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Primary ciliary dyskinesia (PCD) is a genetic disorder causing motile ciliary dysfunction primarily affecting the respiratory and reproductive systems. However, the impact of PCD on the central nervous system remains poorly understood. Rodent models of PCD exhibit marked hydrocephalus leading to early animal mortality, however, most humans with PCD do not develop hydrocephalus for unknown reasons. We hypothesized that patients with PCD exhibit sub-clinical ventriculomegaly related to ependymal motile ciliary dysfunction. We demonstrated highly specific expression levels of known PCD-related genes in human brain multiciliated ependymal cells (p < 0.0001). To assess ventricular size, computed tomography sinus images from patients with PCD (n = 33) and age/sex-matched controls (n = 64) were analysed. Patients with PCD displayed significantly larger ventricular areas (p < 0.0001) and Evans index (p < 0.01), indicating ventriculomegaly that was consistent across all genetic subgroups. Ventricular enlargement correlated positively with increasing age in patients with PCD compared to controls (p < 0.001). Additionally, chart review demonstrated a high prevalence (39%) of neuropsychiatric/neurological disorders in adult PCD patients that did not correlate with degree of ventriculomegaly. Our findings suggest that patients with PCD may have unrecognized, mild ventriculomegaly which correlates with ageing, potentially attributable to ependymal ciliary dysfunction. Further study is required to determine causality, and whether ventricular enlargement contributes to neuropsychiatric/neurological or other morbidity in PCD. [ABSTRACT FROM AUTHOR]

Ependymal cells form a specialized brain–cerebrospinal fluid (CSF) interface and regulate local CSF microcirculation. It is becoming increasingly recognized that ependymal cells assume a reactive state in response to aging and disease, including conditions involving hypoxia, hydrocephalus, neurodegeneration, and neuroinflammation. Yet what transcriptional signatures govern these reactive states and whether this reactivity shares any similarities with classical descriptions of glial reactivity (i.e., in astrocytes) remain largely unexplored. Using single‐cell transcriptomics, we interrogated this phenomenon by directly comparing the reactive ependymal cell transcriptome to the reactive astrocyte transcriptome using a well‐established model of autoimmune‐mediated neuroinflammation (MOG35‐55 EAE). In doing so, we unveiled core glial reactivity‐associated genes that defined the reactive ependymal cell and astrocyte response to MOG35‐55 EAE. Interestingly, known reactive astrocyte genes from other CNS injury/disease contexts were also up‐regulated by MOG35‐55 EAE ependymal cells, suggesting that this state may be conserved in response to a variety of pathologies. We were also able to recapitulate features of the reactive ependymal cell state acutely using a classic neuroinflammatory cocktail (IFNγ/LPS) both in vitro and in vivo. Taken together, by comparing reactive ependymal cells and astrocytes, we identified a conserved signature underlying glial reactivity that was present in several neuroinflammatory contexts. Future work will explore the mechanisms driving ependymal reactivity and assess downstream functional consequences. [ABSTRACT FROM AUTHOR]

Mer tyrosine kinase (MerTK) is a receptor tyrosine kinase that mediates non-inflammatory, homeostatic phagocytosis of diverse types of cellular debris. Highly expressed on the surface of microglial cells, MerTK is of importance in brain development, homeostasis, plasticity and disease. Yet, involvement of this receptor in the clearance of protein aggregates that accumulate with ageing and in neurodegenerative diseases has yet to be defined. The current study explored the function of MerTK in the microglial uptake of alpha-synuclein fibrils which play a causative role in the pathobiology of synucleinopathies. Using human primary and induced pluripotent stem cell-derived microglia, the MerTK-dependence of alpha-synuclein fibril internalization was investigated in vitro. Relevance of this pathway in synucleinopathies was assessed through burden analysis of MERTK variants and analysis of MerTK expression in patient-derived cells and tissues. Pharmacological inhibition of MerTK and siRNA-mediated MERTK knockdown both caused a decreased rate of alpha-synuclein fibril internalization by human microglia. Consistent with the non-inflammatory nature of MerTK-mediated phagocytosis, alpha-synuclein fibril internalization was not observed to induce secretion of pro-inflammatory cytokines such as IL-6 or TNF, and downmodulated IL-1β secretion from microglia. Burden analysis in two independent patient cohorts revealed a significant association between rare functionally deleterious MERTK variants and Parkinson's disease in one of the cohorts (P = 0.002). Despite a small upregulation in MERTK mRNA expression in nigral microglia from Parkinson's disease/Lewy body dementia patients compared to those from non-neurological control donors in a single-nuclei RNA-sequencing dataset (P = 5.08 × 10−21), no significant upregulation in MerTK protein expression was observed in human cortex and substantia nigra lysates from Lewy body dementia patients compared to controls. Taken together, our findings define a novel role for MerTK in mediating the uptake of alpha-synuclein fibrils by human microglia, with possible involvement in limiting alpha-synuclein spread in synucleinopathies such as Parkinson's disease. Upregulation of this pathway in synucleinopathies could have therapeutic values in enhancing alpha-synuclein fibril clearance in the brain. [ABSTRACT FROM AUTHOR]

Background: Activation of the mTOR pathway is pivotal for microglia to induce and sustain neuroprotective functions (Ulland et al., 2017; Wang et al., 2022). mTOR complex 1 (mTORC1) inhibits the translation repressors, eukaryotic translation Initiation Factor 4E (eIF4E)‐Binding Proteins (4E‐BPs), via phosphorylation, which causes their release from eIF4E to promote mRNA translation (Hay and Sonenberg, 2004). mTORC1 promotes mitochondrial biogenesis via inhibition of 4E‐BPs, by preferentially stimulating the translation of mitochondria‐related mRNAs (Gandin et al., 2016; Morita et al., 2013). We investigated the mechanisms at the intersection of 4E‐BP‐dependent translational regulation and metabolism in microglial response to soluble Aβ. Method: We carried out immunoblot analysis to investigate the phosphorylation status of 4E‐BP1, the isoform most abundant in microglia, following exposure to Ab. We manipulated the mTOR pathway by knocking out the downstream effectors, 4E‐BPs, to alleviate translation suppression in microglia in vitro and in vivo. We crossed the microglia‐specific 4E‐BPs knockout mouse with a RiboTag mouse to pull‐down ribosome‐bound mRNAs, providing a genome‐wide pool of actively translating mRNAs in the absence or presence of 4E‐BPs. Finally, we examined the relationship between 4E‐BP1 levels and neuroinflammation markers in cerebrospinal fluid (CSF) of AD patients. Result: We showed that 4E‐BP1 is inhibited acutely upon exposure to soluble Ab, which is dependent on Spleen Tyrosine Kinase (SYK) activation upstream of mTORC1, but is reduced upon chronic exposure. Furthermore, 4E‐BP1 expression is induced during prolonged exposure to Ab. The deletion of 4E‐BPs in microglia in vitro leads to an increase in mitochondrial mass and reliance on oxidative phosphorylation while decreasing expression of pro‐inflammatory mediators and cell death upon exposure to Ab. We observed that increased levels of 4E‐BP1 in the CSF of patients with Aβ pathology are associated with higher neurodegeneration (Nfl) in the presence of microglial activation. Conclusion: We demonstrate that mTORC1 signaling critically impacts microglia physiology and promotes neuroprotective functions via 4E‐BP1 inhibition. 4E‐BP1 activity in microglia engenders a dysfunctional or detrimental state that may lead to increased neurodegeneration. Therefore, 4E‐BP1 is an attractive target for microglia modulation in AD. [ABSTRACT FROM AUTHOR]

Background: Microglia are tissue resident macrophages with a wide range of critically important functions in central nervous system development and homeostasis. Method: In this study, we aimed to characterize the transcriptional landscape of ex vivo human microglia across different developmental ages using cells derived from pre-natal, pediatric, adolescent, and adult brain samples. We further confirmed our transcriptional observations using ELISA and RNAscope. Results: We showed that pre-natal microglia have a distinct transcriptional and regulatory signature relative to their post-natal counterparts that includes an upregulation of phagocytic pathways. We confirmed upregulation of CD36, a positive regulator of phagocytosis, in pre-natal samples compared to adult samples in situ. Moreover, we showed adult microglia have more pro-inflammatory signature compared to microglia from other developmental ages. We indicated that adult microglia are more immune responsive by secreting increased levels of pro-inflammatory cytokines in response to LPS treatment compared to the pre-natal microglia. We further validated in situ up-regulation of IL18 and CXCR4 in human adult brain section compared to the pre-natal brain section. Finally, trajectory analysis indicated that the transcriptional signatures adopted by microglia throughout development are in response to a changing brain microenvironment and do not reflect predetermined developmental states. Conclusion: In all, this study provides unique insight into the development of human microglia and a useful reference for understanding microglial contribution to developmental and age-related human disease. [ABSTRACT FROM AUTHOR]