Biology subjects

Sammler, E.

Publications and source records attributed to Sammler, E..

7 recordsLinked to original sources

bioRxiv2025

Robust analytical methods for bis(monoacylglycero)phosphate profiling in health and disease

Bis(monoacylglycero)phosphate (BMP), a distinct anionic phospholipid predominantly found in late endosomes and lysosomes, plays a pivotal role in supporting lysosomal functions and maintaining metabolic homeostasis. Its impaired function is associated with an array of disorders, notably neurodegenerative diseases. However, the identification and quantitation of BMP remains difficult due to its structural similarity to isomer phosphatidylglycerol (PG), thus necessitating robust analytical methods for accurate and reliable BMP profiling. In this study, we present comprehensive liquid chromatography - tandem mass spectrometry (MS2) methodologies for the precise and systematic analysis of BMP species in biological samples. We detail LC/MS methods for both an untargeted Orbitrap mass spectrometer and a targeted triple quadrupole (QQQ) mass spectrometer. We utilize differences in polarity and structure to annotate BMPs and PGs based on retention time and positive mode MS2 fragmentation patterns, respectively. Further, we propose a new approach for overcoming common challenges in BMP profiling by leveraging the newly discovered biochemical function of CLN5 as the BMP synthase. Since genetic ablation of CLN5 leads to specific depletion of BMPs but not PGs, we use lipid extracts from CLN5 knockout (KO) and wild-type (WT) cells as biological standards to confidently annotate BMPs as targets with significantly low BMP Identification Index (BMPII), defined as BMPII = CLN5 KO / WT. We additionally propose the BMP enrichment score (BMPES) as a secondary validation metric, defined as lysosomal abundance of BMP / whole-cell abundance. Altogether, this approach constitutes a robust method for BMP profiling and annotation, furthering research into health and disease.

biochemistry↗

bioRxiv2024

Tagless LysoIP method for molecular profiling of lysosomal content in clinical samples

Lysosomes are implicated in a wide spectrum of human diseases including monogenic lysosomal storage disorders (LSDs), age-associated neurodegeneration and cancer. Profiling lysosomal content using tag-based lysosomal immunoprecipitation (LysoTagIP) in cell and animal models allowed major discoveries in the field, however studying lysosomal dysfunction in human patients remains challenging. Here, we report the development of the "tagless LysoIP method" to enable rapid enrichment of lysosomes, via immunoprecipitation, using the endogenous integral lysosomal membrane protein TMEM192, directly from clinical samples and human cell lines (e.g. induced Pluripotent Stem Cell (iPSCs) derived neurons). Isolated lysosomes are intact and suitable for subsequent multimodal omics analyses. To validate our approach, we employed the tagless LysoIP to enrich lysosomes from peripheral blood mononuclear cells (PBMCs) derived from fresh blood from patients with CLN3 disease, a neurodegenerative LSD. Metabolic profiling of isolated lysosomes showed massive accumulation of glycerophosphodiesters (GPDs) in patients lysosomes. Interestingly, a patient with a milder phenotype and genotype displayed lower accumulation of lysosomal GPDs, consistent with their potential role as disease biomarkers. Altogether, the tagless LysoIP provides a framework to study native lysosomes from patient samples, identify novel biomarkers and discover human-relevant disease mechanisms.

cell biology↗

bioRxiv2024

Regulation of Leucine-Rich Repeat Kinase 2 by inflammation and IL-4

Mutations in Leucine-Rich Repeat protein Kinase 2 (LRRK2) are associated with Parkinsons Disease (PD) and Crohns Disease (CD), but the regulation of LRRK2 during inflammation remains relatively unexplored. Here we developed a flow cytometry-based assay to assess LRRK2 activity in individual cells and created an EGFP-Lrrk2-knock-in reporter mouse to analyse cell-specific LRRK2 expression. Using these tools, we catalogued LRRK2 levels and activity in splenic and intestinal immune cells. Inflammation increased LRRK2 expression and activity in B-cells, immature neutrophils and immature monocytes, but decreased these in dendritic cells and eosinophils. In mature neutrophils, inflammation stimulated activity but reduced LRRK2 expression. A kinase-activating PD-associated LRRK2-R1441C mutation exacerbated inflammation-induced activation of LRRK2 specifically in monocytes and macrophages without affecting LRRK2 levels. Finally, we identified IL-4 as a novel factor that upregulated LRRK2 expression and activity in B-cells, replicating inflammatory effects observed in vivo. Our findings provide valuable new insights into the regulation of the LRRK2 pathway in immune cells, crucial for understanding LRRK2 and its therapeutic potential in inflammatory diseases such as CD.

immunology↗

bioRxiv2023

The R1441C-LRRK2 mutation induces myeloid immune cell exhaustion in an age- and sex-dependent manner

Considering age is the greatest risk factor for many neurodegenerative diseases, aging, in particular aging of the immune system, is the most underappreciated and understudied contributing factor in the neurodegeneration field. Genetic variation around the LRRK2 gene affects risk of both familial and sporadic Parkinsons disease (PD). The leucine-rich repeat kinase 2 (LRRK2) protein has been implicated in peripheral immune signaling, however, the effects of an aging immune system on LRRK2 function have been neglected to be considered. We demonstrate here that the R1441C mutation induces a hyper-responsive phenotype in macrophages from young female mice, characterized by increased effector functions, including stimulation-dependent antigen presentation, cytokine release, phagocytosis, and lysosomal function. This is followed by age-acquired immune cell exhaustion in a Lrrk2-kinase-dependent manner. Immune-exhausted macrophages exhibit suppressed antigen presentation and hypophagocytosis, which is also demonstrated in myeloid cells from R1441C and Y1699C-PD patients. Our novel findings that LRRK2 mutations confer immunological advantage at a young age but may predispose the carrier to age-acquired immune cell exhaustion have significant implications for LRRK2 biology and therapeutic development. Indeed, LRRK2 has become an appealing target in PD, but our findings suggest that more research is required to understand the cell-type specific consequences and optimal timing of LRRK2-targeting therapeutics. One Sentence SummaryThe R1441C-LRRK2 mutation causes an age-acquired immune cell exhaustion in macrophages in a sex-dependent manner

immunology↗

bioRxiv2022

Impact of 98 LRRK2 variants linked to Parkinson's Disease on kinase activity and microtubule binding

Mutations enhancing the kinase activity of LRRK2 cause Parkinsons disease (PD) and therapies that reduce LRRK2 kinase activity are being tested in clinical trials. Numerous rare variants of unknown clinical significance have been reported, but how the vast majority impact on LRRK2 function is unknown. Here, we investigate 100 LRRK2 variants linked to PD, including previously described pathogenic mutations. We identify 23 LRRK2 variants that robustly stimulate kinase activity, including variants within the N-terminal non-catalytic regions [ARM (E334K, A419V), ANK(R767H), LRR (R1067Q, R1325Q)], as well as variants predicted to destabilise the ROC:CORB interface [ROC (A1442P, V1447M), CORA (R1628P) CORB (S1761R, L1795F)] and COR:COR dimer interface [CORB (R1728H/L)]. Most activating variants decrease LRRK2 biomarker site phosphorylation (pSer935/pSer955/pSer973), consistent with the notion that the active kinase conformation blocks their phosphorylation. We conclude that the impact of variants on kinase activity is best evaluated by deploying a cellular assay of LRRK2-dependent Rab10 substrate phosphorylation, compared to a biochemical kinase assay, as only a minority of activating variants [CORB (Y1699C, R1728H/L, S1761R) and kinase (G2019S, I2020T, T2031S)], enhance in vitro kinase activity of immunoprecipitated LRRK2. Twelve variants including several that activate LRRK2 and have been linked to PD, suppressed microtubule association in the presence of a Type I kinase inhibitor [ARM(M712V), LRR(R1320S), ROC (A1442P, K1468E, S1508R), CORA(A1589S), CORB (Y1699C, R1728H/L) and WD40(R2143M, S2350I, G2385R)]. Our findings will stimulate work to better understand the mechanisms by which variants impact biology and provide rationale for variant carrier inclusion or exclusion in ongoing and future LRRK2 inhibitor clinical trials.

neuroscience↗

bioRxiv2022

A mitochondrial blood-based patient stratification candidate biomarker for Parkinson's disease

Parkinsons disease (PD) is the most common neurodegenerative movement disorder and neuroprotective interventions remain elusive. High throughput biomarkers aimed to stratify patients based on shared etiology is one critical path to the success of disease-modifying therapies in clinical trials. Mitochondrial dysfunction plays a prominent role in the pathogenesis of PD. Previously, we found brain region-specific mitochondrial DNA (mtDNA) damage accumulation in neuronal and in vivo PD models, as well as human PD postmortem brain tissue. In this study, to investigate mtDNA damage as a potential blood biomarker for PD, we describe a novel Mito DNADX assay that allows for the accurate real-time quantification of mtDNA damage in a 96-well platform, compatible with assessing large cohorts of patient samples. We found that levels of mtDNA damage were increased in blood derived from early-stage idiopathic PD patients or those harboring the pathogenic LRRK2 G2019S mutation compared to age-matched healthy controls. Given that increased mtDNA damage was also found in non-manifesting LRRK2 mutation carriers, mtDNA damage may begin to accumulate prior to a clinical PD diagnosis. LRRK2 kinase inhibition mitigated mtDNA damage in idiopathic PD models and patient-derived cells. The latter observations further substantiate a mechanistic role for wild-type LRRK2 kinase activity in idiopathic PD and support mtDNA damage reversal as a suitable approach to slow PD-related pathology. In light of recent advances in the field of precision medicine, the analysis of mtDNA damage as a blood-based patient stratification biomarker should be included in future clinical trials. One Sentence SummaryBlood test identifies Parkinsons patients most likely to respond to mitochondria-targeted therapeutics facilitating a precision medicine approach.

neuroscience↗

bioRxiv2020

Development of a multiplexed targeted mass spectrometry assay for LRRK2 phosphorylated Rabs and Ser910/Ser935 biomarker sites

Mutations that increase the protein kinase activity of LRRK2 are one of the most common causes of familial Parkinsons disease. LRRK2 phosphorylates a subset of Rab GTPases within their Switch-II motif, impacting interaction with effectors. We describe and validate a new, multiplexed targeted mass spectrometry assay to quantify endogenous levels of LRRK2-phosphorylated Rab substrates (Rab1, Rab3, Rab8, Rab10, Rab35 and Rab43) as well as total levels of Rabs, LRRK2 and LRRK2-phosphorylated at the Ser910 and Ser935 biomarker sites. Exploiting this assay, we quantify for the first time the relative levels of each of the pRab proteins in different cells (mouse embryonic fibroblasts, human neutrophils) and mouse tissues (brain, kidney, lung and spleen). We define how these components are impacted by Parkinsons pathogenic mutations (LRRK2[R1441C] and VPS35[D620N]) and LRRK2 inhibitors. We find that the VPS35[D620N], but not LRRK2[R1441C] mutation, enhances Rab1 phosphorylation in a manner blocked by administration of an LRRK2 inhibitor, providing the first evidence that endogenous Rab1 is a physiological substrate for LRRK2. We exploit this assay to demonstrate that in Parkinsons patients with VPS35[D620N] mutations, phosphorylation of multiple Rab proteins (Rab1, Rab3, Rab8, Rab10 and Rab43) is elevated. We highlight the benefits of this assay over immunoblotting approaches currently deployed to assess LRRK2 Rab signalling pathway.

neuroscience↗