measurements of tissue urea from nine neuropathologically confirmed regions of the brain in PDD and post-mortem delay (PMD)-matched controls, in regions including the cerebellum, motor cortex (MCX), sensory cortex, hippocampus (HP), substantia nigra (SN), middle temporal gyrus (MTG), medulla oblongata (MED), cingulate gyrus, and pons, by applying ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Urea concentrations were found to be substantively elevated in all nine regions, with average increases of 3–4-fold. Urea concentrations were remarkably consistent across regions in both cases and controls, with no clear distinction between regions heavily affected or less severely affected by neuronal loss in PDD. These urea elevations mirror those found in uraemic encephalopathy, where equivalent levels are generally considered to be pathogenic, and those previously reported in AD and HD. Increased urea is a widespread metabolic perturbation in brain metabolism common to PDD, AD, and HD, at levels equal to those seen in uremic encephalopathy. This presents a novel pathogenic mechanism in PDD, which is shared with two other neurodegenerative diseases.

Proteolytic processing of the amyloid precursor protein (APP) by β-secretase, β-site APP cleaving enzyme (BACE1), is the initial step in the production of the amyloid β (Aβ) peptide, which is involved in the pathogenesis of Alzheimer's disease. The normal cellular function of the prion protein (PrPC), the causative agent of the transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease in humans, remains enigmatic. Because both APP and PrPC are subject to proteolytic processing by the same zinc metalloproteases, we tested the involvement of PrPC in the proteolytic processing of APP. Cellular overexpression of PrPC inhibited the β-secretase cleavage of APP and reduced Aβ formation. Conversely, depletion of PrPC in mouse N2a cells by siRNA led to an increase in Aβ peptides secreted into the medium. In the brains of PrP knockout mice and in the brains from two strains of scrapie-infected mice, Aβ levels were significantly increased. Two mutants of PrP, PG14 and A116V, that are associated with familial human prion diseases failed to inhibit the β-secretase cleavage of APP. Using constructs of PrP, we show that this regulatory effect of PrPC on the β-secretase cleavage of APP required the localization of PrPC to cholesterol-rich lipid rafts and was mediated by the N-terminal polybasic region of PrPC via interaction with glycosaminoglycans. In conclusion, this is a mechanism by which the cellular production of the neurotoxic Aβ is regulated by PrPC and may have implications for both Alzheimer's and prion diseases.

Bridging integrator 1 (BIN1) has been implicated in sporadic Alzheimer's disease (AD) by a number of genome wide association studies (GWAS) in a variety of populations. Here we measured BIN1 in frontal cortex samples from 24 sporadic AD and 24 age-matched non-dementia brains and correlated the expression of this protein with markers of AD. BIN1 was reduced by 87% (p=0.007) in sporadic AD compared to non-dementia controls, but BIN1 in sporadic AD did not correlate with soluble Aβ (r(s)=-0.084, p=0.698), insoluble Aβ (r(s)=0.237, p=0.269), Aβ plaque load (r(s)=0.063, p=0.771) or phospho-tau load (r(s)=-0.160, p=0.489). In contrast to our findings in sporadic AD, BIN1 was unchanged in the hippocampus from 6 cases of familial AD compared to 6 age-matched controls (p=0.488). BIN1 declined with age in a cohort of non-dementia control cases between 25 and 88 years but the correlation was not significant (rs=-0.449, p=0.081). Although BIN1 is known to have a role in endocytosis, and the processing of the amyloid precursor protein (APP) to form amyloid-β (Aβ) peptides is dependent on endocytosis, knockdown of BIN1 by targeted siRNA or the overexpression of BIN1 in a human neuroblastoma cell line (SH-SY5Y) had no effect on APP processing. These data suggest that the alteration in BIN1 is involved in the pathogenesis of sporadic, but not familial AD and is not a consequence of AD neurodegeneration or the ageing process, a finding in keeping with the numerous GWAS that implicate BIN1 in sporadic AD. However, the mechanism of its contribution remains to be established.

Pantothenic acid (vitamin B5) is an essential trace nutrient required for the synthesis of coenzyme A (CoA). It has previously been shown that pantothenic acid is significantly decreased in multiple brain regions in both Alzheimer’s disease (ADD) and Huntington’s disease (HD). The current investigation aimed to determine whether similar changes are also present in cases of Parkinson’s disease dementia (PDD), another age-related neurodegenerative condition, and whether such perturbations might occur in similar regions in these apparently different diseases. Brain tissue was obtained from nine confirmed cases of PDD and nine controls with a post-mortem delay of 26 h or less. Tissues were acquired from nine regions that show high, moderate, or low levels of neurodegeneration in PDD: the cerebellum, motor cortex, primary visual cortex, hippocampus, substantia nigra, middle temporal gyrus, medulla oblongata, cingulate gyrus, and pons. A targeted ultra–high performance liquid chromatography—tandem mass spectrometry (UHPLC-MS/MS) approach was used to quantify pantothenic acid in these tissues. Pantothenic acid was significantly decreased in the cerebellum (p = 0.008), substantia nigra (p = 0.02), and medulla (p = 0.008) of PDD cases. These findings mirror the significant decreases in the cerebellum of both ADD and HD cases, as well as the substantia nigra, putamen, middle frontal gyrus, and entorhinal cortex of HD cases, and motor cortex, primary visual cortex, hippocampus, middle temporal gyrus, cingulate gyrus, and entorhinal cortex of ADD cases. Taken together, these observations indicate a common but regionally selective disruption of pantothenic acid levels across PDD, ADD, and HD.

In prion diseases, the cellular form of the prion protein, PrP(C), undergoes a conformational conversion to the infectious isoform, PrP(Sc). PrP(C) associates with lipid rafts through its glycosyl-phosphatidylinositol (GPI) anchor and a region in its N-terminal domain which also binds to heparan sulfate proteoglycans (HSPGs). We show that heparin displaces PrP(C) from rafts and promotes its endocytosis, suggesting that heparin competes with an endogenous raft-resident HSPG for binding to PrP(C). We then utilised a transmembrane-anchored form of PrP (PrP-TM), which is targeted to rafts solely by its N-terminal domain, to show that both heparin and phosphatidylinositol-specific phospholipase C can inhibit its association with detergent-resistant rafts, implying that a GPI-anchored HSPG targets PrP(C) to rafts. Depletion of the major neuronal GPI-anchored HSPG, glypican-1, significantly reduced the raft association of PrP-TM and displaced PrP(C) from rafts, promoting its endocytosis. Glypican-1 and PrP(C) colocalised on the cell surface and both PrP(C) and PrP(Sc) co-immunoprecipitated with glypican-1. Critically, treatment of scrapie-infected N2a cells with glypican-1 siRNA significantly reduced PrP(Sc) formation. In contrast, depletion of glypican-1 did not alter the inhibitory effect of PrP(C) on the beta-secretase cleavage of the Alzheimer's amyloid precursor protein. These data indicate that glypican-1 is a novel cellular cofactor for prion conversion and we propose that it acts as a scaffold facilitating the interaction of PrP(C) and PrP(Sc) in lipid rafts.

β-Secretase (BACE, Asp-2) is a transmembrane aspartic proteinase responsible for cleaving the amyloid precursor protein (APP) to generate the soluble ectodomain sAPPβ and its C-terminal fragment$CTF\\beta.\\>CTF\\beta$is subsequently cleaved by β-secretase to produce the neurotoxic/synaptotoxic amyloid-β peptide (Aβ) that accumulates in Alzheimer's disease. Indirect evidence has suggested that amyloidogenic APP processing may preferentially occur in lipid rafts. Here, we show that relatively little wild-type BACE is found in rafts prepared from a human neuroblastoma cell line (SH-SY5Y) by using Triton X-100 as detergent. To investigate further the significance of lipid rafts in APP processing, a glycosylphosphatidylinositol (GPI) anchor has been added to BACE, replacing the transmembrane and C-terminal domains. The GPI anchor targets the enzyme exclusively to lipid raft domains. Expression of GPI-BACE substantially up-regulates the secretion of both sAPPβ and amyloid-β peptide over levels observed from cells overexpressing wild-type BACE. This effect was reversed when the lipid rafts were disrupted by depleting cellular cholesterol levels. These results suggest that processing of APP to the amyloid-β peptide occurs predominantly in lipid rafts and that BACE is the rate-limiting enzyme in this process. The processing of the APP695isoform by GPI-BACE was up-regulated 20-fold compared with wild-type BACE, whereas only a 2-fold increase in the processing of APP751/770was seen, implying a differential compartmentation of the APP isoforms. Changes in the local membrane environment during aging may facilitate the cosegregation of APP and BACE leading to increased β-amyloid production.

Neurovascular dysfunction is a central process in the pathogenesis of stroke and most neurodegenerative diseases, including Alzheimer’s disease. The multicellular neurovascular unit (NVU) combines the neural, vascular and extracellular matrix (ECM) components in an important interface whose correct functioning is critical to maintain brain health. Tissue engineering is now offering new tools and insights to advance our understanding of NVU function. Here, we review how the use of novel biomaterials to mimic the mechanical and functional cues of the ECM, coupled with precisely layered deposition of the different cells of the NVU through 3D bioprinting, is revolutionising the study of neurovascular function and dysfunction. Advances in 3D biomaterials known as hydrogels, with the structural properties of a solid and the capability to contain a high water content (∼99%), allow for 3D culture of NVU cells. Development of hydrogels with bioprintable properties, referred to as bioinks, allows for deposition of layered 3D NVU models. Biofabrication strategies utilising bioprinting and bioassembly can be used to develop 3D NVU models. NVU models could incorporate channels seeded with vascular cells to mimic the BBB. Attachment of fluidic pumps to a vascular channel can aid functionalisation of endothelial cells and the BBB as a whole.