Biology subjects

Healy, L. M.

Publications and source records attributed to Healy, L. M..

5 recordsLinked to original sources

bioRxiv2023

An adapted stem cell-derived microglia protocol for the study of microgliopathies and other neurological disorders

BackgroundAdult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is a primary microgliopathy caused by pathogenic variants in the colony-stimulating factor 1 receptor (CSF1R) gene. Since CSF1R signaling is crucial for microglia development, survival and function, induced pluripotent stem cell-derived microglia (iMGL) represent an excellent tool in studying microglial defects caused by ALSP patient-specific CSF1R variants. MethodsSerial modifications to an existing iMGL protocol were made, including but not limited to changes in growth factor combination to drive microglial differentiation, until successful derivation of microglia-like cells from an ALSP patient carrying a c.2350G > A (p.V784M) CSF1R variant. Using healthy control lines, the quality of the new iMGL protocol was validated through cell yield assessment, measurement of microglia marker expression, transcriptomic comparison to primary microglia, and evaluation of inflammatory and phagocytic activities. Similarly, molecular and functional characterization of the ALSP patient-derived iMGL was carried out in comparison to healthy control iMGL. ResultsThe newly devised protocol allowed the generation of iMGL with enhanced transcriptomic similarity to primary human microglia and with higher phagocytic and inflammatory competence at [~]3-fold greater yield compared to the original protocol. Using this protocol, decreased CSF1R autophosphorylation and cell surface expression was observed in iMGL derived from the ALSP patient compared to those derived from healthy controls. Additionally, ALSP patient-derived iMGL presented a migratory defect accompanying a temporal reduction in purinergic receptor P2Y12 (P2RY12) expression. Finally, ALSP patient-derived cells showed surprisingly high phagocytic capacity, which was associated with higher lysosomal content. ConclusionsWe optimized a pre-existing iMGL protocol, generating a powerful tool to study microglial involvement in human neurological diseases. Using the optimized protocol, we have generated for the first time iMGL from an ALSP patient carrying a pathogenic CSF1R variant, with preliminary characterization pointing toward functional alterations in migratory and phagocytic activities.

neuroscience↗

bioRxiv2023

MerTK mediates the immunologically silent uptake of alpha-synuclein fibrils by human microglia

MerTK is a receptor tyrosine kinase that mediates the immunologically silent phagocytic uptake of diverse types of cellular debris. Highly expressed on the surface of microglial cell, 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 aging 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 to synucleinopathies was assessed by analyzing 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 immunologically silent nature of MerTK-mediated phagocytosis, alpha-synuclein fibril internalization did not induce secretion of pro-inflammatory cytokines from microglia. In addition, burden analysis in two independent patient cohorts revealed a significant association between rare functionally deleterious MERTK variants and Parkinsons disease in one of the cohorts (p = 0.002). Accordingly, MERTK expression was significantly upregulated in nigral microglia from Parkinsons disease/Lewy body dementia patients compared to those from non-neurological control donors in a single-nuclei RNA-sequencing dataset (p = 5.08x10-21), and MerTK protein expression positively correlated with alpha-synuclein level in human cortex lysates (p = 0.0029). Taken together, our findings define a novel role for MerTK in mediating the uptake of alpha-synuclein aggregates by human microglia, with possible involvement in limiting alpha-synuclein spread in synucleinopathies such as Parkinsons disease.

cell biology↗

bioRxiv2022

Systematic comparison of culture media uncovers phenotypic shift of human microglia defined by reduced reliance to CSF1R signaling

Efforts to understand microglia function in health and diseases have been hindered by the lack of culture models that recapitulate in situ cellular properties. In recent years, the use of serum-free media with brain-derived growth factors (CSF1R ligands and TGF-{beta}1/2) have been favored for the maintenance of rodent microglia as they promote morphological features observed in situ. Here we study the functional and transcriptomic impacts of such media on human microglia. Media formulation had little impact on microglia transcriptome assessed by RNA sequencing which was sufficient to significantly alter microglia capacity to phagocytose myelin debris and to elicit an inflammatory response to lipopolysaccharide. When compared to immediately ex vivo microglia from the same donors, the addition of fetal bovine serum to culture media, but not growth factors, was found to aid in the maintenance of key signature genes including those involved in phagocytic processes. A phenotypic shift characterized by CSF1R downregulation in culture correlated with a lack of reliance on CSF1R signaling for survival. Consequently, no improvement in cell survival was observed following culture supplementation with CSF1R ligands. Our study provides better understanding of human microglia in culture, with observations that diverge from those previously made in rodent microglia. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=181 SRC="FIGDIR/small/500101v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@16ea011org.highwire.dtl.DTLVardef@1cef01dorg.highwire.dtl.DTLVardef@f621aborg.highwire.dtl.DTLVardef@11c9f97_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2020

Single-cell RNA sequencing reveals time- and sex-specific responses of spinal cord microglia to peripheral nerve injury and links ApoE to neuropathic pain

Activation of microglia in the spinal cord following peripheral nerve injury is critical for the development of long-lasting pain hypersensitivity. However, it remains unknown whether distinct microglia subpopulations or states contribute to different stages of pain development and maintenance. We show, using single-cell RNA-sequencing, that nerve injury induces the generation of a male-specific inflammatory microglia subtype, and demonstrate increased proliferation of microglia in males as compared to females. We also show time- and sex-specific transcriptional changes in different microglial subpopulations following injury. Apolipoprotein E (Apoe) is the top upregulated gene in microglia at chronic time points after nerve injury in mice and polymorphisms in the APOE gene in humans are associated with chronic pain. Single-cell analysis of human spinal cord microglia reveals a subpopulation with a disease-related transcriptional signature. Our data provide a detailed analysis of transcriptional states of mouse and human spinal cord microglia, and identify a previously unrecognized role for ApoE in neuropathic pain.

neuroscience↗

bioRxiv2020

Vitamin D regulates MerTK-dependent phagocytosis in human myeloid cells

Vitamin D deficiency is a major environmental risk factor for the development of multiple sclerosis (MS). The major circulating metabolite of vitamin D (25OHD) is converted to the active form (calcitriol) by the hydroxylase enzyme CYP27B1. In MS lesions the tyrosine kinase MerTK expressed by microglia and macrophages regulates phagocytosis of myelin debris and apoptotic cells that can accumulate and inhibit tissue repair and remyelination. We show that calcitriol downregulates MerTK mRNA and protein expression in adult human microglia and monocyte-derived macrophages, thereby inhibiting myelin phagocytosis and apoptotic cell clearance. Proinflammatory myeloid cells express high levels of CYP27B1 compared to homeostatic (TGF{beta}-treated) myeloid cells. Only proinflammatory cells in the presence of TNF- generate calcitriol from 25OHD, resulting in repression of MerTK expression and function. The selective production of calcitriol in proinflammatory myeloid cells leading to downregulation of MerTK-mediated phagocytosis has the potential to reduce the risk for auto-antigen presentation while retaining the phagocytic ability of homeostatic myeloid cells, thereby contributing to inflammation reduction and enhanced tissue repair.

immunology↗