Biology subjects

Durcan, T.

Publications and source records attributed to Durcan, T..

At least 19 recordsLinked to original sources

bioRxiv2026

Extracellular vesicles derived from cells overexpressing HGSNAT rescue defects in Mucopolysaccharidosis IIIC neurons

Mucopolysaccharidosis III type C (MPS IIIC) is a rare neurological lysosomal storage disorder caused by genetic deficiency of the lysosomal membrane enzyme, heparan--glucosaminide N-acetyltransferase (HGSNAT). To assess the feasibility of therapeutic strategies based on cross-correction of neurons by HGSNAT secreted from transplanted cells overexpressing the enzyme, we generated induced cortical neurons (iCN) from induced pluripotent stem cells (iPSCs) derived from MPS IIIC patients. The neurons were treated with extracellular vesicles (EV) purified from the culture medium conditioned by human endothelial cells transduced with a lentiviral vector encoding EGFP-tagged HGSNAT (LV-HGSNAT-EGFP). The isolated EV showed supraphysiologic HGSNAT activity levels and efficiently delivered the enzyme to the lysosomes of MPS IIIC iCN reducing lysosomal size and restoring normal synaptic protein levels. EV-mediated delivery of HGSNAT to neurons was further confirmed by the analysis of MPS IIIC iCN either co-cultured with iPSC-derived MPS IIIC microglia (iMGL) transduced with LV-HGSNAT-EGFP or treated with the iMGL conditioned medium. MPS IIIC iCN co-cultured with iMGL overexpressing HGSNAT achieved a complete phenotypic rescue, including normalization of lysosomal size, and the levels of heparan sulfate, GM2-ganglioside, synaptic proteins and brain-derived neurotropic factor. Treatment of MPS IIIC iCNs with conditioned medium led to a partial defects correction. Our findings reveal the translational potential of EV-mediated enzyme delivery in MPS IIIC patients treated with LV-mediated haematopoietic progenitor stem cell gene therapy.

biochemistry↗

bioRxiv2026

Applications of adeno-associated virus for 3D single-cell morphometric analysis in iPSC-derived midbrain organoids.

Human midbrain organoids (hMBOs) are emerging in vitro models to mirror the cellular diversity and the structural complexity of the developing human brain. However, the dense neural network, limits the investigation of individual cells morphology or cell-cell connectivity, which is mostly restricted to fixed organoids following extensive optical clearing techniques. To better resolve individual cells within a brain organoid and for longitudinal tracking of its growth and development, we turned to adeno-associated virus (AAVs) for targeted gene delivery. In particular, we applied AAVs for expressing specific markers that provide the foundation to image individual cells within 3D hMBOs. Thus, we developed a phenotypic platform to specifically inspect the neuronal and astrocytic cytoarchitecture and to examine their connectivity in living hMBOs derived from two genetically unrelated control iPSC lines. We demonstrate that through AAV transduction, we could capture and reconstruct the 3D architecture of both neurons and astrocytes within the hMBO as a whole. Transduced cells exhibited an intrinsic heterogeneity in term of soma volume, arbor complexity and territory covered, regardless of both genetic background, age, and cell-type. Yet, these cellular morphometrics remained equivalent between the two cell lines, indicative of homogeneity in hMBO cellular development. We were able to establish longitudinal profiling of transduced cells, demonstrating how neurons and astrocytes could expand their network over time. Lastly, we describe time-lapse studies to track cellular motility and morphology fluctuations in neurons and astrocytes over time, highlighting the dynamic nature of these cells within the ramified architecture of the neural network in the developing hMBOs. Overall, our platform underscores the versatility of AAVs in studying single cell-morphometrics and cellular connectivity for longitudinal monitoring of cellular dynamics in live 3D hMBOs instead of a static snapshot.

neuroscience↗

bioRxiv2026

Integration of iPSC-derived microglia into human midbrain organoids enhances microglial maturation and inflammatory signaling

Microglia are the resident immune cells of the central nervous system and play key roles in the healthy brain during development and adulthood, as well as during neurodegenerative diseases - including Parkinsons disease (PD). Yet the role of microglia in PD pathogenesis has not been fully elucidated. Limitations of 2D cell culture and animal models in simulating human microglia in the brain parenchyma have contributed to this knowledge gap. Human midbrain organoids (hMOs) provide a promising model that can recapitulate elements of PD pathology but lack microglial cells. Here we adapt protocols for the differentiation of hMOs and human iPSC-derived microglia (iMG) to generate iMG-hMO assembloids. Within assembloids, integrated iMG (intMG) express canonical microglia markers and induce the release of cytokines and chemokines. Transcriptomic profiling by single cell RNA sequencing reveals that intMG adopt a more mature and inflammation-responsive state compared to 2D iMG. The integration of microglia results in increased signaling through inflammatory and trophic pathways that drive altered transcriptional signatures of dopaminergic neurons and astrocytes within assembloids. Overall, iMG-hMO assembloids have the potential to more faithfully model the role of microglia and neuroinflammation in PD pathogenesis.

neuroscience↗

bioRxiv2026

Microbiota-derived extracellular vesicles link intestinal dysbiosis to neuroimmune activation in long COVID

Post COVID-19 condition (Long COVID, LC) is frequently accompanied by persistent neurological symptoms, but the mechanisms linking intestinal dysbiosis to neuroinflammation remain unclear. Here we identify gut microbiota-derived extracellular vesicles (GMEVs) as functional mediators linking LC-associated dysbiosis to systemic and neuroimmune inflammation. In a longitudinally characterized cohort, individuals with LC and neurological symptoms exhibit a persistent intestinal microbiome signature. Transplantation of LC-associated microbiota into germ-free mice induces intestinal barrier disruption and neuroinflammatory phenotypes. GMEVs from individuals with LC activate inflammasome-associated programs and impair epithelial barrier function, promote inflammatory responses in macrophages, and induce coordinated pro-inflammatory transcriptional programs in human induced pluripotent stem cell (iPSC)-derived microglia. Chronic oral administration of LC-derived GMEVs remodels the microbiota and induces intestinal and systemic inflammation with glial activation in vivo. Together, these findings support a vesicle-centered framework in which microbiota-derived extracellular vesicles translate dysbiosis into sustained immune and neuroimmune activation in a post-viral inflammatory state.

microbiology↗

bioRxiv2026

Synthetic analogue of adrenocorticotropic hormone, ACTH(4-7)PGP delays neurological manifestations in diseases of mucopolysaccharidosis III spectrum by reducing neuroinflammation and rescuing neurotransmission, synaptogenesis, and axonal demyelination

Mucopolysaccharidosis III (MPS III or Sanfilippo disease) is a spectrum of 4 genetic disorders (MPS IIIA-D), caused by defects in the genes SGSH, NAGLU, HGSNAT and GNS encoding enzymes involved in degradation of heparan sulfate (HS). HS accumulates in brain tissues and causes neuronal dysfunction and neurodegeneration leading to neuropsychiatric problems, developmental delays, childhood dementia, blindness and death during the second decade of life. Previously, we demonstrated that pathophysiological mechanisms, underlying MPS IIIC in mouse models, involves functional pathological changes, affecting synaptogenesis and synaptic transmission and leading to learning and memory deficits. These results suggested that a treatment for MPS III could be developed by using compounds inducing synaptogenesis. In the current study, we tested the efficacy of a synthetic peptide ACTH(4-7)PGP, an analog of adrenocorticotropic hormone fragment, previously used as a neuroprotective and anti-inflammatory medication for treatment of acute neurological conditions, including stroke. We show that intranasal administration of ACTH(4-7)PGP restores defective synaptic transmission in CA1 pyramidal neurons of MPS IIIA and MPS IIIC mouse models and rescues the decrease in synaptic proteins in cultured MPS IIIC mouse hippocampal neurons and iPSC-derived neurons of human MPS IIIA, MPS IIIB and MPS IIIC patients. Furthermore, daily intranasal administration of ACTH(4-7)PGP to MPS IIIC and MPS IIIA mice reduces hyperactivity and rescues defects in working and spatial memory, delays progression of CNS pathology including neuroinflammation and axonal demyelination, and increases the lifespan. Together with the absence of any adverse reactions to ACTH(4-7)PGP in the MPS III and WT mice, our results justify testing the drugs efficacy in clinical settings.

genetics↗

bioRxiv2026

Stress-adaptive biomaterials with tunable yielding architectures regulate organoid morphogenesis

The yield stress at which biomaterials undergo plastic deformation limits the stresses that can be developed in encapsulated growing tissues. While mechanical properties of the matrix such as stiffness and viscoelasticity have a profound effect on cells, the role of yield stress has remained challenging to define. Here we design a self-healing granular hydrogel platform with supramolecular host-guest dynamic crosslinkers to precisely and quantitatively tune the stress at which the matrix repeatedly yields and reconfigures around tissues as they grow. Designed to provide similar mechanical constraints as a mesh stress ball, matrix yield stresses can be tuned between 12 and 370 Pa, while maintaining a storage modulus below [~]0.1kPa. We show that this range of yield stress is sufficient to promote or limit peripheral shedding in a model of non-adhesive cancer migration; and that early development of midbrain organoids is exquisitely sensitive to yield stress. Optimal yield stresses of only 25 Pa promoted budlike protrusions and large, lumenized neural rosettes, while variations as small as 10 Pa limited these phenotypes. These studies demonstrate that morphogenesis and tissue organization are exquisitely sensitive to yield stress, suggesting a new material property to target in designing biomaterials for disease modeling and regenerative medicine.

biophysics↗

bioRxiv2025

Therapeutic modulation of the calpastatin/calpain pathway restores calpain-mediated synaptic proteolysis and preserves motor neurons survival and function in C9orf72 ALS

A hexanucleotide repeat expansion (GGGGCC) in the C9orf72 gene is the most prevalent genetic cause of ALS, with early neuromuscular junction (NMJ) dysfunction being a key pathological feature. Current therapies provide only limited symptomatic relief, underscoring the need for targeted, mechanism-based interventions. Using a C9orf72 ALS zebrafish model (C9-miR) and patient-derived induced pluripotent stem cell (iPSC) motor neurons, we identified significant downregulation of calpastatin, the endogenous inhibitor of calpains, a calcium-dependent protease family implicated in neurodegeneration. We demonstrate that restoring calpastatin activity with a cell-permeable calpastatin-derived peptide or the small molecule, calpeptin, ameliorates locomotor deficits and NMJ dysfunction in the C9-miR zebrafish model. These interventions enhance synaptic vesicle turnover and quantal release at the NMJ while improving motor neuron excitability and synaptic integrity in iPSC-derived motor neurons. N-terminomic/TAILS mass spectrometry revealed direct calpain-mediated cleavage of synaptic proteins in motor neurons derived from C9orf72 patients. Proteolysis of novel ALS-relevant synaptic and axonal proteins is prevented by calpeptin and calpastatin peptide treatments. Our findings establish the calpastatin as a pivotal regulator of synaptic function in C9orf72-associated ALS and identify it as a promising therapeutic target, offering a novel strategy to restore synaptic transmission and potentially halt disease progression.

neuroscience↗

bioRxiv2025

N-terminal acetylation reduces α-synuclein pathology in models of Parkinson's disease

The -synuclein protein, encoded by SNCA gene, is a major constituent of pathological intracellular inclusions such as Lewy bodies found in the brains of patients with Parkinsons disease and other synucleinopathies. Whereas -synuclein phosphorylation has been much studied, comparatively less work has been devoted to other post-translational modifications such as acetylation, especially given that N-terminally acetylated -synuclein is the most abundant endogenous form of the protein in the brain. In this study, using multiple in vitro and in vivo models, we sought to better understand the role of N-terminal acetylation in the pathogenesis of synucleinopathies. We found that N-terminal acetylation slowed aggregation of both -synuclein monomers and pre-formed fibrils in vitro. Uptake of acetylated -synuclein pre-formed fibrils into both immortalized cell lines and iPSC-derived dopamine neurons was also slowed compared non-acetylated fibrils. In addition, exposure to acetylated pre-formed fibrils induced less seeding of endogenous -synuclein, as measured by the accumulation of Serine129-phosphorylated -synuclein inclusions in both iPSC-derived dopamine neurons and mouse brain. Finally, mice injected with N-terminally acetylated -synuclein pre-formed fibrils survived significantly longer than mice injected with non-acetylated fibrils. Taken together, our study indicates that N-terminal acetylation reduces -synuclein aggregation, uptake into cells, seeding of endogenous -synuclein, and toxicity in vivo, suggesting that this prevalent post-translational modification represents a potent, physiologically relevant protective mechanism, which has thus far largely not been taken into consideration in most experimental paradigms of Parkinsons disease and synucleinopathies.

molecular biology↗

bioRxiv2025

SNCA triplication disrupts proteostasis and extracellular architecture prior to neurodegeneration in human midbrain organoids

Synucleinopathies, including Parkinsons disease, are characterized by -synuclein (SNCA) aggregation and progressive neurodegeneration, yet the early molecular events linking SNCA gene dosage to disrupted proteostasis remain poorly understood. Here, we used human midbrain organoids derived from induced pluripotent stem cells (iPSC) carrying an SNCA triplication (SNCA Trip) and the isogenic corrected line (SNCA Isog) to dissect early pathogenic mechanisms in a 3D human model of synucleinopathy. We combined immunohistochemistry, immunoblotting, tandem mass tag proteomics, bulk RNA sequencing, and ribosome profiling to systematically characterize molecular alterations in SNCA Trip organoids at day 50 (D50) and day 100 (D100) of maturation. SNCA Trip organoids exhibited increased -synuclein accumulation, neuromelanin deposition, and activation of mTORC1 (p-rpS6), ERK1/2, AKT and p-eIF2 signalling pathways by D100. Proteomic and transcriptomic analyses revealed upregulation of cytoskeletal, synaptic, and axonal development pathways, alongside significant downregulation of extracellular matrix (ECM) components and upregulation of perineuronal net (PNN) genes. Ribosome profiling showed minimal global translational changes but uncovered selective translational buffering of neuronal and ECM-associated transcripts. Confocal imaging confirmed progressive disorganization of pericellular and interstitial ECM structures around neurons in SNCA Trip organoids. Our findings demonstrate that SNCA triplication induces early proteostatic disruption and extracellular matrix remodelling prior to neurodegeneration and suggest that altered gene expression and ECM homeostasis may contribute to disease initiation and progression. Targeting these early aberrant mechanisms may offer new therapeutic opportunities for synucleinopathies, such as Parkinsons Disease.

neuroscience↗

bioRxiv2025

α-Synuclein purification significantly impacts seed amplification assay performance and consistency

-Synuclein seed amplification assays are a promising diagnostic tool for synucleinopathies such as Parkinsons disease and multiple system atrophy. Standardized conditions are required to ensure a high degree of inter- and intra-laboratory reproducibility when performing these assays. A significant issue that hinders the utility of seed amplification assays is the de novo aggregation propensity of the -synuclein substrate as well as inter-batch heterogeneity. While much work has focused on determining appropriate seed amplification assay buffer compositions as well as the type and amount of seed used, a robust comparison of -synuclein substrate purification methods has not been reported. We therefore compared the utility of recombinant -synuclein purified using four different methods as seed amplification assay substrates across two laboratories. Osmotic shock-purified -synuclein monomer substrate showed the lowest propensity for de novo aggregation, which translated into being the best substrate for seed amplification assay reactions seeded with -synuclein preformed fibrils or patient brain homogenates. Furthermore, osmotic shock -synuclein monomer showed the best inter-batch reproducibility compared to all other substrates tested. As -synuclein seed amplification assays continue to evolve and move towards adoption in the clinical realm, this work showcases the vital importance of standardizing the production and characterization of recombinant -synuclein substrate. We encourage the widespread adoption of osmotic shock -synuclein monomer as the universal substrate for seed amplification assays to maximize intra- and inter-laboratory reproducibility.

biochemistry↗

bioRxiv2025

Midbrain organoids with an SNCA gene triplication display dopamine-dependent alterations in network activity

Human midbrain organoids (hMOs) show promise as a patient-derived model for the study of Parkinsons disease (PD). Yet, much remains unknown about how accurately hMOs recapitulate key features of PD in the human brain. In both PD patients and animal models, disease progression leads to characteristic changes in neural activity throughout the basal ganglia. Here we demonstrate that patient-derived induced pluripotent stem cell (iPSC) hMOs harboring a triplication in the SNCA gene, encoding -synuclein, a key protein in PD pathogenesis, can recapitulate PD-associated changes in neural activity. Namely, we observe hyperactive network activity in SNCA triplication hMOs, but not in isogenic, CRISPR-corrected iPSC hMOs. These changes are characterized by an increase in the number of bursts and network-wide bursts. Moreover, SNCA triplication hMOs exhibit an increase in network synchrony and burst/network burst strength similar to observations in animal and human PD brains. Subsequently, we show that the observed changes in neuronal activity are attributed to dopamine D2 receptor hypoactivity due to dopamine depletion, which could be reversed by the D2 receptor agonist quinpirole. Thus, hMOs faithfully model network wide electrophysiological changes associated with PD progression and serve as a promising tool for PD research and personalized medicine.

neuroscience↗

bioRxiv2025

STIP1/HOP Promotes the Formation of Cytotoxic α-Synuclein Oligomers

The accumulation of alpha-synuclein (a-Syn) as toxic oligomers, and subsequently in Lewy bodies, is a pathological hallmark of Parkinsons disease (PD) and other synucleinopathies. Molecular chaperones and co-chaperones are expected to act in concert to maintain physiological activities of proteins, including a-Syn, but in neurodegeneration this process can become mal-adaptive. Transcript levels of Stress inducible phosphoprotein 1 (STIP1), a co-chaperone of Hsp90/Hsp70, are elevated in brain samples from PD patients. In synucleinopathy mouse models, STIP1 has unexpected bidirectional effects on a-Syn, with overexpression of STIP1 aggravating a-Syn toxicity, whereas knockdown of STIP1 improves toxicity and behavioural phenotypes. However, it is unclear how STIP1 enhances the toxicity of a-Syn. Here we unravel the mechanisms by which the direct interaction between STIP1/HOP and a-Syn regulates the neurotoxicity of a-Syn. Specifically, two binding motifs in the C-terminus of a-Syn directly interact with the TPR2A domain of STIP1/HOP in a dynamic manner, competing for a shared interface on TPR2A. Binding of STIP1/HOP to a-Syn attenuates the formation of a-Syn fibrils while promoting the accumulation of high molecular weight amorphous a-Syn species. Samples of a-Syn aggregated in the presence of STIP1/HOP contain significantly more A11-positive oligomeric species and cause a greater reduction in cell viability than a-Syn aggregated in the absence of STIP1/HOP in neuronal cells. Our results provide a mechanism by which the direct interaction between STIP1/HOP and the C-terminus of a-Syn promotes the formation of cytotoxic, non-amyloidogenic, high molecular weight a-Syn species. Our model offers an explanation for the unexpected pathological link between STIP1 and a-Syn toxicity, thus opening new therapeutic avenues for the treatment of synucleinopathies.

biochemistry↗

bioRxiv2024

Secondary deficiency of neuraminidase 1 contributes to CNS pathology in neurological mucopolysaccharidoses via hypersialylation of brain glycoproteins

Mucopolysaccharidoses (MPS) are lysosomal storage diseases caused by defects in catabolism of glycosaminoglycans. MPS I, II, III and VII are associated with lysosomal accumulation of heparan sulphate and manifest with neurological deterioration. Most of these neurological MPS currently lack effective treatments. Here, we report that, compared to controls, neuraminidase 1 (NEU1) activity is drastically reduced in brain tissues of neurological MPS patients and in mouse models of MPS I, II, IIIA, IIIB and IIIC, but not of other neurological lysosomal disorders not presenting with heparan sulphate storage. We further show that accumulated heparan sulphate disrupts the lysosomal multienzyme complex of NEU1 with cathepsin A (CTSA), {beta}-galactosidase (GLB1) and glucosamine-6-sulfate sulfatase (GALNS) necessary to maintain enzyme activity, and that NEU1 deficiency is linked to partial deficiencies of GLB1 and GALNS in cortical tissues and iPSC-derived cortical neurons of neurological MPS patients. Increased sialylation of N-linked glycans in brain samples of human MPS III patients and MPS IIIC mice implicated insufficient processing of brain N-linked sialylated glycans, except for polysialic acid, which was reduced in the brains of MPS IIIC mice. Correction of NEU1 activity in MPS IIIC mice by lentiviral gene transfer ameliorated previously identified hallmarks of the disease, including memory impairment, behavioural traits, and reduced levels of the excitatory synapse markers VGLUT1 and PSD95. Overexpression of NEU1 also restored levels of VGLUT1-/PSD95-positive puncta in cortical neurons derived from iPSC of an MPS IIIA patient. Together, our data demonstrate that heparan sulphate-induced secondary NEU1 deficiency and aberrant sialylation of glycoproteins implicated in synaptogenesis, memory, and behaviour constitute a novel pathological pathway in neurological MPS spectrum crucially contributing to CNS pathology. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/587986v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@3905c3org.highwire.dtl.DTLVardef@1a9672corg.highwire.dtl.DTLVardef@b4911forg.highwire.dtl.DTLVardef@a3b190_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

14-3-3ζ regulates adipogenesis by modulating chromatin accessibility during the early stages of adipocyte differentiation

We previously established the scaffold protein 14-3-3{zeta} as a critical regulator of adipogenesis and adiposity, but the temporal specificity of its action during adipocyte differentiation remains unclear. To decipher if 14-3-3{zeta} exerts its regulatory functions on mature adipocytes or on adipose precursor cells (APCs), we generated Adipoq14-3-3{zeta}KO and Pdgfra14-3-3{zeta}KO mouse models. Our findings revealed a pivotal role for 14-3-3{zeta} in APC differentiation in a sex-dependent manner, whereby male and female Pdgfra14-3-3{zeta}KO mice display impaired or potentiated weight gain, respectively, as well as fat mass. To better understand how 14-3-3{zeta} regulates the adipogenic transcriptional program in APCs, CRISPR-Cas9 was used to generate TAP-tagged 14-3-3{zeta}-expressing 3T3-L1 preadipocytes. Using these cells, we examined if the 14-3-3{zeta} nuclear interactome is enriched with adipogenic regulators during differentiation. Regulators of chromatin remodeling, such as DNMT1 and HDAC1, were enriched in the nuclear interactome of 14-3-3{zeta}, and their activities were impacted upon 14-3-3{zeta} depletion. The interactions between 14-3-3{zeta} and chromatin-modifying enzymes suggested that 14-3-3{zeta} may control chromatin remodeling during adipogenesis, and this was confirmed by ATAC-seq, which revealed that 14-3-3{zeta} depletion impacted the accessibility of up to 1,244 chromatin regions corresponding in part to adipogenic genes, promoters, and enhancers during the initial stages of adipogenesis. Moreover, 14-3-3{zeta}-dependent chromatin accessibility was found to directly correlate with the expression of key adipogenic genes. Altogether, our study establishes 14-3-3{zeta} as a crucial epigenetic regulator of adipogenesis and highlights the usefulness of deciphering the nuclear 14-3-3{zeta} interactome to identify novel pro-adipogenic factors and pathways.

physiology↗

bioRxiv2024

Transcriptome-based screening in TARDBP/TDP-43 knock-in motor neurons identifies the NEDD8-activating enzyme inhibitor MLN4924

A growing body of knowledge implicates perturbed RNA homeostasis in amyotrophic lateral sclerosis (ALS), a neurodegenerative disease that currently has no cure and few available treatments. Dysregulation of the multifunctional RNA-binding protein TDP-43 is increasingly regarded as a convergent feature of this disease, evidenced at the neuropathological level by the detection of TDP-43 pathology in most patient tissues, and at the genetic level by the identification of disease-associated mutations in its coding gene TARDBP. To characterize the transcriptional landscape induced by TARDBP mutations, we performed whole-transcriptome profiling of motor neurons differentiated from two knock-in iPSC lines expressing the ALS-linked TDP-43 variants p.A382T or p.G348C. Our results show that the TARDBP mutations significantly altered the expression profiles of mRNAs and microRNAs of the 14q32 cluster in MNs. Using mutation-induced gene signatures and the Connectivity Map database, we identified compounds predicted to restore gene expression toward wild-type levels. Among top-scoring compounds selected for further investigation, the NEDD8-activating enzyme inhibitor MLN4924 effectively improved cell viability and neuronal activity, highlighting a possible role for protein post-translational modification via NEDDylation in the pathobiology of TDP-43 in ALS.

neuroscience↗

bioRxiv2023

The Use of a SOX10 Reporter Towards Ameliorating Oligodendrocyte Lineage Differentiation from Human Induced Pluripotent Stem Cells

Oligodendrocytes (OLs) are key players in the central nervous system, critical for the formation and maintenance of the myelin sheaths insulating axons, ensuring efficient neuronal communication. In the last decade, the use of human induced pluripotent stem cells (iPSCs) has become essential for recapitulating and understanding the differentiation and role of OLs in vitro. Current methods include overexpression of transcription factors for rapid OL generation, neglecting the complexity of OL lineage development. Alternatively, growth factor-based protocols offer physiological relevance but struggle with efficiency and cell heterogeneity. To address these issues, we created a novel SOX10-P2A-mOrange iPSC reporter line to track and purify oligodendrocyte precursor cells (OPCs). Using this reporter cell line, we analyzed an existing differentiation protocol and shed light on the origin of glial cell heterogeneity. Additionally, we have modified the differentiation protocol, towards enhancing reproducibility, efficiency, and terminal maturity. Our approach not only advances OL biology but holds promise to accelerate research and translational work with iPSC-derived OLs. Main PointsO_LIThe differentiation of iPSCs in Oligodendrocyte Precursor Cells (OPCs) and Oligodendrocytes (OLs) is a notoriously difficult technique and often displays variable efficiency and cellular heterogeneity. C_LIO_LIWe engineered a novel reporter line carrying the fluorescent protein mOrange under the control of the OL-specific transcription factor SOX10 to track, purify and characterize OLs. C_LIO_LIBy experimenting with diverse differentiation media, we improved the generation of SOX10-positive cells. Consequently, these cells exhibited increased consistency and effectiveness in evolving into myelinating OLs. C_LI

cell biology↗

bioRxiv2023

Early nuclear phenotypes and reactive transformation in human iPSC-derived astrocytes from ALS patients with SOD1 mutations

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease characterized by the progressive death of motor neurons (MNs). MN degeneration in ALS involves both cell-autonomous and non-cell autonomous mechanisms, with glial cells playing important roles in the latter. More specifically, astrocytes with mutations in the ALS-associated gene Cu/Zn superoxide dismutase 1 (SOD1) promote MN death. The mechanisms by which SOD1-mutated astrocytes reduce MN survival are incompletely understood. In order to characterize the impact of SOD1 mutations on astrocyte physiology, we generated astrocytes from human induced pluripotent stem cell (iPSC) derived from ALS patients carrying SOD1 mutations, together with control isogenic iPSCs. We report that astrocytes harbouring SOD1(A4V) and SOD1(D90A) mutations exhibit molecular and morphological changes indicative of reactive astrogliosis when compared to matching isogenic astrocytes. We show further that a number of nuclear phenotypes precede, or coincide with, reactive transformation. These include increased nuclear oxidative stress and DNA damage, and accumulation of the SOD1 protein in the nucleus. These findings reveal early cell-autonomous phenotypes in SOD1-mutated astrocytes that may contribute to the acquisition of a reactive phenotype involved in alterations of astrocyte-MN communication in ALS.

neuroscience↗

bioRxiv2023

A genome-wide CRISPR/Cas9 screen identifies genes that regulate the cellular uptake of α-synuclein fibrils by modulating heparan sulfate proteoglycans

Synucleinopathies are characterized by the accumulation and propagation of -synuclein (-syn) aggregates throughout the brain, leading to neuronal dysfunction and death. Understanding how these aggregates propagate from cell to cell in a prion-like fashion thus holds great therapeutic promises. Here, we focused on understanding the cellular processes involved in the entry and accumulation of pathological -syn aggregates. We used an unbiased FACS-based genome-wide CRISPR/Cas9 knockout (KO) screening to identify genes that regulate the accumulation of -syn preformed fibrils (PFFs) in cells. We identified key genes and pathways specifically implicated in -syn PFFs intracellular accumulation, including heparan sulfate proteoglycans (HSPG) biosynthesis and Golgi trafficking. We show that all confirmed hits affect heparan sulfate (HS), a post-translational modification known to act as a receptor for proteinaceous aggregates including of -syn and tau. Intriguingly, KO of SLC39A9 and C3orf58 genes, encoding respectively a Golgi-localized exporter of Zn2+, and the Golgi-localized putative kinase DIPK2A, specifically impaired the uptake of -syn PFFs uptake but not of tau oligomers, by preventing the binding of PFFs to the cell surface. Mass spectrometry-based analysis of HS chains indicated major defects in HS maturation in SLC39A9 and C3orf58 KO cells, explaining the cell surface binding deficit. Our findings now clearly establish these two genes as HSPG-modulating factors. Interestingly, C3orf58 KO human iPSC-derived microglia exhibited a strong reduction in their ability to internalize -syn PFFs. Altogether, our data establish HSPGs as major receptors for -syn PFFs binding on the cell surface and identifies new players in -syn PFFs cell surface binding and uptake.

cell biology↗