Acta Neuropathologica Communications最新文献

Drug discovery efforts in neurological diseases, such as Alzheimer's disease (AD), have had particularly poor outcomes due to the lack of models that recapitulate drug interactions at the cerebral vasculature. There is an unmet need to develop physiologically relevant models to study the impacts of blood flow-induced shear stress. In this work, we use a microfluidic platform to model the cerebral vasculature in AD using patient-derived brain endothelial-like cells (BECs). Induced pluripotent stem cells derived from a patient with familial AD (PSEN-2 N141I) and an unaffected control line were differentiated into BECs (AD2-BEC and fControl-BEC, respectively). BECs were exposed to static conditions or 12 dynes/cm² of shear stress for 72 h prior to assessment of barrier permeability using fluorescent tracer assays, monocyte adhesion, and efflux transport function using receptor-inhibition assays. Upon shear conditioning, BECs demonstrated shear responsiveness through greater cell alignment in the direction of flow. AD2-BECs demonstrated reduced capacity for efflux transport by p-glycoprotein (P-gp), breast cancer resistant protein (BCRP), and multidrug resistant protein (MRP1) compared to controls (fControl-BECs, p = 0.0017, p = 0.0004, p = 0.0002, respectively). Upon application of shear conditioning, impairments to efflux transport in AD2-BECs were ameliorated. AD2-BECs also exhibited increased monocyte adhesion (2.2 ± 0.4-fold; p < 0.0001) which was further reduced by the application of shear stress in both lines. Taken together, these observations suggest the lack of shear stress exacerbates altered BEC phenotype in fAD. To our knowledge, we present the first in depth functional characterization of in vitro AD patient-derived BECs in both static and physiologically relevant shear conditions in which lack of shear reveals dysfunction of the cerebral endothelium in AD relevant to drug transport and immune cell trafficking.

Cerebral ischemia increases the risk of post-stroke cognitive impairment (PSCI), but the underlying molecular mechanisms remain unclear. Emerging evidence suggests that hypoxia/ischemia-induced oxidative and endoplasmic reticulum (ER) stress may contribute to protein misfolding and α-Synuclein (α-Syn) aggregation, potentially triggering the unfolded protein response (UPR) to alleviate ER stress. Using bimolecular fluorescence complementation in Drosophila melanogaster and HEK-293 cells, we investigated the effect of acute, repetitive and chronic hypoxia on α-Syn aggregation, UPR activation, mortality, longevity, locomotor function, sleep, and cognition. Furthermore, we evaluated the post-hypoxic in vivo biodistribution and therapeutic efficacy of the aggregation inhibitor anle138b. Acute severe hypoxia induced more α-Syn aggregation than chronic or repetitive hypoxia, resulting in higher mortality, reduced longevity, delayed motor recovery, cognitive impairment, and activation of the detrimental PERK branch of the UPR. Anle138b significantly reduced α-Syn aggregation, repressing post-hypoxic PERK activation and improving survival and decision-making. Our findings demonstrate the effectiveness of anle138b in mitigating hypoxia-induced α-Syn aggregation and cognitive impairment, paving the way for future studies on its potential as a therapeutic strategy for PSCI.

Background: Accumulated levels of mutant huntingtin protein (mHTT) and its fragments are considered contributors to the pathogenesis of Huntington's disease (HD). Stimulating autophagy may enhance clearance of mHTT and its aggregates which has been considered as a possible therapeutic strategy. However, the role and competence of the autophagy-lysosomal pathway (ALP) during HD progression in the human disease remains largely unknown.

Methods: Here, we used multiplex confocal and ultrastructural immunocytochemical analyses of ALP functional markers in relation to mHTT aggresome pathology in striatum and the less affected cortex or cerebellum of HD brains staged from Grade HD2 to HD4 by Vonsattel neuropathological criteria compared to controls.

Results: Immunolabeling revealed the localization of HTT/mHTT in ALP vesicular compartments labeled by autophagy-related adaptor proteins sequestosome 1 (p62/SQSTM1) and ubiquitin, and cathepsin D (CTSD) as well as HTT-positive inclusions. Although comparatively normal at HD2, neurons at later HD stages exhibited progressive enlargement and clustering of CTSD-immunoreactive autolysosomes/lysosomes and, ultrastructurally, autophagic vacuole/lipofuscin granules accumulated progressively, more prominently in striatum than cortex. These changes were accompanied by rises in levels of HTT/mHTT and p62/SQSTM1, particularly their fragments, in striatum but not in the cortex, and by increases of LAMP1 and LAMP2 RNA and LAMP1 protein. In addition, cargo-loaded autophagosomes and cathepsin-positive autolysosomes were readily observed, implying a lack of significant blockage in autophagosome formation and autophagosome-lysosome fusion.

Conclusions: The findings collectively suggest that upregulated lysosomal biogenesis and preserved proteolysis maintain autophagic clearance in early-stage HD, but the observed progressive HTT build-up and AL accumulation at advanced disease stages may signify a failure in autophagy substrate clearance. These findings support the prospect that ALP stimulation applied at early disease stages, when clearance machinery is fully competent, could lead to therapeutic benefits in HD patients.

It is currently understood that the characteristic loss of the repressive histone mark H3K27me3 in PFA ependymoma and diffuse midline glioma (DMG) are caused by complementary mechanisms mediated by EZHIP and the oncohistone H3K27M, respectively. To support the complementarity of these mechanisms, rare H3K27M-negative DMGs express EZHIP. Interestingly, EZHIP is one of the few genes recurrently mutated in PFA. The significance of EZHIP mutations in PFA, and whether EZHIP has wider functions in addition to repression of H3K27me3 deposition, are not known. Here, we investigated the mutational landscape of EZHIP in pediatric brain tumors. We found that EZHIP mutations occur not only in PFA, but also in rare medulloblastoma and pediatric high-grade glioma (HGG), including in H3K27-positive DMG. Contrary to current expectations, we show that mutant EZHIP is expressed in H3K27M-positive DMG. All the EZHIP-mutated HGG cases also have EGFR mutations. Further, we pursued better understanding of the function of EZHIP by expressing it in human-derived neural models. Our transcriptomic analyses indicate that EZHIP expression potentiates neuronal-like gene programs associated with synaptic function. Metabolomics data indicate that EZHIP leads to repression of methionine and polyamine metabolism, suggesting links between metabolic and epigenetic changes that are observed in PFA. Collectively, our results expand the repertoire of tumor types known to harbor EZHIP mutations and shed light on EZHIP-dependent metabolic and transcriptional programs in relevant neural models.

α-Synuclein seed amplification assays are a promising diagnostic tool for synucleinopathies such as Parkinson's disease and multiple system atrophy. Standardized conditions are required to ensure a high degree of inter- and intra-laboratory reproducibility when performing these assays. A significant issue that hinders the utility of seed amplification assays is the de novo aggregation propensity of the α-synuclein substrate as well as inter-batch heterogeneity. While much work has focused on determining appropriate seed amplification assay buffer compositions as well as the type and amount of seed used, a robust comparison of α-synuclein substrate purification methods has not been reported. We therefore compared the utility of recombinant α-synuclein purified using four different methods as seed amplification assay substrates across two laboratories. Osmotic shock-purified α-synuclein monomer substrate showed the lowest propensity for de novo aggregation, which translated into being the best substrate for seed amplification assay reactions seeded with α-synuclein preformed fibrils or patient brain homogenates. Furthermore, osmotic shock α-synuclein monomer showed the best inter-batch reproducibility compared to all other substrates tested. As α-synuclein seed amplification assays continue to evolve and move towards adoption in the clinical realm, this work showcases the vital importance of standardizing the production and characterization of recombinant α-synuclein substrate. We encourage the widespread adoption of osmotic shock α-synuclein monomer as the universal substrate for seed amplification assays to maximize intra- and inter-laboratory reproducibility.

Alzheimer's Disease (AD) is marked by the accumulation of pathology, neuronal loss, and gliosis and frequently accompanied by decline in cognition. Understanding brain cell interactions is key to identifying new therapeutic targets to slow its progression. Here, we used systems biology methods to analyze single-nucleus RNA sequencing (snRNASeq) data generated from dorsolateral prefrontal cortex (DLPFC) tissues of 424 participants in the Religious Orders Study or the Rush Memory and Aging Project (ROSMAP). We identified modules of co-regulated genes in seven major cell types and assigned them to coherent cellular processes. We showed that coexpression structure was conserved in the majority of modules across cell types, but we also found distinct communities with altered connectivity, especially when compared to bulk RNASeq, suggesting cell-specific gene co-regulation. These coexpression modules can also capture signatures of cell subpopulations and be influenced by cell proportions. Finally, we performed associations of modules with AD traits such as amyloid-β deposition, tangle density, and cognitive decline, and showed replications in an independent single-nucleus dataset. Using a Bayesian network framework, we modeled the direction of relationships between the modules and AD progression. We highlight one key module, the astrocytic module 19 (ast_M19), associated with cognitive decline through a subpopulation of stress-response cells. Our work provides cell-specific molecular networks modeling the molecular events leading to AD.

Parkinson's disease (PD) progression involves dopaminergic neurodegeneration and pathological α-synuclein aggregation, processes linked to metabolic dysregulation and autophagy-lysosomal pathway (ALP) impairment. Transaldolase1 (TAL1) is a key enzyme of the pentose phosphate pathway. While elevated TAL1 protein levels have been observed in postmortem substantia nigra of PD patients, the enzyme's functional role in disease pathogenesis remains undefined. In this study, we explored the role of TAL1 in PD-related pathologies using MPTP-induced and AAV-A53T mouse models. We demonstrate that TAL1 upregulation is associated with dopaminergic neuron degeneration across both experimental models. TAL1 knockdown activated TFEB-mediated transcription of autophagy-lysosomal genes (Ctsb, Ctsd, Lamp1, Becn1, and Map1Lc3b). In addition, targeted metabolomics revealed that TAL1 knockdown modulates the energy pathways, especially in the TCA cycle, and glycolysis. The neuroprotective effects were mediated through AMPK/mTORC1 pathway activation, evidenced by increased AMP levels, p-AMPK/AMPK ratios, and downstream ALP enhancement. Importantly, TAL1 inhibition improved locomotor function in AAV-A53T mice and normalized stride length in footprint analysis. Pathological experiments confirmed reduced phospho-α-synuclein level and preserved the neuron loss in substantia nigra. Our findings highlight TAL1 as a regulator of autophagy-lysosomal function and energy metabolism in PD-related experimental models, where its inhibition restores the degradation of α-synuclein through coordinated activation of autophagy-lysosomal clearance and energetic reprogramming. These results suggest that targeting TAL1 may offer a potential therapeutic approach to mitigate PD-associated neuropathology.