Tau depletion in human neurons mitigates Aß-driven toxicity.

Alzheimer's disease (AD) is an age-related neurodegenerative condition and the most common type of dementia, characterised by pathological accumulation of extracellular plaques and intracellular neurofibrillary tangles that mainly consist of amyloid-ß (Aß) and hyperphosphorylated tau aggregates, respectively. Previous studies in mouse models with a targeted knock-out of the microtubule-associated protein tau (Mapt) gene demonstrated that Aß-driven toxicity is tau-dependent. However, human cellular models with chronic tau lowering remain unexplored. In this study, we generated stable tau-depleted human induced pluripotent stem cell (iPSC) isogenic panels from two healthy individuals using CRISPR-Cas9 technology. We then differentiated these iPSCs into cortical neurons in vitro in co-culture with primary rat cortical astrocytes before conducting electrophysiological and imaging experiments for a wide range of disease-relevant phenotypes. Both AD brain derived and recombinant Aß were used in this study to elicit toxic responses from the iPSC-derived cortical neurons. We showed that tau depletion in human iPSC-derived cortical neurons caused considerable reductions in neuronal activity without affecting synaptic density. We also observed neurite outgrowth impairments in two of the tau-depleted lines used. Finally, tau depletion protected neurons from adverse effects by mitigating the impact of exogenous Aß-induced hyperactivity, deficits in retrograde axonal transport of mitochondria, and neurodegeneration. Our study established stable human iPSC isogenic panels with chronic tau depletion from two healthy individuals. Cortical neurons derived from these iPSC lines showed that tau is essential in Aß-driven hyperactivity, axonal transport deficits, and neurodegeneration, consistent with studies conducted in Mapt-/- mouse models. These findings highlight the protective effects of chronic tau lowering strategies in AD pathogenesis and reinforce the potential in clinical settings. The tau-depleted human iPSC models can now be applied at scale to investigate the involvement of tau in disease-relevant pathways and cell types.

Post-translational proteomics platform identifies neurite outgrowth impairments in Parkinson's disease GBA-N370S dopamine neurons.

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.

PDMS Organ-On-Chip Design and Fabrication: Strategies for Improving Fluidic Integration and Chip Robustness of Rapidly Prototyped Microfluidic In Vitro Models.

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.

Toward three-dimensional in vitro models to study neurovascular unit functions in health and disease.

The high metabolic demands of the brain require an efficient vascular system to be coupled with neural activity to supply adequate nutrients and oxygen. This supply is coordinated by the action of neurons, glial and vascular cells, known collectively as the neurovascular unit, which temporally and spatially regulate local cerebral blood flow through a process known as neurovascular coupling. In many neurodegenerative diseases, changes in functions of the neurovascular unit not only impair neurovascular coupling but also permeability of the blood-brain barrier, cerebral blood flow and clearance of waste from the brain. In order to study disease mechanisms, we need improved physiologically-relevant human models of the neurovascular unit. Advances towards modeling the cellular complexity of the neurovascular unit in vitro have been made using stem-cell derived organoids and more recently, vascularized organoids, enabling intricate studies of non-cell autonomous processes. Engineering and design innovations in microfluidic devices and tissue engineering are progressing our ability to interrogate the cerebrovasculature. These advanced models are being used to gain a better understanding of neurodegenerative disease processes and potential therapeutics. Continued innovation is required to build more physiologically-relevant models of the neurovascular unit encompassing both the cellular complexity and designed features to interrogate neurovascular unit functionality.

An in vitro bioengineered model of the human arterial neurovascular unit to study neurodegenerative diseases.

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.

A single-cell atlas of the human substantia nigra reveals cell-specific pathways associated with neurological disorders.

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.

Cerebrovascular amyloid Angiopathy in bioengineered vessels is reduced by high-density lipoprotein particles enriched in Apolipoprotein E.

BACKGROUND: Several lines of evidence suggest that high-density lipoprotein (HDL) reduces Alzheimer's disease (AD) risk by decreasing vascular beta-amyloid (Aß) 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 Aß deposition within the vasculature and attenuates endothelial inflammation as measured by monocyte binding. RESULTS: We demonstrate that HDL reduces vascular Aß accumulation independently of its principal binding protein, scavenger receptor (SR)-BI, in contrast to the SR-BI-dependent mechanism by which HDL prevents Aß-induced vascular inflammation. We describe multiple novel mechanisms by which HDL acts to reduce CAA, namely: i) altering Aß binding to collagen-I, ii) forming a complex with Aß that maintains its solubility, iii) lowering collagen-I protein levels produced by smooth-muscle cells (SMC), and iv) attenuating Aß 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 Aß and shed new light on a potential role of peripherally-acting apoE in AD.

HDL from an Alzheimer's disease perspective.

PURPOSE OF REVIEW: We review current knowledge regarding HDL and Alzheimer's disease, focusing on HDL's vasoprotective functions and potential as a biomarker and therapeutic target for the vascular contributions of Alzheimer's disease. RECENT FINDINGS: Many epidemiological studies have observed that circulating HDL levels associate with decreased Alzheimer's disease risk. However, it is now understood that the functions of HDL may be more informative than levels of HDL cholesterol (HDL-C). Animal model studies demonstrate that HDL protects against memory deficits, neuroinflammation, and cerebral amyloid angiopathy (CAA). In-vitro studies using state-of-the-art 3D models of the human blood-brain barrier (BBB) confirm that HDL reduces vascular Aß accumulation and attenuates Aß-induced endothelial inflammation. Although HDL-based therapeutics have not been tested in clinical trials for Alzheimer's disease , several HDL formulations are in advanced phase clinical trials for coronary artery disease and atherosclerosis and could be leveraged toward Alzheimer's disease . SUMMARY: Evidence from human studies, animal models, and bioengineered arteries supports the hypothesis that HDL protects against cerebrovascular dysfunction in Alzheimer's disease. Assays of HDL functions relevant to Alzheimer's disease may be desirable biomarkers of cerebrovascular health. HDL-based therapeutics may also be of interest for Alzheimer's disease, using stand-alone or combination therapy approaches.

Haplotype-specific MAPT exon 3 expression regulated by common intronic polymorphisms associated with Parkinsonian disorders.

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.

MAPT Genetic Variation and Neuronal Maturity Alter Isoform Expression Affecting Axonal Transport in iPSC-Derived Dopamine Neurons.

The H1 haplotype of the microtubule-associated protein tau (MAPT) locus is genetically associated with neurodegenerative diseases, including Parkinson's disease (PD), and affects gene expression and splicing. However, the functional impact on neurons of such expression differences has yet to be fully elucidated. Here, we employ extended maturation phases during differentiation of induced pluripotent stem cells (iPSCs) into mature dopaminergic neuronal cultures to obtain cultures expressing all six adult tau protein isoforms. After 6 months of maturation, levels of exon 3+ and exon 10+ transcripts approach those of adult brain. Mature dopaminergic neuronal cultures display haplotype differences in expression, with H1 expressing 22% higher levels of MAPT transcripts than H2 and H2 expressing 2-fold greater exon 3+ transcripts than H1. Furthermore, knocking down adult tau protein variants alters axonal transport velocities in mature iPSC-derived dopaminergic neuronal cultures. This work links haplotype-specific MAPT expression with a biologically functional outcome relevant for PD.

Induced pluripotent stem cell (iPSC)-derived dopaminergic models of Parkinson's disease.

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 carrying mutations 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.

The role of MAPT sequence variation in mechanisms of disease susceptibility.

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.

Haplotype-specific expression of the N-terminal exons 2 and 3 at the human MAPT locus.

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.

Functional MAPT haplotypes: bridging the gap between genotype and neuropathology.

The microtubule-associated protein tau (MAPT) locus has long been associated with sporadic neurodegenerative disease, notably progressive supranuclear palsy and corticobasal degeneration, and more recently with Alzheimer's disease and Parkinson's disease. However, the functional biological mechanisms behind the genetic association have only now started to emerge. The genomic architecture in the region spanning MAPT is highly complex, and includes a approximately 1.8 Mb block of linkage disequilibrium (LD). The region is divided into two major haplotypes, H1 and H2, defined by numerous single nucleotide polymorphisms and a 900 kb inversion which suppresses recombination. Fine mapping of the MAPT region has identified sub-clades of the MAPT H1 haplotype which are specifically associated with neurodegenerative disease. Here we briefly review the role of MAPT in sporadic and familial neurodegenerative disease, and then discuss recent work which, for the first time, proposes functional mechanisms to link MAPT haplotypes with the neuropathology seen in patients.

Haplotype-specific expression of exon 10 at the human MAPT locus.

Neurofibrillary tangles composed of exon 10+ microtubule associated protein tau (MAPT) deposits are the characteristic feature of the neurodegenerative diseases progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). PSP, CBD and more recently Alzheimer's disease and Parkinson's disease, are associated with the MAPT H1 haplotype, but the relationship between genotype and disease remains unclear. Here, we investigate the hypothesis that H1 expresses more exon 10+ MAPT mRNA compared to the other haplotype, H2, leading to a greater susceptibility to neurodegeneration in H1 carriers. We performed allele-specific gene expression on two H1/H2 heterozygous human neuronal cell lines, and 14 H1/H2 heterozygous control individual post-mortem brain tissue from two brain regions. In both tissue culture and post-mortem brain tissue, we show that the MAPT H1 haplotype expresses significantly more exon 10+ MAPT mRNA than H2. In post-mortem brain tissue, we show that the total level of MAPT expression from H1 and H2 is not significantly different, but that the H1 chromosome expresses up to 1.43-fold more exon 10+ MAPT mRNA than H2 in the globus pallidus, a brain region highly affected by tauopathy (maximum exon 10+ MAPT H1:H2 transcript ratio=1.425, SD=0.205, P<0.0001), and up to 1.29-fold more exon 10+ MAPT mRNA than H2 in the frontal cortex (maximum exon 10+ MAPT H1:H2 transcript ratio=1.291, SD=0.315, P=0.006). These data may explain the increased susceptibility of H1 carriers to neurodegeneration and suggest a potential mechanism between MAPT genetic variability and the pathogenesis of neurodegenerative disease.