bioRxiv Science⌕ Search

Biology subjects

Konrad, C.

Publications and source records attributed to Konrad, C..

12 recordsLinked to original sources

A mouse model of CHCHD10 p.R15L familial ALS presents mild, age-related motor neuron degeneration without protein instability or mitochondrial dysfunction

Mutations in the mitochondrial protein CHCHD10 (D10) cause a spectrum of hereditary neurodegenerative disorders. Among these, the p.R15L variant is linked to a slowly progressive, late-onset familial form of amyotrophic lateral sclerosis (ALS) with unclear pathogenic mechanisms. To better understand this, we investigated a knock-in (KI) mouse model carrying the p.R15L mutation in the endogenous protein. Unlike previously described mutant D10 KI models, p.R15L KI mice exhibited normal D10 protein levels, with no evidence of large protein aggregates. Mitochondrial respiration and hydrogen peroxide emission in mitochondria isolated from muscle and brain were unaltered. Similarly, fibroblasts from human p.R15L carriers exhibited normal D10 levels and unchanged oxidative phosphorylation function. Histochemical analyses of p.R15L KI muscle revealed mild increases in mitochondrial enzymatic activity in a subset of muscle fibers and muscle transcriptomics showed elevated expression of PGC-1, suggesting enhanced mitochondrial biogenesis. p.R15L KI mice developed subtle, late-onset phenotypes, including reduced body weight and motor activity and increased anxiety-like behavior. Importantly, in aged mice electrophysiological studies demonstrated decreased amplitude of the compound muscle action potential, commensurate with a moderate loss of spinal cord motor neurons and elevated serum neurofilament light levels, indicative of neurodegeneration. Together, these results indicate that the p.R15L mutation produces a mild, late-onset motor neuron phenotype in mice, partially recapitulating the human disease, without mitochondrial functional or morphological alterations. The findings indicate that p.R15L D10 selectively impairs mouse motor neurons through a gain-of-function mechanism, providing a genetically accurate yet mild in vivo model of familial ALS.

neuroscience↗

Mitochondrial dysfunction precedes neurodegeneration in DRPLA patient-derived neurons, and phenylbutyrate improves survival.

Dentatorubral-pallidoluysian atrophy (DRPLA) is a progressive autosomal-dominant neurodegenerative disorder caused by a CAG repeat expansion in the ATN1 gene, which encodes the transcriptional corepressor Atrophin-1. The temporal sequence of molecular mechanisms driving neuronal dysfunction and degeneration in DRPLA is poorly understood, limiting therapeutic development. We generated patient-derived induced pluripotent stem cells and differentiated them into cortical excitatory glutamatergic neurons to model early pathogenic processes. Early pathological features of DRPLA patient-derived neurons included mitochondrial dysfunction and oxidative stress. These alterations occurred before overt neuronal loss, highlighting bioenergetic stress as a key early driver of disease progression toward neurodegeneration. Pharmacological treatment with phenylbutyrate significantly improved neuronal survival and reduced mitochondrial reactive oxygen species production, demonstrating the therapeutic potential of targeting mitochondrial dysfunction and oxidative stress. These findings challenge the conventional aggregation-centric model of polyglutamine disease pathogenesis and position mitochondrial stress as a central and early promoter of neuronal degeneration in DRPLA. By providing mechanistic insight into early-stage disease processes, our study lays the foundation for therapeutic strategies targeting mitochondrial dysfunction in DRPLA and related polyglutamine disorders.

neuroscience↗

Rescuing Ischemic Brain Injury by Rewiring Mitochondrial Electron Flow

Mitochondrial metabolic flux alterations are critical drivers of acute ischemia-reperfusion (IR) brain injury. Reverse electron transfer (RET), defined as the upstream flow of electrons from the quinone pool to complex I, is a major source of pathological reactive oxygen species (ROS) under stress conditions. In an in vivo brain IR model, oxygen deprivation induces the buildup of RET-supporting substrates, with glycerol 3-phosphate identified as the dominant contributor in addition to succinate. Rapid oxidation of these substrates by brain mitochondria upon reoxygenation drives massive ROS production, while also leading to over-reduction and dissociation of the complex I flavin mononucleotide (FMN) cofactor. The resulting FMN-deficient complex I becomes catalytically impaired, unable to oxidize NADH or to produce ROS. To mitigate RET and preserve complex I function, we used transgenic mice xenotopically expressing alternative oxidase (AOX). This enzyme bypasses complexes III and IV by directly oxidizing the reduced quinone pool and passing electrons onto molecular oxygen. AOX expression did not alter complex I abundance, supercomplexes assembly, or basal respiration rates, but effectively diverted electrons from the quinone pool, decreasing RET flux via complex I and limiting ROS generation during IR. For the first time we showed that AOX expression and attenuation of RET preserved complex I FMN binding, suppressed oxidative stress, and conferred neuroprotection in vivo. Our findings reveal a novel strategy for rewiring mitochondrial electron flux to mitigate initial IR brain injury, highlighting modulation of the quinone pool by AOX as a potential therapeutic strategy for IR.

biochemistry↗

Disease Severity Across Psychiatric Disorders Is Linked to Pro-Inflammatory Cytokines

ImportanceNumerous studies indicate that the traditional categorical classification of severe mental disorders (SMD), such as schizophrenia, bipolar disorders, and major depressive disorders, does not align with the underlying biology of those disorders as they frequently overlap in terms of symptoms and risk factors. ObjectiveThis study aimed to identify transdiagnostic patient clusters based on disease severity and explore the underlying biological mechanisms independently of the traditional categorical classification. DesignWe utilized data from 443 participants diagnosed with SMD of the PsyCourse Study, a longitudinal study with deep phenotyping across up to four visits. We performed longitudinal clustering to group patients based on symptom trajectories and cognitive performance. The resulting clusters were compared on cross-sectional variables, including independent measures of severity as well as polygenic risk scores, serum protein quantification, miRNA expression, and DNA methylation. ResultsWe identified two distinct clusters of patients that exhibited marked differences in illness severity but did not differ significantly in age, sex, or diagnostic proportions. We found 19 serum proteins significantly dysregulated between the two clusters. Functional enrichment pointed to a convergence of immune system dysregulation and neurodevelopmental processes. ConclusionThe observed differences in serum protein expression suggest that disease severity is associated with the convergence of immune system dysregulation and neurodevelopmental alterations, particularly involving pathways related to inflammation and brain plasticity. The identification of pro-inflammatory proteins among the differentially expressed markers underscores the potential role of systemic inflammation in the pathophysiology of SMD. These results highlight the importance of considering illness severity as a core dimension in psychiatric research and clinical practice and suggest that targeting immune-related mechanisms may offer promising new therapeutic avenues for patients with SMD. Key points QuestionCan analyzing symptom trajectories and cognitive profiles across diagnostic categories reveal clinically relevant subgroups in severe mental disorders? FindingsIn this longitudinal study of 443 individuals with severe mental disorders, two distinct clusters emerged, differing significantly in illness severity, with the more severe group displaying elevated pro-inflammatory serum proteins, suggesting an association between disease severity and inflammation. MeaningThese findings suggest that transdiagnostic clustering clarifies shared mechanisms, underscores the importance of inflammation in severe mental disorders, and highlights a promising avenue for novel therapeutic approaches.

immunology↗

Investigation of mitochondrial phenotypes in motor neurons derived by direct conversion of fibroblasts from familial ALS subjects

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease of motor neurons, leading to fatal muscle paralysis. Familial forms of ALS (fALS) account for approximately 10% of cases and are associated with mutations in numerous genes. Alterations of mitochondrial functions have been proposed to contribute to disease pathogenesis. Here, we employed a direct conversion (DC) technique to generate induced motor neurons (iMN) from skin fibroblasts to investigate mitochondrial phenotypes in a patient-derived disease relevant cell culture system. We converted 7 control fibroblast lines and 17 lines harboring the following fALS mutations, SOD1A4V, TDP-43N352S, FUSR521G, CHCHD10R15L, and C9orf72 repeat expansion. We developed new machine learning approaches to identify iMN, analyze their mitochondrial function, and follow their fate longitudinally. Mitochondrial and energetic abnormalities were observed, but not all fALS iMN lines exhibited the same alterations. SOD1A4V, C9orf72, and TDP-43N352S iMN had increased mitochondrial membrane potential, while in CHCHD10R15L cells membrane potential was decreased. TDP-43N352S iMN displayed changes in mitochondrial morphology and increased motility. SOD1A4V, TDP-43N352S, and CHCHD10R15L iMN had increased oxygen consumption rates and altered extracellular acidification rates, reflecting a hypermetabolic state similar to the one described in sporadic ALS fibroblasts. FUSR521G mutants had decreased ATP/ADP ratio, suggesting impaired energy metabolism. We then tested the viability of iMN and found decreases in survival in SOD1A4V, C9orf72, and FUSR521G, which were corrected by small molecules that target mitochondrial stress. Together, our findings reinforce the role of mitochondrial dysfunction in ALS and indicate that fibroblast-derived iMN may be useful to study fALS metabolic alterations. Strengths of the DC iMN approach include low cost, speed of transformation, and the preservation of epigenetic modifications. However, further refinement of the fibroblasts DC iMN technique is still needed to improve transformation efficiency, reproducibility, the relatively short lifespan of iMN, and the senescence of the parental fibroblasts.

neuroscience↗

RPPA survey of cancer hotspot panel proteins and cell markers in matched tumor-normal human breast and kidney samples reveals a weak correlation between proteins expression and public transcriptome repositories

Despite the tremendous global effort in discovering and cataloguing somatic mutations in cancer-related genes and the impact they exert on human cancers, the effects of these mutations on the levels of protein expression are inadequately addressed. Here, we semi-quantitated the expression of 48 (out of 50) proteins represented by the most widely used cancer hotspot panel, the Ion AmpliSeq Cancer Hotspot Panel v2, and 14 cell markers commonly used as house-keeping proteins, using reverse phase protein array (RPPA) technology in 586 human breast- and 192 kidney matched tumor-normal samples. We further correlated our RPPA data with gene chip data from the Gene Expression Omnibus repository fetched through the TNMplot portal. Nearly half of proteins expression exhibited a negative correlation from their gene expression, while several of the remaining were only moderately correlated. Our data complement the vast information obtained elsewhere by transcriptomic analysis of the respective genes and gene products, potentially assisting in harmonizing proteomic with NGS outputs.

cancer biology↗

Machine learning approaches based on fibroblast morphometry confidently identify stress but have limited ability to predict ALS

ObjectiveAmyotrophic lateral sclerosis (ALS) is a devastating neuromuscular disease with limited therapeutic options. Diagnostic and surrogate endpoint biomarkers are needed for early disease detection, clinical trial design, and personalized medicine. MethodsWe tested the predictive power of a large set of primary skin fibroblast (n=443) from sporadic and familial ALS patients and healthy controls. We measured morphometric features of endoplasmic reticulum, mitochondria, and lysosomes by imaging with vital dyes. We also analysed immunofluorescence images of ALS-linked proteins, including TDP-43 and stress granule components. We studied fibroblasts under basal conditions and under metabolic (galactose medium), oxidative (arsenite), and heat stress conditions. We then employed machine learning (ML) techniques on the dataset to develop biomarkers. ResultsStress perturbations caused robust changes in the measured features, such as organellar morphology, stress granule formation, and TDP-43 mislocalization. ML approaches were able to predict the perturbation with near perfect performance (ROC-AUC > 0.99). However, when trying to predict disease state or disease groups (e.g., sporadic, or familial ALS), the performance of the ML algorithm was more modest (ROC-AUC Control vs ALS = 0.63). We also detected modest but significant scores when predicting clinical features, such as age of onset (ROC-AUC late vs early = 0.60). ConclusionsOur findings indicate that the ML morphometry we developed can accurately predict if human fibroblasts are under stress, but the differences between ALS and controls, while statistically significant, are small and pose a challenge for the development of biomarkers for clinical use by these approaches.

neuroscience↗

S1P controls endothelial sphingolipid homeostasis via ORMDL

Sphingolipids (SL) are both membrane building blocks and potent signaling molecules regulating a variety of cellular functions in both physiological and pathological conditions. Under normal physiology, sphingolipid levels are tightly regulated, whereas disruption of sphingolipid homeostasis and signaling has been implicated in diabetes, cancer, cardiovascular and autoimmune diseases. Yet, mechanisms governing cellular sensing of SL, and according regulation of their biosynthesis remain largely unknown. In yeast, serine palmitoyltransferase (SPT), catalyzing the first and rate limiting step of sphingolipid de novo biosynthesis, is negatively regulated by Orosomucoid 1 and 2 (Orm) proteins. Lowering sphingolipid levels triggers Orms phosphorylation, resulting in the removal of the inhibitory brake on SPT to enhance sphingolipid de novo biosynthesis. However, mammalian orthologs ORMDLs lack the N-terminus hosting the phosphosites. Thus, which sphingolipid(s) are sensed by the cells, and mechanisms of homeostasis remain largely unknown. This study is aimed at filling this knowledge gap. Here, we identify sphingosine-1-phosphate (S1P) as the key sphingolipid sensed by endothelial cells via S1PRs. The increase of S1P-S1PR signaling stabilizes ORMDLs, which downregulates SPT activity to maintain SL homeostasis. These findings reveal the S1PR/ORMDLs axis as the sensor-effector unit regulating SPT activity accordingly. Mechanistically, the hydroxylation of ORMDLs at Pro137 allows a constitutive degradation of ORMDLs via ubiquitin-proteasome pathway, therefore preserving SPT activity at steady state. The disruption of the S1PR/ORMDL axis results in ceramide accrual, mitochondrial dysfunction, and impaired signal transduction, all leading to endothelial dysfunction, which is an early event in the onset of cardio- and cerebrovascular diseases. The disruption of S1P-ORMDL-SPT signaling may be implicated in the pathogenesis of conditions such as diabetes, cancer, cardiometabolic disorders, and neurodegeneration, all characterized by deranged sphingolipid metabolism. Our discovery may provide the molecular basis for a therapeutic intervention to restore sphingolipid homeostasis.

cell biology↗

Tau interactome mapping reveals dynamic processes in synapses and mitochondria associated with neurodegenerative disease

Tau (MAPT) drives neuronal dysfunction in Alzheimers disease (AD) and other tauopathies. To dissect the underlying mechanisms, we combined an engineered ascorbic acid peroxidase (APEX) approach with quantitative affinity purification mass spectrometry (AP-MS) followed by proximity ligation assay (PLA) to characterize Tau interactomes modified by neuronal activity and mutations that cause frontotemporal dementia (FTD) in human induced pluripotent stem cell (iPSC)-derived neurons. We established activity-dependent interactions of Tau with presynaptic vesicle proteins during Tau secretion and mapped the exact APEX-tau-induced biotinylated tyrosines to the cytosolic domains of the interacting vesicular proteins. We showed that FTD mutations impair bioenergetics and markedly diminished Taus interaction with mitochondria proteins, which were downregulated in AD brains of multiple cohorts and correlated with disease severity. These multi-modal and dynamic Tau interactomes with unprecedented spatiotemporal resolution shed novel insights into Taus role in neuronal function and disease-related processes with potential therapeutic targets to block Tau-mediated pathogenesis.

neuroscience↗

Targeting the Id1-Kif11-Aurka axis in triple negative breast cancer using combination therapy

Evidence points to breast cancer following a hierarchical model, with Cancer Stem Cells (CSCs) driving critical phenotypes of the bulk tumor. Chemoresistant CSCs are not an abstract concept but have clinical consequences as they drive relapse and ultimately lead to mortality in patients, making it imperative to understand how these subpopulations of cells survive. Our previous work (1-2) has demonstrated that the bHLH transcription factor, Inhibitor of Differentiation 1 (Id1) and its closely related family member Id3, have an important role in maintaining the CSC phenotype in the Triple Negative breast cancer (TNBC) subtype. A genetic screen conducted to further elucidate the molecular mechanism underlying the Id (Id1/3) mediated CSC phenotypes in TNBC revealed critical cell cycle genes such as Kif11 and Aurka as putative Id targets. We take this work forward by investigating how alteration in Kif11 and Aurka via Id proteins promotes the CSC phenotype in TNBC. Cells lacking Id are poised in a state of G0/G1 arrest from which they can re-enter the cell cycle. Intriguingly, depletion of Kif11 and Aurka independently did not phenocopy the G0/G1 arrest observed in Id knockdown (Id KD) cells. We have further explored the hypothesis that we can deplete the chemo resistant Id expressing CSC population by combining chemotherapy with targeted therapy using existing small molecule inhibitors (against Id target Kif11) to more effectively debulk the entire tumor. This work opens up exciting new possibilities of targeting Id targets like Kif11, in the TNBC subtype which is currently refractory to chemotherapy.

cancer biology↗

Id proteins promote a cancer stem cell phenotype in triple negative breast cancer via Robo1-dependent c-Myc activation

Breast cancers display phenotypic and functional heterogeneity and several lines of evidence support the existence of cancer stem cells (CSCs) in certain breast cancers, a minor population of cells capable of tumor initiation and metastatic dissemination. Identifying factors that regulate the CSC phenotype is therefore important for developing strategies to treat metastatic disease. The Inhibitor of Differentiation Protein 1 (Id1) and its closely related family member Inhibitor of Differentiation 3 (Id3) (collectively termed Id) are expressed by a diversity of stem cells and are required for metastatic dissemination in experimental models of breast cancer. In this study, we show that ID1 is expressed in rare neoplastic cells within ER-negative breast cancers. To address the function of Id1 expressing cells within tumors, we developed two independent murine models of Triple Negative Breast Cancer (TNBC) in which a genetic reporter permitted the prospective isolation of Id1+ cells. Id1+ cells are enriched for self-renewal in tumorsphere assays in vitro and for tumor initiation in vivo. Conversely, depletion of Id1 and Id3 in the 4T1 murine model of TNBC demonstrates that Id1/3 are required for cell proliferation and self-renewal in vitro, as well as primary tumor growth and metastatic colonization of the lung in vivo. Using combined bioinformatic analysis, we have defined a novel mechanism of Id protein function via negative regulation of the Roundabout Axon Guidance Receptor Homolog 1 (Robo1) leading to activation of a Myc transcriptional programme.

cancer biology↗

Identification of a Distinct Metabolomic Subtype of Sporadic ALS Patients

Sporadic amyotrophic lateral sclerosis (sALS) is a progressive motor neuron disease resulting in paralysis and death. Genes responsible for familial ALS have been identified, however the molecular basis for sALS is unknown. To discover metabotypic biomarkers that inform on disease etiology, untargeted metabolite profiling was performed on 77 patient-derived dermal fibroblast lines and 45 age/sex-matched controls. Surprisingly, 25% of sALS lines showed upregulated methionine-derived homocysteine, channeled to cysteine and glutathione (GSH). Stable isotope tracing of [U-13C]-glucose showed activation of the trans-sulfuration pathway, associated with accelerated glucose flux into the TCA cycle, glutamate, GSH, alanine, aspartate, acylcarnitines and nucleotide phosphates. A four-molecule support vector machine model distinguished the sALS subtype from controls with 97.5% accuracy. Plasma metabolite profiling identified increased taurine as a hallmark metabolite for this sALS subset, suggesting systemic perturbation of cysteine metabolism. Furthermore, integrated multiomics (mRNAs/microRNAs/metabolites) identified the super-trans-sulfuration pathway as a top hit for the sALS subtype. We conclude that sALS can be stratified into distinct metabotypes, providing for future development of personalized therapies that offer new hope to sufferers.

neuroscience↗