An adapted protocol to derive microglia from stem cells and its application in the study of CSF1R-related disorders.

BACKGROUND: Induced pluripotent stem cell-derived microglia (iMGL) represent an excellent tool in studying microglial function in health and disease. Yet, since differentiation and survival of iMGL are highly reliant on colony-stimulating factor 1 receptor (CSF1R) signaling, it is difficult to use iMGL to study microglial dysfunction associated with pathogenic defects in CSF1R. METHODS: Serial 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 adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (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. RESULTS: The newly devised protocol allowed the generation of iMGL with enhanced transcriptomic similarity to cultured primary human microglia and with higher scavenging and inflammatory competence at ~ threefold 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, a heightened capacity to internalize myelin, as well as heightened inflammatory response to Pam3CSK4. Poor P2RY12 expression was confirmed to be a consequence of CSF1R haploinsufficiency, as this feature was also observed following CSF1R knockdown or inhibition in mature control iMGL, and in CSF1RWT/KO and CSF1RWT/E633K iMGL compared to their respective isogenic controls. CONCLUSIONS: We 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, phagocytic and inflammatory activities.

Regulation of long non-coding RNA expression by aryl hydrocarbon receptor activation.

The aryl hydrocarbon receptor (AhR) is a cytosolic transcription factor that can be activated by endogenous or xenobiotic ligands. Upon activation, the AhR translocates to the nucleus, dimerizes with the AhR nuclear translator (ARNT), and binds to specific DNA sequences called xenobiotic response elements (XRE) to promote target gene transcription, including cytochrome P450 (e.g., CYP1A1) expression. In addition to mRNA, the AhR may also regulate long non-coding RNA (lncRNA) expression. lncRNA are transcripts more than 200 nucleotides in length that do not encode a protein. Herein, we tested whether AhR activation regulates the expression of lncRNA in response to benzo[a]pyrene (B[a]P) using RNA sequencing (RNA-seq). We found that many lncRNA (e.g., SATB1-AS1, MIR4290HG, AC008969.1, LINC01533, VIPR1-AS1) and protein-coding RNA (e.g., CYP1A1, BX005266.2, AQP3, BTG2, DCX, and AhRR) were differentially expressed (DE) in A549 cells treated with B[a]P; many of these genes were dependent on AhR expression including CYP1A1, CYP1B1 and TiPARP. GO analyses indicated that DE protein-coding RNAs in A549WT cells are associated with distinct molecular functions compared to A549KO cells. KEGG analyses showed the hsa01100 pathway was associated with DE lncRNA only in A549WT cells. A549KO cells treated with B[a]P exhibited a distinct set of differentially-regulated lncRNA including upregulation of HOTAIR. We further confirmed that despite AhR activation in A549WT cells, B[a]P did not alter the expression of many well-characterized lncRNA including NEAT1, HOTTIP, SOX2OT, MALAT1, H19, and Linc00673. Thus, there is control over select lncRNA expression in A549 cells exposed to B[a]P, a finding which could yield insight into the molecular function of the AhR.

Dynamic relationship between the aryl hydrocarbon receptor and long noncoding RNA balances cellular and toxicological responses.

The aryl hydrocarbon receptor (AhR) is a cytosolic transcription factor activated by endogenous ligands and xenobiotic chemicals. Once the AhR is activated, it translocates to the nucleus, dimerizes with the AhR nuclear translator (ARNT) and binds to xenobiotic response elements (XRE) to promote gene transcription, notably the cytochrome P450 CYP1A1. The AhR not only mediates the toxic effects of environmental chemicals, but also has numerous putative physiological functions. This dichotomy in AhR biology may be related to reciprocal regulation of long non-coding RNA (lncRNA). lncRNA are defined as transcripts more than 200 nucleotides in length that do not encode a protein but are implicated in many physiological processes such as cell differentiation, cell proliferation, and apoptosis. lncRNA are also linked to disease pathogenesis, particularly the development of cancer. Recent studies have revealed that AhR activation by environmental chemicals affects the expression and function of lncRNA. In this article, we provide an overview of AhR signaling pathways activated by diverse ligands and highlight key differences in the putative biological versus toxicological response of AhR activation. We also detail the functions of lncRNA and provide current data on their regulation by the AhR. Finally, we outline how overlap in function between AhR and lncRNA may be one way in which AhR can be both a regulator of endogenous functions but also a mediator of toxicological responses to environmental chemicals. Overall, more research is still needed to fully understand the dynamic interplay between the AhR and lncRNA.

Aberrant Post-Transcriptional Regulation of Protein Expression in the Development of Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is an incurable and prevalent respiratory disorder that is characterized by chronic inflammation and emphysema. COPD is primarily caused by cigarette smoke (CS). CS alters numerous cellular processes, including the post-transcriptional regulation of mRNAs. The identification of RNA-binding proteins (RBPs), microRNAs (miRNAs), and long non-coding RNAs (lncRNAs) as main factors engaged in the regulation of RNA biology opens the door to understanding their role in coordinating physiological cellular processes. Dysregulation of post-transcriptional regulation by foreign particles in CS may lead to the development of diseases such as COPD. Here we review current knowledge about post-transcriptional events that may be involved in the pathogenesis of COPD.