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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: 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]

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]

Abstract: The microtubule-associated protein tau (MAPT) H1 haplotype shows a strong association to the sporadic neurodegenerative diseases, progressive supranuclear palsy and corticobasal degeneration. The functional biological mechanisms behind the genetic association have started to emerge with differences recently shown in haplotype splicing of the neuropathologically relevant exon 10. Here we investigate the hypothesis that expression of the alternatively spliced N-terminal exons also differs between the two MAPT haplotypes. We performed allele-specific gene expression analysis on a H1/H2 heterozygous human neuronal cell line model and 14 H1/H2 heterozygous human post-mortem brain tissues from two brain regions. In both cell culture and post-mortem brain tissue, we show that the protective MAPT H2 haplotype significantly expresses two-fold more 2N (exons 2+3+) MAPT transcripts than the disease-associated H1 haplotype. We suggest that inclusion of exon 3 in MAPT transcripts may contribute to protecting H2 carries from neurodegeneration. [Copyright &y& Elsevier]

Background: Amyloid‐b (Ab) and tau aggregates are two major pathological hallmarks and known to be dysregulated in Alzheimer's disease (AD), leading to widespread aggregation. We now know from tau‐deficient animal models that tau does not merely facilitate Ab‐induced toxicity but is also essential in that particular toxic cascade. However, a tau‐deficient human in vitro model has not been available to corroborate these studies. Method: Here we generated the first MAPT‐/‐ human induced pluripotent stem cell (iPSC) lines with CRISPR‐Cas9‐mediated genome editing in order to study the effects of tau deficiency in human biological context. We also established a versatile and scalable cortical neuron differentiation protocol which successfully produced a heterogeneous population of functional neurons manifesting cortical identity in co‐culture with rat astrocytes. Furthermore, we extracted brain homogenate from an AD patient to serve as the source of extrinsic Ab in addition to synthetic Ab oligomers. Result: iPSC‐derived MAPT‐/‐ cortical neurons exhibited lower firing amplitude and frequency compared to MAPT+/+ neurons at baseline, while expressing similar number of synapses. On the other hand, MAPT‐/‐ neurons demonstrated impaired axonal outgrowth over 5 days of live imaging. Upon extrinsic AD brain homogenate and/or Ab1‐42 oligomer insults, MAPT‐/‐ neurons appeared to be protected from axonal degeneration, cytotoxicity and hyperactivation as compared to MAPT+/+ neurons. However, the MAPT‐/‐background was unable to prevent Ab‐induced loss of synapses. Conclusion: Taken together, the absence of tau in human neurons resulted in phenotypic changes at baseline and could result in neuroprotection in MAPT‐/‐ neurons from Ab‐induced toxicity. [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]