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The PDMS-based microfluidic organ-on-chip platform represents an exciting paradigm that has enjoyed a rapid rise in popularity and adoption. A particularly promising element of this platform is its amenability to rapid manufacturing strategies, which can enable quick adaptations through iterative prototyping. These strategies, however, come with challenges; fluid flow, for example, a core principle of organs-on-chip and the physiology they aim to model, necessitates robust, leak-free channels for potentially long (multi-week) culture durations. In this report, we describe microfluidic chip fabrication methods and strategies that are aimed at overcoming these difficulties; we employ a subset of these strategies to a blood–brain-barrier-on-chip, with others applied to a small-airway-on-chip. Design approaches are detailed with considerations presented for readers. Results pertaining to fabrication parameters we aimed to improve (e.g., the thickness uniformity of molded PDMS), as well as illustrative results pertaining to the establishment of cell cultures using these methods will also be presented. [ABSTRACT FROM AUTHOR]

Background: Several lines of evidence suggest that high-density lipoprotein (HDL) reduces Alzheimer's disease (AD) risk by decreasing vascular beta-amyloid (Aβ) deposition and inflammation, however, the mechanisms by which HDL improve cerebrovascular functions relevant to AD remain poorly understood. Methods: Here we use a human bioengineered model of cerebral amyloid angiopathy (CAA) to define several mechanisms by which HDL reduces Aβ deposition within the vasculature and attenuates endothelial inflammation as measured by monocyte binding. Results: We demonstrate that HDL reduces vascular Aβ accumulation independently of its principal binding protein, scavenger receptor (SR)-BI, in contrast to the SR-BI-dependent mechanism by which HDL prevents Aβ-induced vascular inflammation. We describe multiple novel mechanisms by which HDL acts to reduce CAA, namely: i) altering Aβ binding to collagen-I, ii) forming a complex with Aβ that maintains its solubility, iii) lowering collagen-I protein levels produced by smooth-muscle cells (SMC), and iv) attenuating Aβ uptake into SMC that associates with reduced low density lipoprotein related protein 1 (LRP1) levels. Furthermore, we show that HDL particles enriched in apolipoprotein (apo)E appear to be the major drivers of these effects, providing new insights into the peripheral role of apoE in AD, in particular, the fraction of HDL that contains apoE. Conclusion: The findings in this study identify new mechanisms by which circulating HDL, particularly HDL particles enriched in apoE, may provide vascular resilience to Aβ and shed new light on a potential role of peripherally-acting apoE in AD. [ABSTRACT FROM AUTHOR]

Background: Genome wide association studies have identified microtubule associated protein tau (MAPT) H1 haplotype single nucleotide polymorphisms (SNPs) as leading common risk variants for Parkinson's disease, progressive supranuclear palsy and corticobasal degeneration. The MAPT risk variants fall within a large 1.8 Mb region of high linkage disequilibrium, making it difficult to discern the functionally important risk variants. Here, we leverage the strong haplotype-specific expression of MAPT exon 3 to investigate the functionality of SNPs that fall within this H1 haplotype region of linkage disequilibrium. Methods: In this study, we dissect the molecular mechanisms by which haplotype-specific SNPs confer allele-specific effects on the alternative splicing of MAPT exon 3. Firstly, we use haplotype-hybrid whole-locus genomic MAPT vectors studies to identify functional SNPs. Next, we characterise the RNA-protein interactions at two loci by mass spectrometry. Lastly, we knockdown candidate splice factors to determine their effect on MAPT exon 3 using a novel allele-specific qPCR assay. Results: Using whole-locus genomic DNA expression vectors to express MAPT haplotype variants, we demonstrate that rs17651213 regulates exon 3 inclusion in a haplotype-specific manner. We further investigated the functionality of this region using RNA-electrophoretic mobility shift assays to show differential RNA-protein complex formation at the H1 and H2 sequence variants of SNP rs17651213 and rs1800547 and subsequently identified candidate trans-acting splicing factors interacting with these functional SNPs sequences by RNA-protein pull-down experiment and mass spectrometry. Finally, gene knockdown of candidate splice factors identified by mass spectrometry demonstrate a role for hnRNP F and hnRNP Q in the haplotype-specific regulation of exon 3 inclusion. Conclusions: We identified common splice factors hnRNP F and hnRNP Q regulating the haplotype-specific splicing of MAPT exon 3 through intronic variants rs1800547 and rs17651213. This work demonstrates an integrated approach to characterise the functionality of risk variants in large regions of linkage disequilibrium. [ABSTRACT FROM AUTHOR]

Variants at the GBA locus, encoding glucocerebrosidase, are the strongest common genetic risk factor for Parkinson's disease (PD). To understand GBA -related disease mechanisms, we use a multi-part-enrichment proteomics and post-translational modification (PTM) workflow, identifying large numbers of dysregulated proteins and PTMs in heterozygous GBA - N370S PD patient induced pluripotent stem cell (iPSC) dopamine neurons. Alterations in glycosylation status show disturbances in the autophagy-lysosomal pathway, which concur with upstream perturbations in mammalian target of rapamycin (mTOR) activation in GBA -PD neurons. Several native and modified proteins encoded by PD-associated genes are dysregulated in GBA -PD neurons. Integrated pathway analysis reveals impaired neuritogenesis in GBA -PD neurons and identify tau as a key pathway mediator. Functional assays confirm neurite outgrowth deficits and identify impaired mitochondrial movement in GBA -PD neurons. Furthermore, pharmacological rescue of glucocerebrosidase activity in GBA -PD neurons improves the neurite outgrowth deficit. Overall, this study demonstrates the potential of PTMomics to elucidate neurodegeneration-associated pathways and potential drug targets in complex disease models. [Display omitted] • Proteomic/PTMomic analysis of GBA patient iPSC neurons identify disease phenotypes • PTM changes are highly abundant on lysosomal proteins and PD GWAS-related proteins • mTOR activation and mitochondrial movement perturbations are identified • GBA patient neurons show neuritogenesis defects that are rescued by a GCase chaperone Bogetofte et al. apply a post-translational proteomics pipeline to iPSC-dopaminergic neurons from patients with Parkinson's with a GBA-N370S mutation. This workflow simultaneously quantifies changes in protein phosphorylation, sialylation, and cysteine modifications, identifying affected pathways and phenotypes including impaired neurite outgrowth in GBA-N370S patient dopaminergic neurons that can be rescued by a GBA chaperone. [ABSTRACT FROM AUTHOR]

iPSCs (induced pluripotent stem cells) are the newest tool used to model PD (Parkinson's disease). Fibroblasts from patients carrying pathogenic mutations that lead to PD have been reprogrammed into iPSCs, which can subsequently be differentiated into important cell types. Given the characteristic loss of dopaminergic neurons in the substantia nigra pars compacta of PD patients, iPSC-derived midbrain dopaminergic neurons have been generated to investigate pathogenic mechanisms in this important cell type as a means of modelling PD. iPSC-derived cultures studied so far have been made from patients carryingmutations in LRRK2 (leucine-rich repeat kinase 2), PINK1 [PTEN (phosphatase and tensin homologue deleted on chromosome 10)-induced putative kinase 1], PARK2 (encodes parkin) or GBA (ß-glucocerebrosidase), in addition to those with SNCA (α-synuclein) multiplication and idiopathic PD. In some cases, isogenic control lines have been created to minimize inherent variability between lines from different individuals. Disruptions in autophagy, mitochondrial function and dopamine biology at the synapse have been described. Future applications for iPSC-derived models of PD beyond modelling include drug testing and the ability to investigate the genetic diversity of PD. [ABSTRACT FROM AUTHOR]

The microtubule-associated protein tau (MAPT or tau) is of great interest in the field of neurodegeneration as there is a well-established genetic link between the MAPT gene locus and tauopathies, a diverse group of neurodegenerative dementias and movement disorders. The genomic architecture in the region spanning the MAPT locus contains a ~1.8 Mb block of linkage disequilibrium characterized by two major haplotypes: H1 and H2. Recent studies have established strong genetic association between the MAPT locus and neurodegenerative disease and uncovered haplotype-specific differences in expression and alternative splicing of MAPT transcripts. Integrating genetic association data and gene expression data to understand how non-coding genetic variation at a gene locus affects gene expression and leads to susceptibility to disease is a high priority in disease genetics, and the MAPT locus provides an excellent paradigm for this. In the absence of protein-coding changes caused by haplotype sequence variation, altered levels of protein expression or altered ratios of isoform expression are excellent candidate mechanisms to link the MAPT genetic disease association with biological function. The use of novel transgenic and endogenous genetic models are required to understand the role of MAPT sequence variation in mechanisms of disease susceptibility. [ABSTRACT FROM AUTHOR]

Introduction: The neurovascular unit (NVU) – the interaction between the neurons and the cerebrovasculature – is increasingly important to interrogate through human-based experimental models. Although advanced models of cerebral capillaries have been developed in the last decade, there is currently no in vitro 3-dimensional (3D) perfusible model of the human cortical arterial NVU. Method: We used a tissue-engineering technique to develop a scaffold-directed, perfusible, 3D human NVU that is cultured in native-like flow conditions that mimics the anatomy and physiology of cortical penetrating arteries. Results: This system, composed of primary human vascular cells (endothelial cells, smooth muscle cells and astrocytes) and induced pluripotent stem cell (iPSC) derived neurons, demonstrates a physiological multilayer organization of the involved cell types. It reproduces key characteristics of cortical neurons and astrocytes and enables formation of a selective and functional endothelial barrier. We provide proof-of-principle data showing that this in vitro human arterial NVU may be suitable to study neurovascular components of neurodegenerative diseases such as Alzheimer's disease (AD), as endogenously produced phosphorylated tau and beta-amyloid accumulate in the model over time. Finally, neuronal and glial fluid biomarkers relevant to neurodegenerative diseases are measurable in our arterial NVU model. Conclusion: This model is a suitable research tool to investigate arterial NVU functions in healthy and disease states. Further, the design of the platform allows culture under native-like flow conditions for extended periods of time and yields sufficient tissue and media for downstream immunohistochemistry and biochemistry analyses. [ABSTRACT FROM AUTHOR]

We describe a human single-nuclei transcriptomic atlas for the substantia nigra (SN), generated by sequencing approximately 17,000 nuclei from matched cortical and SN samples. We show that the common genetic risk for Parkinson's disease (PD) is associated with dopaminergic neuron (DaN)-specific gene expression, including mitochondrial functioning, protein folding and ubiquitination pathways. We identify a distinct cell type association between PD risk and oligodendrocyte-specific gene expression. Unlike Alzheimer's disease (AD), we find no association between PD risk and microglia or astrocytes, suggesting that neuroinflammation plays a less causal role in PD than AD. Beyond PD, we find associations between SN DaNs and GABAergic neuron gene expression and multiple neuropsychiatric disorders. Conditional analysis reveals that distinct neuropsychiatric disorders associate with distinct sets of neuron-specific genes but converge onto shared loci within oligodendrocytes and oligodendrocyte precursors. This atlas guides our aetiological understanding by associating SN cell type expression profiles with specific disease risk. The substantia nigra is important in neurological disease, particularly movement disorders. Here the authors provide a single cell transcriptomic atlas for the human substantia nigra. [ABSTRACT FROM AUTHOR]