Biology subjects

Korneck, M.

Publications and source records attributed to Korneck, M..

4 recordsLinked to original sources

bioRxiv2026

Deep axonal proteomics of human iPSC-derived neurons by microfluidic separation and DIA-MS

One of the defining features of neurons is their compartmentalisation into soma, dendrites and axon. Axons are highly specialised for the transmission of signals over long distances but also exhibit unique vulnerabilities in the context of neurodegeneration, implying a specialised axonal proteome. However, in-depth proteomic characterisation of axons has been limited by the difficulty of isolating pure axonal material in sufficient quantities for conventional mass spectrometry analysis. Here, we combine microfluidic-based axon-soma separation with data-independent acquisition mass spectrometry (DIA-MS) on an Orbitrap Astral mass spectrometer for deep profiling of compartment-resolved proteomes of human induced pluripotent stem cell (iPSC)-derived cortical neurons. We quantify over 9,000 proteins in the somatodendritic compartment and [~]6,000 proteins in the axonal compartment, to our knowledge, representing the deepest human axonal proteome reported to date. Differential abundance analysis identifies 1,250 axon-enriched proteins with strong enrichment of axon-related pathways including vesicle-mediated transport, synaptic vesicle dynamics, and cytoskeletal organisation. Extending this workflow to iPSC-derived lower motor neurons, we reveal a core set of 417 axon-enriched proteins shared between cortical and lower motor neurons, alongside subtype-specific axonal signatures whose functional differences become evident only through compartment-restricted analysis. We show broad coverage of genes for the neurodegenerative diseases hereditary spastic paraplegia and amyotrophic lateral sclerosis, establishing a quantitative reference for interpreting how disease-linked mutations may differentially affect axonal versus somatodendritic proteostasis. This workflow and resource are readily applicable to other neuronal subtypes and disease models, paving the way for studying axonal proteome dynamics in health and disease. HighlightsO_LIDeepest human axonal proteome with [~]6,000 proteins from iPSC-derived neurons C_LIO_LIMicrofluidic separation paired with DIA mass spectrometry C_LIO_LIAxon-specific signatures masked in whole-cell analyses are revealed C_LIO_LICore set of 417 axon-enriched proteins shared across neuron subtypes C_LIO_LIFramework for studying axon-selective vulnerability in neurodegeneration C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/739061v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@191493dorg.highwire.dtl.DTLVardef@15f459forg.highwire.dtl.DTLVardef@1d3e61forg.highwire.dtl.DTLVardef@a9262a_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

Most L1CAM is not associated with extracellular vesicles in human biofluids and iPSC-derived neurons

Transmembrane L1 cell adhesion molecule (L1CAM) is widely used as a marker to enrich for neuron-derived extracellular vesicles (EVs), especially in plasma. However, this approach lacks sufficient robust validation. This study aimed to assess whether human biofluids are indeed enriched for EVs, particularly neuron-derived EVs, by L1CAM immunoaffinity, utilizing multiple sources (plasma, CSF, conditioned media from iPSC-derived neurons [iNCM]) and different methods (mass spectrometry [MS], nanoparticle tracking analysis [NTA]). Following a systematic multi-step validation approach, we confirmed isolation of generic EV populations using size-exclusion chromatography (SEC) and polymer-aided precipitation (PPT) - two most commonly applied EV isolation methods - from all sources. Neurofilament light (NfL) was detected in both CSF and blood-derived EVs, indicating their neuronal origin. However, L1CAM immunoprecipitation did not yield enrichment of L1CAM in EV fractions. Instead, it was predominantly found in its free-floating form. Additionally, MS-based proteomic analysis of CSF-derived EVs also did not show L1CAM enrichment. Our study validates EV isolation from diverse biofluid sources by several isolation approaches and confirms that some EV subpopulations in human biofluids are of neuronal origin. Thorough testing across multiple sources by different orthogonal methods, however, does not support L1CAM as a marker to reliably enrich for a specific subpopulation of EVs, particularly of neuronal origin.

neuroscience↗

bioRxiv2024

Unraveling Axonal Transcriptional Landscapes: Insights from iPSC-Derived Cortical Neurons and Implications for Motor Neuron Degeneration

Neuronal function and pathology are deeply influenced by the distinct molecular profiles of the axon and soma. Traditional studies have often overlooked these differences due to the technical challenges of compartment specific analysis. In this study, we employ a robust RNA-sequencing (RNA-seq) approach, using microfluidic devices, to generate high-quality axonal transcriptomes from iPSC-derived cortical neurons (CNs). We achieve high specificity of axonal fractions, ensuring sample purity without contamination. Comparative analysis revealed a unique and specific transcriptional landscape in axonal compartments, characterized by diverse transcript types, including protein-coding mRNAs, RNAs encoding ribosomal proteins (RPs), mitochondrial-encoded RNAs, and long non-coding RNAs (lncRNAs). Previous works have reported the existence of transcription factors (TFs) in the axon. Here, we detect a set of TFs specific to the axon and indicative of their active participation in transcriptional regulation. To investigate transcripts and pathways essential for central motor neuron (MN) degeneration and maintenance we analyzed KIF1C-knockout (KO) CNs, modeling hereditary spastic paraplegia (HSP), a disorder associated with prominent length-dependent degeneration of central MN axons. We found that several key factors crucial for survival and health were absent in KIF1C-KO axons, highlighting a possible role of these also in other neurodegenerative diseases. Taken together, this study underscores the utility of microfluidic devices in studying compartment-specific transcriptomics in human neuronal models and reveals complex molecular dynamics of axonal biology. The impact of KIF1C on the axonal transcriptome not only deepens our understanding of MN diseases but also presents a promising avenue for exploration of compartment specific disease mechanisms.

neuroscience↗

bioRxiv2023

Identification of post-transcriptional modifications in nucleic acid sequences using propose-designed molecular beacons

Post-transcriptional RNA modifications (PTxMs) present in small RNA species, specifically circulating extracellular RNAs, were recently identified as clinically relevant readouts, often more indicative of disease severity than the classical "up and down" changes in their copy number alone. While identification of PTxMs requires multiple and complex sample preparation steps, microgram-range amounts of RNA, followed by expensive and protracted bioinformatics analyses, the clinically relevant information is usually a yes/no for a particular genetic variant(s), and an up/down answer for relevant biomarkers. We have previously shown that molecular beacons (MBs) can identify specific nucleic acid sequences with picomolar sensitivity and single nucleotide specificity by exploiting the target-dependent change in their electrophoretic mobility profile. We now present a method for direct identification of miRNAs and isomiRs in cells and extracellular vesicles using gel electrophoresis, without the need for RNA isolation and purification. The detection is based on discreet changes in the hydrodynamic surface profile, the overall size, charge and charge distribution of the MB-target hybrid. Furthermore, using an RNA tertiary structure prediction algorithm (iFoldRNA) and a custom molecular dynamics simulation (DMD), we designed modified MBs specific for m6A-modified nucleotides in target RNA sequences. The sample preparation method coupled to the software package affords the design of specific MBs and sensitive, multiplex-type detection of targets in a wide variety of biofluids and cells, in a simple mix and read approach.

molecular biology↗