Biology subjects

Gruenewald, A.

Publications and source records attributed to Gruenewald, A..

6 recordsLinked to original sources

bioRxiv2026

SNCA triplication shapes neuronal extracellular vesicle biology and promotes microglial activation in patient-derived iPSC-based models of Parkinson's disease

Extracellular vesicles (EVs) are emerging as key mediators of intercellular communication and potential biomarkers in Parkinson's disease (PD), yet how disease-causing genetic alterations shape neuronal EV biology remains incompletely understood. Here, we used PD-patient iPSC-derived midbrain dopaminergic neurons (mDANs) harboring SNCA triplication (SNCA-4x), gene-corrected controls (SNCA-GC), and SNCA knockout (SNCA-KO) to investigate the impact of -synuclein overexpression on neuronal-enriched EV (nEV) biology and neuron to microglia communication. SNCA-4x mDANs exhibited marked transcriptional alterations in pathways related to vesicle trafficking and extracellular matrix organization. Using an optimized isolation workflow, SNCA-4x neurons released significantly more and smaller nEVs enriched in -synuclein, mitochondrial DNA (mtDNA), and PARK7/DJ-1 mRNA, while displaying reduced acetylcholinesterase activity. Functionally, SNCA-4x-derived nEVs were taken up more efficiently by isogenic control iPSC-derived microglia than SNCA-GC nEVs and induced stronger pro-inflammatory activation than both SNCA-GC nEVs and untreated microglia, characterized by altered microglial morphology and increased TNF- and IL-1{beta}, consistent with damage-associated molecular pattern (DAMP)-mediated signaling. Together, these findings demonstrate that SNCA-4x reshapes the properties and molecular cargo of nEVs, enhancing their capacity to trigger microglial activation, and identify EV-associated -synuclein, mtDNA, and PARK7 mRNA as candidate mechanistic and biomarker features in PD.

neuroscience↗

bioRxiv2026

Single-cell resolution uncovers cell type-specific dysregulation in Parkin-deficient neuron-microglia co-cultures

BackgroundMutations in the E3 ubiquitin ligase Parkin (encoded by PRKN) are the most frequently known cause of recessively inherited Parkinsons disease. In addition to the loss of dopaminergic neurons, microglial activation is another pathological feature observed in Parkinsons disease. While postmortem brain samples show the end stage of the disease, neurons and glia derived from patients induced pluripotent stem cells (iPSCs) provide a model for detecting early pre-degenerative disease trajectories. However, mixed cell populations often confound these cultures, leading to heterogeneous disease phenotypes. MethodsHere, we tease apart the cell type-specific phenotypes underlying Parkin-linked Parkinsons disease by performing single-nucleus RNA sequencing in iPSC-derived co-cultures of dopaminergic neurons and microglia from PRKN mutation carriers and healthy controls. We validated our transcriptomic key findings through inflammatory cytokine profiling and live-cell calcium imaging. ResultsSingle-nucleus RNA sequencing identified seven major cell types composed of neuronal, glial, and precursor cells, with dopaminergic neurons accounting for the largest cell population. Pathway analysis revealed cell type-specific dysregulated biological processes in Parkin-deficient cells, including gene expression differences in dopaminergic neurons that control mitophagy and dopamine homeostasis, whereas microglia showed changes in calcium homeostasis and inflammatory signaling. Functional analysis verified elevated secretion of monocyte chemotactic protein 1 in PRKN-mutant co-cultures compared with controls, linking Parkin deficiency to increased microglial chemotactic signaling. Furthermore, lower intracellular calcium levels and diminished calcium release following treatment confirmed impaired calcium homeostasis in PRKN-mutant microglia. ConclusionsProfiling at the single-cell level resolved distinct cell subpopulations, enabling us to identify cell type-specific pathway disturbances underlying Parkin deficiency. This unique dataset provides a basis for understanding the impairment of individual cell types and the impact of cellular crosstalk in Parkinsons disease pathology.

molecular biology↗

bioRxiv2024

DJ-1 mediates regulation of metabolism and immune response in Parkinsons disease astrocytes and Glioblastoma cells

An inverse correlation for the expression of Parkinsons disease (PD)- and cancer-associated genes has been previously reported. Genes that are upregulated in cancer are frequently downregulated in PD and vice versa. PARK7, encoding DJ-1, was initially identified as an oncogene, but loss of DJ-1 causes early-onset PD. However, it remains elusive how differential DJ-1 levels contribute to opposite cell fates in cancer and PD. Here, we demonstrate specific effects of differential DJ-1 protein levels on the energy metabolism and cell growth in patient-derived cellular models of PD and glioblastoma (GBM) cell lines. Impaired energy metabolism was associated with an increased immune response upon IL-1{beta} stimulation and increased apoptosis and decreased cell growth in models of PD, whereas in GBM cells increased metabolic activity translated into a reduced immune response and increased cell growth. Furthermore, we found decreased glutathione (GSH) synthesis and therefore increased levels of reactive oxygen species (ROS) and oxidized glutathione (GSSG) in models of DJ-1 deficiency and decreased ROS levels in GBM cell lines. Thus, the mechanism by which DJ-1 modulates these phenotypes is the same in both diseases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/621212v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@4eccb4org.highwire.dtl.DTLVardef@161669corg.highwire.dtl.DTLVardef@197bfc3org.highwire.dtl.DTLVardef@1aecb36_HPS_FORMAT_FIGEXP M_FIG C_FIG DJ-1 levels modulate GSSG/GSH ratio and ROS levels, which results in divergent effects on cell growth and immune response in DJ-1-dependent glial pathologies in glioblastoma and PD. In models of PD, DJ-1 level dependent phenotypes can be rescued by antioxidant treatment that reduces the GSSG/GSH ratio and ROS levels.

neuroscience↗

bioRxiv2022

A conserved isoleucine in the binding pocket of RIG-I controls immune tolerance to mitochondrial RNA

RIG-I is a cytosolic receptor of viral RNA essential for the immune response to numerous RNA viruses. Accordingly, RIG-I must sensitively detect viral RNA yet tolerate abundant self-RNA species. The basic binding cleft and an aromatic amino acid of the RIG-I C-terminal domain(CTD) mediate high-affinity recognition of 5triphosphorylated and 5base-paired RNA(dsRNA). Here, we found that, while 5unmodified hydroxyl(OH)-dsRNA demonstrated residual activation potential, 5-monophosphate(5p)-termini, present on most cellular RNAs, prevented RIG-I activation. Determination of CTD/dsRNA co-crystal structures and mutant activation studies revealed that the evolutionarily conserved I875 within the CTD sterically inhibits 5p-dsRNA binding. RIG-I(I875A) was activated by both synthetic 5p-dsRNA and endogenous long dsRNA within the polyA-rich fraction of total cellular RNA. RIG-I(I875A) specifically interacted with a long, highly structured, polyA-bearing, non-coding mitochondrial(mt) RNA, and depletion of mtRNA from total RNA abolished its activation. Altogether, our study demonstrates that avoidance of 5p-RNA recognition is crucial to preventing mtRNA-triggered RIG-I-mediated autoinflammation.

immunology↗

bioRxiv2021

Analysis of mitochondrial genome methylation using Nanopore single-molecule sequencing

The level and the biological significance of mitochondrial DNA (mtDNA) methylation in human cells is a controversial topic. Using long-read third-generation sequencing technology, mtDNA methylation can be detected directly from the sequencing data, which overcomes previously suggested biases, introduced by bisulfite treatment-dependent methods. We investigated mtDNA from whole blood-derived DNA and established a workflow to detect CpG methylation with Nanopolish. In order to obtain native mtDNA, we adjusted a whole-genome sequencing protocol and performed ligation library preparation and Nanopore sequencing. To validate the workflow, 897bp of methylated and unmethylated synthetic DNA samples at different dilution ratios were sequenced and CpG methylation was detected. Interestingly, we observed that reads with higher methylation in the synthetic DNA did not pass Guppy calling, possibly affecting conclusions about DNA methylation in Nanopore sequencing. We detected in all blood-derived samples overall low-level methylation across the mitochondrial genome, with exceptions at certain CpG sites. Our results suggest that Nanopore sequencing is capable of detecting low-level mtDNA methylation. However, further refinement of the bioinformatical pipelines including Guppy failed reads are recommended.

genetics↗

bioRxiv2017

A dementia-associated risk variant near TMEM106B alters chromatin architecture and gene expression

Neurodegenerative diseases pose an extraordinary threat to the worlds aging population, yet no disease-modifying therapies are available. While genome-wide association studies (GWAS) have identified hundreds of novel risk loci for neurodegeneration, the mechanisms by which these loci influence disease risk are largely unknown. Indeed, of the many thousands of SNP-trait associations identified by GWAS over the past ~10 years, very few are understood mechanistically. Here, we investigate the association of common genetic variants at the 7p21 locus with risk for the neurodegenerative disease frontotemporal lobar degeneration. We show that variants associated with disease risk correlate with increased brain expression of the 7p21 gene TMEM106B, and no other genes. Furthermore, incremental increases in TMEM106B levels result in incremental increases in lysosomal phenotypes and cell toxicity. We then combine fine-mapping, bioinformatics, and bench-based approaches to functionally characterize all candidate causal variants at this locus. This approach identified a noncoding variant, rs1990620, which differentially recruits CTCF, influencing CTCF-mediated long-range chromatin looping interactions between multiple cis-regulatory elements, including the TMEM106B promoter. Our findings thus provide an in-depth analysis of the 7p21 locus linked by GWAS to frontotemporal lobar degeneration, nominating a causal variant and a causal mechanism for allele-specific expression and disease association at this locus. Finally, we show that genetic variants associated with risk for neurodegenerative diseases beyond frontotemporal lobar degeneration are enriched in brain CTCF-binding sites genome-wide, implicating CTCF-mediated gene regulation in risk for neurodegeneration more generally.

genetics↗