Biology subjects

Fledrich, R.

Publications and source records attributed to Fledrich, R..

7 recordsLinked to original sources

bioRxiv2026

A Multicenter Confirmatory Randomized-Controlled Study of rhNRGβ1 Protein Replacement Therapy in a Murine Model of NF2-related Schwannomatosis

BackgroundPrevious exploratory studies identified recombinant human Neuregulin-1 {beta} (rhNRG{beta}1) as a promising therapeutic strategy for inhibiting the growth of Nf2-deficient schwannomas by promoting cellular differentiation. Because robust confirmation across independent laboratories is essential for advancing promising preclinical findings toward clinical translation, we conducted a multicenter, randomized, controlled confirmatory study under stringent preclinical standards. MethodsIn a pre-registered trial (DOI: 10.17590/asr.0000304), 216 mice (Nf2-flox;P0-Cre;Nefh- Cre) were randomized at three independent research sites. Following a standardized sciatic nerve crush, mice received systemic rhNRG{beta}1 (10 {micro}g/kg) or vehicle for 13 weeks. Rigorous quality measures included double-blinding, standardized surgery, centralized data management, and an automated Fiji macro for objective nerve thickness quantification (Primary Outcome). Secondary molecular outcomes included Western blot and in-depth, quantitative proteomics and phosphoproteomics. All methods were SOP-based for reproducible and comparable results across the three study centers ResultsThe primary confirmatory analysis revealed no reduction in nerve thickness in the rhNRG{beta}1 group (pbest case imputation = 0.076 and pworst case imputation = 0.533). Secondary analyses via quantitative Western blotting and DIA proteomics demonstrated that core biochemical markers of Schwann cell differentiation (MBP, ERBB2) remained unchanged across all centers. Based on the absence of macroscopic or primary biochemical effects, further histological analysis was omitted to avoid scientific redundancy. High-depth profiling of a predefined 60-protein functional marker panel confirmed a remarkably stable tumor proteome across all replication sites and both sexes, with no evidence of coordinated changes in key downstream oncogenic signaling pathways (Hippo/YAP, mTORC1, and RTK-Ras-MAPK) or metabolic signaling cascades. These findings indicate an absence of measurable target engagement under our tested dosing regimen, potentially reflecting pharmacokinetic or tissue-delivery limitations rather than an invalidation of the underlying biological pathway. ConclusionDespite high statistical power and rigorous methodology, this study could not confirm rhNRG{beta}1 as a robust therapeutic candidate for schwannoma growth arrest or shrinkage. These findings suggest that previously reported therapeutic effects were either highly context- dependent or could not be reproduced under adequately powered, rigorously controlled experimental conditions. As underpowered preclinical studies are more susceptible to random biological variation, our results highlight the importance of sufficient sample sizes alongside robust experimental design. Our study underscores the value of trial-like methodological standards in preclinical therapeutic evaluation to identify ineffective interventions (dead ends) early and strengthen translational decision-making. Although we could not confirm the previously reported efficacy of rhNRG{beta}1, the multicenter framework established here provides a methodological benchmark for robust preclinical testing in translational oncology, with the potential to improve reproducibility and the success of therapies progressing to early-phase clinical trials. From a translational perspective, these findings provide a robust foundation for optimizing future rhNRG{beta}1-based therapeutic approaches through improved dosing, delivery routes, and treatment schedules.

cancer biology↗

bioRxiv2026

Red fluorescent labeling of myelin by membrane-targeted tdTomato in transgenic mouse lines

Myelin is a highly complex membranous structure wrapped around axons by oligodendrocytes or Schwann cells in the central and peripheral nervous system, respectively. Fluorescent labeling is widely used to study the structure and dynamics of myelin. Combining structural with functional imaging requires labeling of myelin with red fluorescence, as many functional sensors, including Ca2+ indicators and genetically encoded metabolite sensors, fluoresce in the green spectral range. However, in vivo tools enabling red fluorescent labeling of myelinating cells and their myelin sheaths remain limited. Here, we generated a set of seven transgenic mouse lines expressing a membrane-targeted variant of the red fluorescent protein tdTomato in myelinating oligodendrocytes and Schwann cells throughout the nervous system. The mouse lines provide a variety of expression patterns ranging from wide-spread labeling of myelin to a rather sparse expression, the latter enabling visualization of individual oligodendrocytes and their associated myelin sheaths. In the peripheral nervous system, the pattern of fluorescence in sciatic nerves indicates predominant localization of tdTomato to non-compact myelin compartments including the inner and outer tongues, paranodal loops and Schmidt-Lanterman incisures. In summary, our work provides a set of novel mouse lines with myelin labeled by red fluorescence, which are compatible with diverse imaging modalities in the green spectral range enabling integrated structural and functional imaging. Main PointsO_LITransgenic mouse lines expressing membrane-targeted tdTomato in myelin enable imaging of myelin in the red spectral range C_LIO_LIDistinct expression patterns range from wide-spread labeling to sparse single-cell resolution, supporting diverse imaging applications C_LI

neuroscience↗

bioRxiv2025

High affinity cross-context cellular assays reveal novel protein-protein interactions of peripheral myelin protein of 22 kDa

Peripheral Myelin Protein 22 (PMP22) is a tetraspan membrane protein whose altered dosage causes the most common hereditary neuropathy, Charcot-Marie-Tooth disease type 1A (CMT1A). Despite its clinical significance, the physiological functions of PMP22 and the mechanism behind its tightly controlled gene dosage sensitivity remain unknown since over 30 years, in part due to limited knowledge of its protein-protein interactions (PPIs). In fact, integral membrane proteins such as PMP22 are significantly underrepresented in known cellular interactomes, likely due to limited suitability or technical challenges specific to these hydrophobic molecules in the major PPI discovery approaches. Here, we applied a rigorously optimized co-immunoprecipitation and mass spectrometry workflow using the mild detergent DDM and the high affinity ALFA-tag/anti-ALFA nanobody interaction to identify cellular PMP22-associated proteins. In a cross-context approach, we ran our standardized pipeline across multiple cell types including HEK293T, MDCKII epithelial cells, the Schwann cell line MSC80, and primary rat Schwann cells. We confirm known interactors, and uncover distinct, cell type-specific enrichment patterns following functional annotation analysis. Adhesion-related PPIs dominated in MDCKII cells (e.g., CD47, CLDN1, ATP1B1), while in Schwann cells myelin-associated PPIs were enriched. Importantly, we identified novel PPI candidates that may be highly relevant for PMP22 function including enzymes of the de novo sphingolipid biosynthesis pathway.

neuroscience↗

bioRxiv2024

Multi-omic characterization of human sural nerves acrosspolyneuropathies

Diseases of peripheral nerves termed polyneuropathies (PNPs) are common, mechanistically heterogeneous, and challenging to diagnose. Here, we integrated single nuclei transcriptomics of peripheral nerves from 33 human PNP patients and four controls (365,708 nuclei) with subcellular spatial transcriptomics. We identified novel and human-specific nerve cell type markers including unexpectedly heterogeneous perineurial fibroblasts. All PNPs shared a loss of myelinating and an increase in repair Schwann cells and endoneurial lipid-associated macrophages. Transcriptional changes affected multiple cells outside of the endoneurium across PNPs, suggesting PNPs as pan-nerve diseases. Spatially, PNPs showed a previously unknown perineurial hyperplasia and fibrotic dispersion and this was most pronounced in immune-mediated PNPs. Single cell transcriptomics supported the differential diagnosis of PNPs with potential for future unbiased diagnostic classification. One-sentence summaryThe first large-scale integrated single cell and spatial transcriptomic characterization of human peripheral nerves identifies novel cell markers and unexpected heterogeneity of perineurial cells, reveals polyneuropathies as pan-nerve diseases, and shows that single cell transcriptomics hold potential for unbiased nerve disease classification.

neuroscience↗

bioRxiv2021

Myelin insulation as a risk factor for axonal degeneration in autoimmune demyelinating disease

Axonal degeneration determines the clinical outcome of multiple sclerosis (MS), and is thought to result from exposure of denuded axons to immune-mediated damage. We challenge this view after finding in MS and its mouse models that myelin itself increases the risk of axons to degenerate under inflammatory conditions. We propose a model for demyelinating diseases in which for axons that remain myelinated, and thus shielded from the extracellular milieu, dependence from oligodendroglial support turns fatal in an autoimmune disease environment.

neuroscience↗

bioRxiv2021

Inversely proportional myelin growth due to altered Pmp22 gene dosage identifies PI3K/Akt/mTOR signaling as a novel therapeutic target in HNPP

Duplication of the gene encoding the myelin protein PMP22 causes the hereditary neuropathy Charcot-Marie-Tooth disease 1A (CMT1A), characterized by hypomyelination of medium to large caliber peripheral axons. Conversely, haplo-insufficiency of PMP22 leads to focal myelin overgrowth in hereditary neuropathy with liability to pressure palsies (HNPP). However, the molecular mechanisms of myelin growth regulation by PMP22 remain obscure. Here, we found that the major inhibitor of the myelin growth signaling pathway PI3K/Akt/mTOR, phosphatase and tensin homolog (PTEN) is increased in abundance in CMT1A and decreased in HNPP rodent models. Indeed, treatment of DRG co-cultures from HNPP mice with PI3K/Akt/mTOR pathway inhibitors reduced focal hypermyelination and, importantly, treatment of HNPP mice with the mTOR inhibitor Rapamycin improved motor behavior, increased compound muscle amplitudes (CMAP) and reduced tomacula formation in the peripheral nerve. In Pmp22tg CMT1A mice, we uncovered that the differentiation defect of Schwann cells is independent from PI3K/Akt/mTOR activity, rendering the pathway insufficient as a therapy target on its own. Thus, while CMT1A pathogenesis is governed by dys-differentiation uncoupled from PI3K/Akt/mTOR signaling, targeting the pathway provides novel proof-of-principle for a therapeutic approach to HNPP.

neuroscience↗

bioRxiv2020

Long term AAV2/9-mediated silencing of PMP22 prevents CMT1A disease in rats and validates skin biomarkers as treatment outcome measure

Charcot-Marie-Tooth disease 1A (CMT1A) results from a duplication of the PMP22 gene leading to an excess of PMP22, a deficit of myelination and an instability of the myelin sheath in peripheral nerves. Patients present with reduced nerve conduction velocity, muscle waste, hand and foot deformations and foot drop walking problems. As gene silencing therapy has been shown to be effective in other monogenic neurological disorders, we evaluated the safety and efficacy of recombinant adeno-associated viral vector serotype 9 (AAV2/9)-based gene therapy for CMT1A. AAV2/9-mediated delivery of eGFP and shRNAs targeting PMP22 mRNA in the sciatic nerve allowed widespread gene expression in myelinating Schwann cells in mouse, rat and nonhuman primate. The treatment restored wild-type PMP22 level, increased myelination and prevented motor and sensory impairment over 12 months in a rat model of CMT1A. Intra-nerve injection limited off-target transduction and immune response to barely detectable levels. A combination of previously characterized human skin biomarkers successfully discriminated treated animals from their untreated littermate controls indicating their potential use as part of outcome measures in future clinical trials. Our results support intra nerve injection of AAV2/9 as an effective strategy for the treatment of CMT1A as well as other demyelinating CMT diseases.

neuroscience↗