Tim Vangansewinkel

Synaptic dysfunction is a hallmark of neurodevelopmental disorders (NDDs), often linked to genes involved in cytoskeletal regulation. While the role of these genes has been extensively studied in neurons, microglial functions such as phagocytosis are also dependent on cytoskeletal dynamics. We demonstrate that disturbance of actin cytoskeletal regulation in microglia, modeled by genetically impairing the scaffold protein Disrupted-in-Schizophrenia 1 (DISC1), which integrates actin-binding proteins, causes a shift in actin regulatory balance favoring filopodial versus lamellipodial actin organization. The resulting microglia-specific dysregulation of actin dynamics leads to excessive uptake of synaptic proteins. Genetically engineered DISC1-deficient mice show diminished hippocampal excitatory transmission and associated spatial memory deficits. Reintroducing wild-type microglia-like cells via bone marrow transplantation in adult DISC1-deficient mice restores the synaptic function of neurons and rescues cognitive performance. These findings reveal a pivotal role for microglial actin cytoskeletal remodeling in preserving synaptic integrity and cognitive health. Targeting microglial cytoskeletal dynamics may effectively address cognitive impairments associated with NDDs, even in adulthood.

Charcot–Marie–Tooth disease type 1A (CMT1A) is the most common inherited peripheral neuropathy caused by a 1.5 Mb tandem duplication of chromosome 17 harbouring the PMP22 gene. This dose-dependent overexpression of PMP22 results in disrupted Schwann cell myelination of peripheral nerves. To obtain better insights into the underlying pathogenic mechanisms in CMT1A, we investigated the role of PMP22 duplication in cellular homeostasis in CMT1A mouse models and in patient-derived induced pluripotent stem cells differentiated into Schwann cell precursors (iPSC-SCPs). We performed lipidomic profiling and bulk RNA sequencing (RNA-seq) on sciatic nerves of two developing CMT1A mouse models and on CMT1A patient-derived iPSC-SCPs. For the sciatic nerves of the CMT1A mice, cholesterol and lipid metabolism was downregulated in a dose-dependent manner throughout development. For the CMT1A iPSC-SCPs, transcriptional analysis unveiled a strong suppression of genes related to autophagy and lipid metabolism. Gene ontology enrichment analysis identified disturbances in pathways related to plasma membrane components and cell receptor signalling. Lipidomic analysis confirmed the severe dysregulation in plasma membrane lipids, particularly sphingolipids, in CMT1A iPSC-SCPs. Furthermore, we identified reduced lipid raft dynamics, disturbed plasma membrane fluidity and impaired cholesterol incorporation and storage, all of which could result from altered lipid storage homeostasis in the patient-derived CMT1A iPSC-SCPs. Importantly, this phenotype could be rescued by stimulating autophagy and lipolysis. We conclude that PMP22 duplication disturbs intracellular lipid storage and leads to a more disordered plasma membrane owing to an alteration in the lipid composition, which might ultimately lead to impaired axo-glial interactions. Moreover, targeting lipid handling and metabolism could hold promise for the treatment of patients with CMT1A.

Leukocyte- and Platelet-Rich Fibrin (L-PRF) is a second-generation platelet concentrate that is prepared directly from the patient’s own blood. It is widely used in the field of regenerative medicine, and to better understand its clinical applicability we aimed to further explore the biological properties and effects of L-PRF on cells from the central and peripheral nervous system. To this end, L-PRF was prepared from healthy human donors, and confocal, transmission, and scanning electron microscopy as well as secretome analysis were performed on these clots. In addition, functional assays were completed to determine the effect of L-PRF on neural stem cells (NSCs), primary cortical neurons (pCNs), and peripheral dorsal root ganglion (DRG) neurons. We observed that L-PRF consists of a dense but porous fibrin network, containing leukocytes and aggregates of activated platelets that are distributed throughout the clot. Antibody array and ELISA confirmed that it is a reservoir for a plethora of growth factors. Key molecules that are known to have an effect on neuronal cell functions such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) were slowly released over time from the clots. Next, we found that the L-PRF secretome had no significant effect on the proliferative and metabolic activity of NSCs, but it did act as a chemoattractant and improved the migration of these CNS-derived stem cells. More importantly, L-PRF growth factors had a detrimental effect on the survival of pCNs, and consequently, also interfered with their neurite outgrowth. In contrast, we found a positive effect on peripheral DRG neurons, and L-PRF growth factors improved their survival and significantly stimulated the outgrowth and branching of their neurites. Taken together, our study demonstrates the positive effects of the L-PRF secretome on peripheral neurons and supports its use in regenerative medicine but care should be taken when using it for CNS applications.

Duplication of PMP22 causes Charcot-Marie-Tooth disease type 1A (CMT1A) and is known to disrupt the lipid metabolism in myelinating Schwann cells by unknown mechanisms. By using two CMT1A mouse models overexpressing human PMP22 , we discovered that PMP22 dose-dependently downregulates genes that are involved in lipid and cholesterol metabolism. Lipidomic analysis on CMT1A mouse sciatic nerves confirmed lipid metabolic abnormalities primarily associated with cholesterol and sphingolipids. We observed similar lipidomic profiles and downregulation of genes associated with lipid metabolism in human CMT1A patient induced pluripotent stem cell-derived Schwann cell precursors (iPSC-SCPs). We confirmed these findings by demonstrating altered lipid raft dynamics and plasma membrane fluidity in CMT1A iPSC-SCPs. Additionally, we identified impaired cholesterol incorporation in the plasma membrane due to altered lipid storage homeostasis in CMT1A iPSC-SCPs, which could be modulated by changing the lipid composition of the cell culture medium. These findings suggest that PMP22 plays a role in regulating the lipid composition of the plasma membrane and lipid storage homeostasis. Targeting lipid metabolism may hold promise as a potential treatment for CMT1A patients. Graphical Highlights: PMP22 copy number causes a dose-dependent suppression of cholesterol and lipid biosynthesis in peripheral nerves of CMT1A mice Lipid composition is altered in the sciatic nerves of CMT1A mice and in the membranes of patient derived iPSC-SCPs, with a significant reduction in sphingolipids CMT1A iPSC-SCPs show decreased plasma membrane lipids required for regulating lipid raft dynamics, membrane fluidity, and membrane order Lipid storage misregulation is key in the pathogenesis of CMT1A

Ameloblastomas are benign, mostly intra-osseous, tumours originating from ectodermal odontogenic epithelium and display extracellular matrix remodelling. We determined whether fibroblast activation protein-alpha (FAP-α), a collagenolytic enzyme and lysyl oxidase (LOX), a collagen cross-linker were differentially regulated in ameloblastoma subtypes. Masson’s trichrome staining and immunohistochemistry were performed on human samples from gross mandibular or maxillary ameloblastoma. Co-localization studies in tumorigenic tissue of follicular, plexiform and desmoplastic ameloblastoma showed absence of the mesenchymal cell marker vimentin but high epithelial cell adhesion molecule (EPCAM) expression, an epithelial marker. Strong FAP-α staining was also noted, however, the stellate reticulum of plexiform ameloblastoma contained a higher number of FAP-α positive cells than the follicular subtype. Weak LOX staining was demonstrated in tumorigenic tissue of the follicular subtype but profound reactivity was observed in stromal plexiform and tumorigenic desmoplastic tissue. The desmoplastic staining profile for FAP-α and LOX shared resemblance with the unicystic subtypes. Tumorigenic outgrowths in desmoplastic ameloblastoma were associated with vimentin positive and EPCAM negative cancer-associated fibroblasts suggesting a role in tumour invasion. In conclusion, FAP-α expression in ameloblastoma seems to be involved in tumour growth and invasion whereas LOX expression is differentially regulated in ameloblastoma subtypes offering a new perspective for understanding specific growth patterns of ameloblastoma subtypes.

Traumatic spinal cord injury (SCI) most often leads to permanent paralysis due to the inability of axons to regenerate in the adult mammalian central nervous system (CNS). In the past, we have shown that mast cells (MCs) improve the functional outcome after SCI by suppressing scar tissue formation at the lesion site via mouse mast cell protease 6 (mMCP6). In this study, we investigated whether recombinant mMCP6 can be used therapeutically to improve the functional outcome after SCI. Therefore, we applied mMCP6 locally via an intrathecal catheter in the subacute phase after a spinal cord hemisection injury in mice. Our findings showed that hind limb motor function was significantly improved in mice that received recombinant mMCP6 compared to the vehicle-treated group. In contrast to our previous findings in mMCP6 knockout mice, the lesion size and expression levels of the scar components fibronectin, laminin, and axon-growth-inhibitory chondroitin sulfate proteoglycans were not affected by the treatment with recombinant mMCP6. Surprisingly, no difference in infiltration of CD4+ T cells and reactivity of Iba-1+ microglia/macrophages at the lesion site was observed between the mMCP6-treated mice and control mice. Additionally, local protein levels of the pro- and anti-inflammatory mediators IL-1β, IL-2, IL-4, IL-6, IL-10, TNF-α, IFNγ, and MCP-1 were comparable between the two treatment groups, indicating that locally applied mMCP6 did not affect inflammatory processes after injury. However, the increase in locomotor performance in mMCP6-treated mice was accompanied by reduced demyelination and astrogliosis in the perilesional area after SCI. Consistently, we found that TNF-a/IL-1B-astrocyte activation was decreased, and that oligodendrocyte precursor cell (OPC) differentiation was increased after recombinant mMCP6 treatment in vitro . Mechanistically, this suggests effects of mMCP6 on reducing astrogliosis and improving (re)myelination in the spinal cord after injury. In conclusion, these data show for the first time that recombinant mMCP6 is therapeutically active in enhancing recovery after SCI.

Pathologies of the central nervous system are characterized by loss of brain tissue and neuronal function which cannot be adequately restored by endogenous repair processes. This stresses the need for novel treatment options such as cell-based therapies that are able to restore damaged tissue or stimulate repair. This study investigated the neuroregenerative potential of the conditioned medium of human dental pulp stem cells (CM-hDPSCs) on neural stem cell (NSC) proliferation and migration as well as on neurite outgrowth of primary cortical neurons (pCNs). Additionally, the effect of leukocyte- and platelet-rich fibrin (L-PRF) priming on the neuroregenerative potential of the hDPSC secretome on NSCs and pCNs was evaluated. L-PRF contains factors that enhance stem cell-induced regeneration, but its effect on hDPSC-mediated neuroregeneration is unknown. This study demonstrated that CM-hDPSCs enhanced neuritogenesis. Moreover, CM-hDPSCs had a chemoattractant effect on NSCs. Although priming hDPSCs with L-PRF increased brain-derived neurotrophic factor secretion, no additional effects on the paracrine-mediated repair mechanisms were observed. These data support the neuroregenerative potential of hDPSCs, and although priming had no additional effect, the potential of L-PRF-primed hDPSCs on distinct regenerative mechanisms remains to be clarified.