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Biology subjects

MacDonald, A.

Publications and source records attributed to MacDonald, A..

At least 19 recordsLinked to original sources

Sensitivity of tree species demography to climate and competition across their range

Theory predicts that demographic performance should peak at the core of species ranges and decrease toward their limits. Yet, empirical correlations between population growth rate and species distribution remain weak for most tree species. Part of the problem may arise from the difficulty of integrating multiple demographic processes across the complex life cycle of a forest, and from the significant variability among individuals and locations. It remains unclear if the mismatch between performance and distribution arises from modelling limitations or if climate is simply a poor predictor of species performance across distributions. Here, rather than asking whether demographic performance correlates with species distributions, we ask how climate and competition jointly shape population growth rate for 31 tree species across eastern North America. By combining flexible nonlinear hierarchical models for growth, survival, and recruitment with explicit uncertainty propagation, we use Integral Projection Models to address key gaps in previous studies. Perturbation analyses revealed that population growth rate was consistently more sensitive to mean annual temperature than to conspecific or heterospecific competition across all species. We further examined how sensitivities to climate and competition varied across species thermal ranges. The dominance of climate over competition increased toward both cold and hot range limits, while sensitivity to competition generally declined from cold to hot limits. Notably, these patterns emerged along the continental thermal gradient shared across species rather than within each species individual range, suggesting that range-edge demographic responses may arise as a community-level phenomenon. Across species, the largest source of variability remained the local plot conditions captured by random effects, likely reflecting differences in soil conditions, drainage, and disturbance history. Together, these results may provide a mechanistic pathway underlying the performance declines predicted by range-limit theories, and offer a basis for understanding how forest populations and communities may reorganize in response to ongoing climate change and shifting disturbance regimes.

ecology↗

Myeloid PINK1 represses mtDNA release and immune signaling that impacts neuronal pathology in patient-derived idiopathic PD models

Parkinsons disease (PD) is a neurodegenerative disorder marked by the development of cardinal motor deficits preceded by a protracted prodromal period of non-motor symptoms often involving the gastrointestinal (GI) tract. There is an emerging consensus that both the peripheral immune system and local neuroinflammation play key roles in the etiology of PD. We previously demonstrated a critical function for the Parkinsons related proteins PINK1 and Parkin as repressors of the innate to adaptive immune response in cultured cells and mouse models of infection. However, it remained unclear whether these processes were conserved in patient-derived models, and precisely how immune signaling may ultimately drive the death of dopaminergic neurons. Here we show that GI infection of PINK1 knockout (KO) mice triggered acute neurodegeneration which was evident early in the enteric nervous system. Treating wild type enteric or dopaminergic neurons with conditioned medium from immune-stimulated PINK1 KO macrophages was sufficient to promote neuronal disruption in both mouse and human neurons in vitro. Within immune-activated macrophages, we reveal that loss of PINK1 led to an enhanced release of mitochondrial DNA (mtDNA) within mitochondrial derived vesicles, leading to the activation of cGAS/STING pathways. These changes were seen in both mouse/human in vitro models and in PD patient-derived primary macrophages. Notably, pharmacological modulation using a PINK1 activator with high therapeutic potential attenuated pro-inflammatory profiles elicited by the mtDNA-dependent STING/NF-{kappa}B pathway in idiopathic patient-derived macrophages. Ultimately, our study lays the foundation for understanding PINK1-related peripheral macrophage mechanisms in idiopathic PD and provides a target for further development to treat the disease at early stages. Graphical abstractPINK1-related immune mechanisms of Parkinsons disease and associations with early neurodegenerative events. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/694713v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@6f6488org.highwire.dtl.DTLVardef@13139aaorg.highwire.dtl.DTLVardef@c0d4d1org.highwire.dtl.DTLVardef@1d41e25_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

BATF3 controls the homeostatic maintenance and function of innate-like γδ T cells

{gamma}{delta} T cells compose an evolutionarily conserved lineage of lymphocytes, with both adaptive- and innate-like characteristics, contributing to tissue homeostasis, immune surveillance, and rapid responses to stress and infection. While their functional diversity and tissue-specific roles are tightly regulated by transcriptional networks, the underlying molecular mechanisms remain incompletely understood. The transcription factor basic leucine zipper ATF-like transcription factor 3 (BATF3) plays a central role in the development of conventional type 1 dendritic cells (cDC1s). Here, we unveil BATF3 as a critical cell-intrinsic regulator of the homeostasis, functional specialization, and tissue distribution of {gamma}{delta} T cells. Batf3-deficient mice display an altered composition of {gamma}{delta} T cell subsets, with a marked decrease in the numbers of innate-like {gamma}{delta} T cells across multiple organs when compared to their wild-type counterparts, independently of cDC1s. Loss of BATF3 impacts not only cell survival but also IL-17 production after {gamma}{delta} T cells complete their thymic development. Mechanistically, Batf3-deficient innate-like {gamma}{delta} T cells exhibit transcriptional changes that disrupt pathways governing actin cytoskeleton remodelling, immunological synapse organization and cellular identity. Notably, Batf3-deficient mice present decreased survival in a viral infection model highly dependent on innate-like {gamma}{delta} T cells. Together, our findings uncover a previously unrecognized BATF3-dependent pathway that controls {gamma}{delta} T cell morphology and function, profoundly impacting their biology.

immunology↗

Use of a cytochrome P450 humanised mouse model to refine schistosomiasis drug discovery.

Control of schistosomiasis, a neglected tropical disease caused by infection with Schistosoma spp., remains reliant on a single chemotherapy, praziquantel (PZQ). This strategy presents a risk to global health should PZQ-resistant schistosomes establish in endemic areas and justifies the search for new drugs. However, species-specific metabolic differences between humans and preclinical models hinder the optimisation of next-generation anti-schistosomal therapeutics. Here, to bypass these species-specific limitations, we exploited a humanised mouse model, 8HUM, engineered to express the principal human Phase I cytochrome P450 enzymes (CYP1A1/2, CYP2C9, CYP2D6, CYP3A4/7) as well as the transcription factors constitutive androstane receptor (CAR) and pregnane X receptor (PXR) in place of 35 murine orthologues. We characterised S. mansoni development, immunopathology, hepatic transcriptomic responses, intestinal microbiome changes and PZQ metabolism as well as PZQ efficacy in 8HUM versus wild-type (WT) mice. 8HUM mice supported normal S. mansoni maturation, infection-associated microbiome dysbiosis, Th2-dominant immune responses and characteristic hepatic pathology. PZQ intrinsic clearance in 8HUM hepatic microsomes mirrored human levels and was >10-fold lower than that found for WT microsomes. Oral dosing revealed human-like PZQ exposures of (R)-PZQ and 4OH-PZQ in 8HUM mice at 25 mg/kg bodyweight and >90% reductions in worm burdens at 100 mg/kg bodyweight (equivalent to that seen in WT mice administered PZQ at 400 mg/kg bodyweight). Our results revealed that 8HUM mice recapitulate key features of murine schistosomiasis while exhibiting human-relevant drug metabolism. These findings establish 8HUM as a refined translational platform for anti-schistosomal drug development, improving predictive accuracy and accelerating therapeutic discovery. One Sentence SummaryA cytochrome P450 humanised mouse model is used to study Schistosoma mansoni development, schistosomiasis, drug metabolism and drug efficacy.

pharmacology and toxicology↗

Shell-bound archives: uncovering nematode encapsulations in the Galapagos' largest radiation

How macroevolution interacts with ecological networks remains a major question in eco-evolutionary science. We investigate this interplay in the Naesiotus snail radiation of the Galapagos Islands, which encapsulates parasitic nematodes within the shell--a recently discovered gastropod defense. Using a natural history collection, we examined dry shells from 47 species across 12 islands, quantified encapsulations, and sequenced nematode DNA to reconstruct a host-parasite network. Encapsulations were widespread and revealed high nematode diversity, including in snail hosts presumed extinct. Nematode diversity was shaped by habitat, while encapsulation load was better explained by host species identity, suggesting species-specific defenses. Neither trait showed phylogenetic signal, and shell brightness was unrelated to nematode interactions. Similarly, host diversification rate did not predict network position, suggesting that macroevolution may leave a weak or obscured imprint on this host-parasite network. This snail-nematode system in islands readily enables integration of ecological networks, phylogeny, functional traits, and biogeography.

ecology↗

Mgl2+ cDC2 triggering of fungal allergic inflammation depends on a spore induced glycolytic shift fuelled by local availability of glucose

Fungal spores are a major cause of severe asthmatic disease. However, the precise events that cause individuals to become sensitised to spores are poorly understood. Mgl2+ type 2 conventional dendritic cells (Mgl2+ cDC2s) are critical in coordinating allergic airway inflammation in mice following repeated exposure to inhaled spores. Yet, whether these DCs are directly acquiring spores from the airway, and the downstream mechanism(s) upon fungal uptake causing DCs to trigger allergic inflammation are unknown. Here we find that spores are acquired by lung DCs after inhalation although these events are rare ([~] 0.5% of the cDC2 population). Transcriptomics on isolated spore+ Mgl2+ cDC2s, compared to spore- Mgl2+ cDC2s from the same environment, revealed that a major consequence of fungal uptake was a boost in metabolic activity. Single-cell metabolic profiling revealed this increase in Mgl2+ cDC2 metabolism upon spore acquisition was fuelled by a glycolytic shift. To pinpoint if nutrient availability and acquisition is an important determinant of this response, mass spectrometry-based metabolomics revealed that, during fungal allergic inflammation, the local airway nutrient environment is altered. To ascertain which of these could be fuelling DC responses, we identified which substrates that feed into glycolysis were crucial. Of these, we found that glucose availability acts as a key rheostat in shaping cDC2 responses to spores. These data highlight a crucial role for glycolytic metabolism in driving cDC2 responses to spores, which is governed by glucose availability, defining novel targets for future therapeutic development of fungal allergic inflammation.

immunology↗

VasoTracker 2: An Open-source Platform for Quantitative Analysis of Vascular Reactivity and Function

VasoTracker 2 is an open-source platform for studying blood vessel dynamics, featuring versatile diameter-tracking software and complementary low-cost hardware components. This system surpasses existing tools through accessible, high-resolution analysis across multiple imaging modalities, enabling comprehensive assessment of vascular dynamics in both real-time and pre-recorded experiments. Advanced algorithms enable multi-point diameter tracking in branched vessels, automated pressure-response protocols, and reliable edge detection. The software can assess vessels imaged by brightfield microscopy, fluorescence imaging, and in ultrasound recordings, supporting diverse applications from isolated vessel studies to in vivo assessment. For ex vivo applications, VasoTracker 2 includes modular open-source hardware components that can be used to create a low-cost pressure myograph system: a confocal-compatible vessel chamber and a programmable pressure controller, VasoMoto. By combining powerful analytical capabilities with an open-access approach, VasoTracker 2 provides free software and low-cost hardware alternatives to commercial systems, democratizing access to advanced vascular research tools for scientists worldwide.

physiology↗

Changing food conditions and size declines in a North Sea forage fish

Declining body sizes are prevalent in marine fish. While these declines have been suggested to be a response to increasing temperatures, the evidence is mixed and the underlying causes of observed declines often unknown. Here, we explore drivers of spatio-temporal patterns in size in lesser sandeel (Ammodytes marinus), an important prey for seabirds and marine mammals, focusing on ongoing size declines in the North Sea. We combine experimental and field data with ecological theory to develop a biologically realistic dynamic energy budget model that explicitly models feeding, metabolism and energy allocation to produce daily predictions of size during the juvenile growth season from 1979 to 2016. When forced with daily temperature estimates and zooplankton data from the Continuous Plankton Recorder, model predictions reproduce observed spatio-temporal patterns in size well. Our results suggest that the most plausible driver of observed size declines in the western North Sea is declining prey densities. In contrast, the direct effect of temperature on sandeel size is small, but interacts with local prey availability so that the effect varies in both size and direction over space. Our results thus suggest that to understand effects of climate change on fish size we need to account for both direct physiological effects and changes in resource availability. Finally, we use the model to show that early-life phenology and turbidity (via its impact on intake rates in the visually foraging sandeel) may also impact sandeel size, highlighting the importance of broadening our view of potential drivers of size declines.

ecology↗

SOX9 plays an essential role in myofibroblast driven hepatic granuloma integrity and parenchymal repair during schistosomiasis-induced liver damage

Schistosomiasis is a neglected, and potentially lethal, parasitic disease that affects hundreds of millions of people worldwide. As part of the schistosome lifecycle, parasite eggs accumulate within the liver where they evoke intense granulomatous pathology, typified by a dense extracellular matrix (ECM) barrier, which serves to contain toxic egg secretions. In severe cases, this progressive and irreversible egg-evoked ECM deposition can lead to pathological scarring, impaired liver function and lethality. Thus, identifying the core regulators that govern ECM deposition may aid discovery of new therapeutic targets for schistosomiasis. The transcription factor Sex determining region Y-box 9 (Sox9) is a known regulator of pathological scaring. We found that, following Schistosoma mansoni infection, SOX9 was ectopically expressed in myofibroblasts within the granuloma and in surrounding hepatocytes. In the absence of SOX9, granuloma size was significantly diminished, and mice failed to produce a robust ECM barrier around eggs, resulting in more diffuse liver injury and scattered distribution of immune cells. Immunologically, SOX9 loss in both naive and infected mice led to an increase in hepatic neutrophil and monocyte proportions, with the expansion of Ly6clo monocyte populations in infected SOX9 deficient mice only. Infected SOX9-deficient mice also displayed exaggerated Type 2 inflammation, including pronounced eosinophilia. These data highlight the importance of SOX9 for intact hepatic granuloma formation during schistosomiasis and suggest SOX9 or its related factors may provide attractive future targets for meeting the clinical need to limit and/or reverse fibrotic disease. Author SummaryMammalian infection with schistosome worms results in the deposition of parasite eggs in the liver, where they secrete organ damaging toxins. In response, the liver generates a cellular granuloma barrier rich in extracellular matrix to limit these secretions and protect the overall organ. As in other liver injuries, SOX9 becomes progressively expressed in multiple cell types during the time course of schistosome infection. To understand the role of SOX9 in the liver response to schistosomes we utilised a global SOX9 deficient mouse model. These mice show reduced and disorganised granuloma formation during schistosome infection, with disrupted hepatic immune profiles. This suggests that SOX9 is required to form a robust and coordinated granuloma barrier that limits liver damage in this important but neglected parasitic disease.

immunology↗

Global diversity of soil-transmitted helminths reveals population-biased genetic variation that impacts diagnostic targets

Soil-transmitted helminths (STHs) are intestinal pathogens affecting over a billion people worldwide. STH control relies on microsopy-based diagnostics to monitor parasite prevalence and enable post-treatment surveillance, however, molecular diagnostics are rapidly being developed due to increased sensitivity, particularly in low-STH-prevalence settings. The genetic diversity of helminths and its potential impact on molecular diagnostics remains unclear. Using low-coverage genome sequencing, we assessed the presence of STHs in worm, faecal and purified egg samples from 27 countries, identifying differences in the genetic connectivity and diversity of STH-positive samples across regions and cryptic diversity between closely related human- and pig-infective species. We defined substancial copy number and sequence variants in current diagnostic targets and validated the impact of genetic variants on qPCR diagnostics using in vitro assays. Our study provides insights into the diversity and genomic epidemiology of STHs, highlighting challenges and opportunities for developing molecular diagnostics needed to support STH control efforts.

genomics↗

A rewiring of the earliest immune events leading to T-cell mediated disease following intestinal infection in a PINK1KO mouse model of Parkinsons disease

Parkinsons disease (PD) is characterized by a protracted period of non-motor symptoms, including gastrointestinal (GI) dysfunction, which can precede the development of the cardinal motor deficits by decades. This long prodrome of disease is highly suggestive of immune cell involvement in the initiation of disease, but currently the field lacks robust model systems to study such mechanisms. It has been hypothesized that pathology may be first initiated in the periphery due to environmental triggers, such as pathogens that enter the GI tract. We further speculate that the impact of such pathogens on the immune system could be exacerbated in genetically predisposed individuals. Our group has developed a GI-targeted pathogen-induced PD mouse model system in PINK1 KO mice with Gram-negative bacterial infections and found that T cells are a major player in driving PD-like motor symptoms at late stages following infection. Herein, we now map the initiating immune events at the site of infection at the earliest stages with the goal of shedding light on the earliest mechanisms triggering immune-mediated pathological processes relevant to PD. Using unbiased single cell sequencing, we demonstrate that myeloid cells are the earliest dysregulated immune cell type in PINK1 KO infected mice at 1-week post-infection, followed by a dysregulated T cell response shortly after, at 2 weeks post-infection. We find that these myeloid cells have an enhanced proinflammatory profile, are more mature, and develop enhanced capacity for antigen presentation. Using unbiased prediction analysis, our data suggest that cytotoxic T cells and myeloid cells are particularly poised for interacting with each other, and we identify possible direct cell-cell interaction pathways that might be implicated. Taken together, deciphering the earliest immune mechanisms in the periphery underpinning PD autoimmunity will be instrumental in the development of effective therapeutic targeting strategies before irrevocable neuronal damage ensues.

immunology↗

Hydrocarbon-degrading microbial populations in permanently cold deep-sea sediments in the NW Atlantic

Permanently cold deep-sea sediments (2500-3500 m water depth) with or without indications of thermogenic hydrocarbon seepage were exposed to naphtha to examine the presence and potential of aerobic hydrocarbon-degrading microbial populations. Monitoring these microcosms for volatile hydrocarbons by GC-MS revealed sediments without in situ hydrocarbons responded more rapidly to naphtha amendment than hydrocarbon seep sediments overall, but seep sediments removed BTEX compounds more readily. Naphtha-driven aerobic respiration was more evident in surface sediment (0-20 cmbsf) than deeper anoxic layers (>130 cmbsf) that responded less rapidly. In all cases, enrichment of Gammaproteobacteria included lineages of Oleispira, Pseudomonas, and Alteromonas known to be associated with marine oil spills. On the other hand, taxa known to be prevalent in situ and diagnostic for thermogenic hydrocarbon seepage in deep sea sediment did not respond to naphtha amendment. This suggests a limited role for seep-associated populations in the context of oil spill biodegradation.

microbiology↗

Modeling gene-environment interactions in Parkinson's Disease: Helicobacter pylori infection of Pink1-/- mice induces CD8 T cell-dependent motor and cognitive dysfunction.

Parkinsons disease (PD) is a chronic neurodegenerative disorder characterized by progressive loss of motor function. Diagnosis occurs late: after motor symptom development downstream of the irreparable loss of a large proportion of the dopaminergic neurons in the substantia nigra of the brain. Understanding PD pathophysiology in its pre-motor prodromal phase is needed for earlier diagnosis and intervention. Genetic risk factors, environmental triggers, and dysregulated immunity have all been implicated in PD development. Here, we demonstrate in a mouse model deficient in the PD-associated gene Pink, that infection with the human PD-associated gastric bacterium Helicobacter pylori leads to development of motor and cognitive signs resembling prodromal features of PD. This was also associated with proliferation and activation of primary mitochondria-reactive CD8 T cells and infiltration of CD8 T cells into the brain. Development of the motor and cognitive phenotypes in the infected Pink1-/- mice was abrogated when CD8 T cells were depleted prior to infection. We anticipate that this new model, which integrates genetic PD susceptibility, a PD-relevant environmental trigger, and specific immune changes that are required for symptom development, will be a valuable tool for increasing our understanding of this complex disease.

immunology↗

The breath shape controls intonation of mouse vocalizations

Intonation in speech is the control of vocal pitch to layer expressive meaning to communication, like increasing pitch to indicate a question. Also, stereotyped patterns of pitch are used to create distinct sounds with different denotations, like in tonal languages and, perhaps, the ten sounds in the murine lexicon. A basic tone is created by exhalation through a constricted laryngeal voice box, and it is thought that more complex utterances are produced solely by dynamic changes in laryngeal tension. But perhaps, the shifting pitch also results from altering the swiftness of exhalation. Consistent with the latter model, we describe that intonation in most vocalization types follows deviations in exhalation that appear to be generated by the re-activation of the cardinal breathing muscle for inspiration. We also show that the brainstem vocalization central pattern generator, the iRO, can create this breath pattern. Consequently, ectopic activation of the iRO not only induces phonation, but also the pitch patterns that compose most of the vocalizations in the murine lexicon. These results reveal a novel brainstem mechanism for intonation.

neuroscience↗

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↗

REPRODUCTIVE COMPATIBILITY OF TWO LINES OF DELIA PLATURA (DIPTERA: ANTHOMYIIDAE)

Accurate identification of agricultural pests is a major component of integrated pest management. The seedcorn maggot, Delia platura (Diptera: Anthomyiidae), is a cosmopolitan polyphagous pest species which may be found in high numbers in numerous crops. Two morphologically identical genetic lines of D. platura (H- and N- lines) with distinct distributions were recently identified. However, no study to date has investigated the reproductive compatibility of the two lines and thus the possibility that they may actually be two unique biological entities. A previous study described the reproductive traits of the two lines and suggested that H-line females are highly selective towards the male with which they mate, pointing to a possible pre-mating isolation mechanism between the lines. Using laboratory-reared colonies originating from the Monteregie region in Quebec, this study investigates the reproductive compatibility of the two D. platura lines. We found that only one of thirty H-line females was inseminated by an N-line male, further suggesting mate choice as a pre-mating isolation mechanism between the lines. However, N-line females were readily inseminated by H-line males, suggesting a lack of pre-mating isolation in this type of cross. The eggs laid by N-line females mated with H-line males had a lower hatching rate than the ones laid by females of intra-line crosses suggesting either post-mating pre-zygotic or post-zygotic partial isolation. However, the larvae that did hatch had a comparable developmental success to those from intra-line crosses in terms of survival and developmental time from larval hatching to adult emergence, pupal weight, and adult sex ratio, suggesting a lack of post-zygotic isolation for these life stages. Considering the different biological traits of the two lines, we suggest the use of the biotype terminology to designate the two biological entities and discuss their implications for integrated pest management.

ecology↗

Comparative Analysis of Methods to Reduce Activation Signature Gene Expression in PBMCs

Preserving the in vivo cell transcriptome is essential for accurate profiling, yet factors during cell isolation including time ex vivo and temperature induce artifactual gene expression, particularly in stress-responsive immune cells. In this study, we investigated two methods to mitigate ex vivo activation signature gene (ASG) expression in peripheral blood mononuclear cells (PBMCs): transcription and translation inhibitors (TTis) and cold temperatures during isolation. Comparative analysis of PBMCs isolated with TTis revealed reduced ASG expression. However, TTi treatment impaired responsiveness to LPS stimulation in subsequent in vitro experiments. In contrast, cold isolation methods also prevented ASG expression; up to a point where the addition of TTis during cold isolation offered minimal additional advantage. These findings highlight the importance of considering the advantages and drawbacks of different isolation methods to ensure accurate interpretation of PBMC transcriptomic profiles. HighlightsO_LITraditional room temperature isolation methods trigger activation signature gene expression in PBMCs, even when rapidly isolated, whereas 4{degrees}C isolation methods do not. C_LIO_LITranscription and translation inhibitors and cold processing techniques reduce activation signature gene expression via shared mechanisms. C_LIO_LIPBMCs treated with transcription and translation inhibitors lose responsiveness to external stimuli. C_LIO_LICold isolation methods offer a suitable and inexpensive alternative to mitigate activation signature gene expression in PBMCs. C_LI

immunology↗

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↗