Biology subjects

Ueffing, M.

Publications and source records attributed to Ueffing, M..

At least 19 recordsLinked to original sources

bioRxiv2026

VCP inhibition prevents cone photoreceptor degeneration in the cpfl1 mouse model of achromatopsia

Achromatopsia (ACHM) is a rare autosomal recessive retinal disorder characterized by absent cone photoreceptor function from early life, leading to severe visual impairment. Mutations in genes involved in the cone phototransduction cascade frequently result in elevated cyclic guanosine monophosphate (cGMP) levels and activation of stress pathways, including endoplasmic reticulum (ER) stress and the unfolded protein response. Targeting common downstream mechanisms rather than individual mutations may provide a broadly applicable therapeutic strategy. Here, we investigated whether pharmacological inhibition of valosin-containing protein (VCP), a key regulator of ER and protein homeostasis, can prevent cone degeneration in the spontaneous cone photoreceptor function loss 1 (cpfl1) mouse model of ACHM. Organotypic culture of retinal explants from cpfl1 mice were treated with the selective VCP inhibitor ML240. Cone survival, cell death, opsin expression and localization were assessed by TUNEL assay, immunohistochemistry, and quantitative image analysis. ML240 treatment significantly increased cone density and improved cone opsin expression and trafficking to the outer segments (OSs) in cpfl1 explants compared to controls. Importantly, rhodopsin trafficking in rod photoreceptors was unaffected, indicating that VCP inhibition did not impair normal rod phototransduction. These findings demonstrate that VCP inhibition by ML240 effectively preserves cone photoreceptors and improves cone-specific functional markers in the cpfl1 model. Targeting VCP may represent a mutation-independent therapeutic strategy for preventing cone death in ACHM.

neuroscience↗

bioRxiv2026

VCP inhibition preserves photoreceptor integrity under hydroquinone-induced oxidative stress in a human iPSC-RPE/porcine neuroretina co-culture model

Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss in older adults. It is characterised by early retinal pigment epithelium (RPE) dysfunction followed by progressive photoreceptor degeneration. Cigarette smoking is a major environmental risk factor for AMD, and hydroquinone (HQ), a redox-active cigarette smoke component, induces oxidative stress and apoptosis in RPE cells. To analyse how RPE stress contributes to photoreceptor degeneration, we employed a retinal co-culture model composed of human induced pluripotent stem cell-derived RPE (iPSC-RPE) cells in conjunction with porcine neuroretina explants. Exposure to HQ induced oxidative stress in iPSC-RPE cells as well as retinal photoreceptors (RPR), resulting in apoptosis, executed at least in part by caspase activation. Concomitantly, HQ caused endoplasmic reticulum (ER) stress (ERAD) in RPR followed by their degeneration, evidenced by reduced outer nuclear layer (ONL) rows and shortened RPR outer segments (OS). Based on earlier results, which suggest a perturbation of proteostasis due to HQ, we tested whether ML240, a bona fide inhibitor of valosin-containing protein (VCP), would influence the degree of degenerative activities. ML240 did not prevent HQ-induced apoptosis in iPSC-RPE cells. However, it significantly preserved photoreceptor integrity, retaining OS length and cone density in HQ-stressed co-cultures. Proteomic analysis suggested that ML240 reshapes stress response patterns of the HQ-exposed neuroretina, as evidenced by a reduction in ERAD-associated markers, increased levels of antioxidant response proteins, and the preservation of cytochrome c enrichment in photoreceptor inner segments, which indicates improved mitochondrial integrity consistent with the observed preservation of photoreceptor structure. Together, these findings establish the iPSC-RPE/neuroretina co-culture as a platform to analyse pathophysiological features of AMD, dissect cell type-specific retinal responses to environmental stress and test neuroprotective pharmacological approaches to protect photoreceptors in oxidative stress-associated retinal degeneration.

cell biology↗

bioRxiv2025

Ocular delivery of different VCP inhibitory formulations prevents retinal degeneration in rhodopsin 255 isoleucine deletion mice

Rhodopsin-mediated autosomal dominant retinitis pigmentosa (RHO-adRP) is a progressive inherited retinal degenerative disorder currently lacking effective treatments. A recurrent 3-base pair deletion in the RHO gene, resulting in the loss of isoleucine at codon 255 or 256 (RHO{Delta}I255 or RHO{Delta}I256), has been identified in patients from the United Kingdom, Germany, Belgium, China, and Korea, suggesting a broad geographic distribution. This mutation leads to rhodopsin (RHO) misfolding, its retention in the endoplasmic reticulum (ER), and aggregation with wild-type (WT) RHO, ultimately triggering ER stress and photoreceptor degeneration. These aggregates are primarily cleared via the ER-associated degradation (ERAD) pathway, with valosin-containing protein (VCP) playing a key role in their retrotranslocation and proteasomal degradation. Pharmacological or genetic inhibition of VCP has shown neuroprotective effects in other models of adRP, but the poor aqueous solubility of VCP inhibitors and challenges in retinal drug delivery hinders clinical translation. To overcome these limitations, we evaluated and compared three VCP-targeted therapeutic strategies in Rho{Delta}I255 knock-in mouse retinae: (1) small-molecule inhibitors (ML240, NMS-873) solubilized in DMSO, (2) ML240 encapsulated in monomethoxy-polyethylene glycol (mPEG)-cholane nanoparticles, and (3) small interfering RNA (siRNA) targeting VCP, delivered via magnetic nanoparticles. Neuroprotective effects were assessed in vitro in retinal explants and in vivo following intravitreal injection. Our findings provide the first evidence that VCP inhibition restores RHO trafficking to the outer segments and prevents photoreceptor cell death in the Rho{Delta}I255 model. Among the three approaches, nanocarrier-encapsulated ML240 exhibited superior efficacy, enabling sustained drug delivery and enhanced photoreceptor protection. These results establish a preclinical proof-of-concept for nanocarrier-mediated VCP inhibition as a promising therapeutic strategy for RHO-adRP and potentially other ER-stress-related retinal degenerations. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=191 HEIGHT=200 SRC="FIGDIR/small/661245v1_ufig1.gif" ALT="Figure 1"> View larger version (73K): org.highwire.dtl.DTLVardef@12f9366org.highwire.dtl.DTLVardef@76437dorg.highwire.dtl.DTLVardef@48dd6corg.highwire.dtl.DTLVardef@1c11812_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

The AMD-associated genetic polymorphism CFH Y402H confers vulnerability to Hydroquinone-induced stress in iPSC-RPE cells

Age-related macular degeneration (AMD), a degenerative disease of the macula, is caused by an interplay of diverse risk factors (genetic predisposition, age and lifestyle habits). One of the main genetic risks includes the Y402H polymorphism in complement Factor H (FH), an inhibitor of complement system activation. There has been, and continues to be, much discussion around the functional consequences of this Y402H polymorphism, whether the soluble FH protein confers its risk association, or if the cells expressing the protein themselves are affected by the genetic alteration. In our study, we examined the cell characteristics of the retinal pigment epithelium (RPE) cells, which play a major role in retinal homeostasis and stability and which are synonymously linked to AMD. Here, we employ RPE cells derived from induced pluripotent stem cells (iPSC) generated from donors, carrying either homozygous 402Y (low risk) or 402H (high risk) variants of the CFH gene. RPE cells were treated with Hydroquinone (HQ), a component of cigarette smoke, to induce oxidative damage. Intriguingly, RPE cells carrying high genetic risk proved more vulnerable to oxidative insult when exposed to HQ, as demonstrated by increased cytotoxicity, caspase activation, compared to the low-risk RPE cells. The exposure of RPE cells to RPE conditioned medium, normal human serum (NHS) and inactivated NHS (iNHS) had minimal impact on cell cytotoxicity and caspase activation, nor did the presence of purified soluble FH rescue the observed effects. This suggests that the degree of cellular susceptibility to oxidative stress is not conferred by soluble FH protein and other complement sources, but intercellularly because of the corresponding genetic risk predisposition. Considering the known connection of oxidative stress to proteotoxic stress and degrading processes, we investigated the unfolded protein response (UPR) and autophagy. When exposed to HQ, RPE cells showed an increase in autophagy markers, however iPSC-RPE cells carrying high genetic risk showed an overall reduced autophagic flux. Our data support the hypothesis that RPE cells carrying high genetic risk are less resilient to oxidative stress.

cell biology↗

bioRxiv2024

Proximity proteomics reveals a co-evolved LRRK2-regulatory network linked to centrosomes

Leucine-rich repeat kinase 2 (LRRK2) not only plays a vital role in familial forms of Parkinsons disease (PD) but also represents a risk factor for idiopathic PD. Its multi-domain architecture enables fine-tuned regulation of its biological function by orchestrating intra- and intermolecular interactions. Here, we present BioID proximity proteomes of LRRK2 that reveal new interactors, which we further characterize using a novel evolutionary and structural bioinformatics pipeline. Co-evolutionary analysis of the protein-protein interaction network identifies a structural and functional module enriched in cytoskeletal components associated with the centrosome and microtubules. In addition, structural modelling of binary interactions using AlphaFold-Multimer reveals distinct groups of interactors that engage LRRK2 in a manner dependent on specific conformations and epitopes. Furthermore, we identify distinct changes in the LRRK2 proximity proteome that are induced by the type I kinase inhibitor MLi-2 or by co-expression of the LRRK2 upstream effector RAB29. Depending on its activity state and conformation, these protein-protein interactions link LRRK2 to defined cellular sub-compartments, including centriolar satellites and vesicular sub-compartments. SummaryThis study defines a comprehensive, LRRK2 interaction network by combining proximity proteomics with evolutionary and structural analyses, revealing how LRRK2 conformation and activity state dictate distinct protein-protein interactions. These findings link LRRK2 to conserved centrosomal, cytoskeletal, and vesicular pathways, providing mechanistic insight into its role in Parkinsons disease. Key resultsO_LICentrosome-linked LRRK2 network: BioID combined with co-evolutionary analysis identifies a highly conserved module enriched in centrosomal and microtubule-associated proteins, including CYLD as a top co-evolved interactor. C_LIO_LIConformation-specific interactions: AlphaFold-Multimer modelling reveals distinct classes of LRRK2 interactors that bind different domains and conformations ("locked" vs. "unlocked"), corresponding to specific cellular functions. C_LIO_LIState-dependent rewiring of the interactome: The type I inhibitor MLi-2 selectively redirects LRRK2 to centriolar satellite proteins, whereas RAB29 co-expression shifts LRRK2 interactions toward vesicular and lysosomal pathways. C_LI

neuroscience↗

bioRxiv2024

Autosomal dominant Retinitis Pigmentosa caused by the rhodopsin isoleucine 255 deletion features rapid neuroretinal degeneration, decreased synaptic connectivity, and neuroinflammation.

Retinitis Pigmentosa (RP) is a group of inherited retinal diseases that initially affects rod photoreceptors and causes progressive vision loss and blindness. Mutations in rhodopsin (RHO) can cause both autosomal recessive (ar) and dominant (ad) forms of RP, yet, the underlying degenerative mechanisms remain largely unknown, rendering the disease untreatable. Here, we focus on an in-frame, 3-base pair deletion, eliminating the isoleucine residue at codon 255 (i.e., RHO{Delta}I255) and resulting in adRP. We generated a novel knock-in mouse homologous to the human RHO{Delta}I255 mutation. This new mouse model displays a severe disruption of photoreceptor structure and function, as is seen in human patients. Our results indicate that this form of RP is a systems disease of the neuroretina that also impacts neuronal connectivity of bipolar- and horizontal cells, initiates neuroinflammation, and reduces the structural and functional integrity of the retina. Typical for adRP, Rho{Delta}I255 mice exhibit primary rod photoreceptor loss, followed by secondary cone degeneration, rhodopsin protein (RHO) mislocalization, progressive shortening of outer segments (OS), and disorganized OS structures. Subsequently, increasing gliosis, morphologic abnormalities of the inner retina, and impaired cone-driven visual function developed. In adRP, a single mutated allele is sufficient to cause the disease, as confirmed here in Rho{Delta}I255/+ heterozygous animals, where most photoreceptors were lost within two months after birth. Compared to this, homozygous Rho{Delta}I255/{Delta}I255 mutants exhibit an accelerated onset and even faster progression of retinal degeneration. The degeneration of Rho{Delta}I255-mutant photoreceptors was linked to the activation of both caspase- and calpain-type proteases, as well as poly(ADP-ribose) polymerase (PARP), indicating a parallel execution of both apoptotic and non-apoptotic processes. In conclusion, our data indicate that this form of RP affects the neuroretina beyond photoreceptor cell loss sharing features typical for other degenerative central nervous systems diseases, an insight, which may bear critical impact to understand and eventually develop treatment for these currently untreatable forms of blindness. Author summaryDominant mutations in the human rhodopsin gene are among the most common causes for the blinding disease retinitis pigmentosa (RP). To date, the underlying pathophysiological mechanisms are still largely unknown and dominant RP remains untreatable. Here, we introduce a new knock-in mouse model carrying the dominant human Rho{Delta}I255 mutation. As in humans, the Rho{Delta}I255 mouse suffers from a rapid degeneration of rod photoreceptors followed by subsequent cell death of cone photoreceptors and complete loss of visual function. The new mouse model displays sign of neuroinflammation and the concomitant activation of both apoptotic and non-apoptotic cell death mechanisms. These results will likely stimulate further studies into the degenerative processes governing dominant RP and may facilitate future therapy development for inherited retinal diseases that are still untreatable to this day.

pathology↗

bioRxiv2023

De-suppression of mesenchymal cell identities and variable phenotypic outcomes associated with knockout of Bbs1.

Bardet-Biedl syndrome (BBS) is an archetypal ciliopathy caused by dysfunction of primary cilia. BBS affects multiple tissues, including the kidney, eye and hypothalamic satiety response. Understanding pan-tissue mechanisms of pathogenesis versus those which are tissue specific, and gauging their associated inter-individual variation owing to genetic background and stochastic processes, is of paramount importance in syndromology. The BBSome is a membrane trafficking and intraflagellar transport (IFT) adaptor protein complex formed by 8 BBS proteins, including BBS1, which is the most commonly mutated gene in BBS. To investigate disease pathogenesis we generated a series of clonal renal collecting duct IMCD3 cell lines carrying defined biallelic nonsense or frameshift mutations in Bbs1, as well as a panel of matching wild-type CRISPR control clones. Using a phenotypic screen and an unbiased multi-omics approach we note significant clonal variability for all assays, emphasising the importance of analysing panels of genetically-defined clones. Our results suggest that BBS1 is required for suppression of mesenchymal cell identities as IMCD3 cell passage number increases. This was associated with a failure to express epithelial cell markers and tight junction formation, which was variable amongst clones. Transcriptomic analysis of hypothalamic preparations from BBS mutant mice, and BBS patient fibroblasts, suggested that dysregulation of epithelial-to-mesenchymal transition (EMT) genes is a general predisposing feature of BBS across tissues. Collectively this work suggests that the dynamic stability of the BBSome is essential for suppression of mesenchymal cell identities as epithelial cells differentiate.

genetics↗

bioRxiv2023

Ciliopathy-associated missense mutations in IFT140 are hypomorphic and have edgetic effects on protein interaction networks

The mechanisms underlying recessive Mendelian diseases and the interplay between genotype and phenotype still need to be better understood. It is therefore necessary to characterise the functional effects of missense mutations at the protein level. Here we focus on missense mutations in the intraflagellar transport protein IFT140, which forms part of the IFT complex A (IFT-A), a crucial component of the ciliary machinery. Mutations in IFT140 can cause a vast spectrum of diseases belonging to the group of ciliopathies, reaching from isolated retinal dystrophy to severe skeletal abnormalities and multi-organ diseases such as Mainzer-Saldino and Jeune syndrome. We hypothesise that missense mutations in IFT140 are hypomorphic leading to quantitative effects on a subset of protein-protein interactions. This may affect complex stability as well as perturbations of protein interaction networks. In this work we assessed how 24 missense mutations in IFT140 affect interactions with other IFT and effector proteins using affinity purification coupled to mass spectrometry. Our data reveals that several mutations in IFT140 are hypomorphic and disrupt the stability of the IFT-A complex to varying degrees in a quantitative way. Allelic combination and the degree of IFT-A complex disruption in analysed missense mutations correlates with the severity of the observed phenotype in a subset of patients. In addition, we show that a distinct subset of mutations in IFT140 shows edgetic effects by disrupting specific PPIs rather than causing a total loss of IFT-A binding. This is the case e.g. with the disease-associated protein TULP3 which is involved in cilia-dependent sonic hedgehog signalling.

cell biology↗

bioRxiv2023

Probing the sub-cellular mechanisms of LCA5-Leber Congenital Amaurosis and associated gene therapy with expansion microscopy

Leber Congenital Amaurosis (LCA) is a group of Inherited Retinal Diseases (IRDs) characterized by the early onset and rapid loss of photoreceptor cells. Despite the discovery of a growing number of genes associated with this disease, the molecular mechanisms of photoreceptor cell degeneration of most LCA subtypes remain poorly understood. Here, using retina-specific affinity proteomics combined with Ultrastructure Expansion Microscopy (U-ExM), we revealed the structural and molecular defects underlying LCA type 5 (LCA5) with unprecedented resolution. We showed that LCA5-encoded lebercilin, together with Retinitis Pigmentosa 1 protein (RP1) and the intraflagellar transport (IFT) proteins IFT81 and IFT88, localize at the bulge region of the photoreceptor outer segment (OS), a region crucial for OS membrane disc formation. Next, we demonstrated that mutant mice deficient for lebercilin exhibit early axonemal defects at the bulge region and the distal OS, accompanied by reduced level of RP1 and IFT proteins, affecting membrane disc formation and presumably leading to photoreceptor death. Finally, we probed the LCA5 gene augmentation therapy strategy using U-ExM by monitoring its subcellular outcome. We found that, expression of LCA5 partially restores the bulge region, preserves OS axoneme structure and membrane disc formation, as well as photoreceptor survival.

molecular biology↗

bioRxiv2022

Super-resolution STED imaging in the inner and outer whole-mount mouse retina

Since its invention in 1994, super-resolution microscopy has become a popular tool for advanced imaging of biological structures, allowing visualisation of subcellular structures at a spatial scale below the diffraction limit. Thus, it is not surprising that recently, different super-resolution techniques are being applied in neuroscience, e.g. to resolve the clustering of neurotransmitter receptors and protein complex composition in presynaptic terminals. Still, the vast majority of these experiments were carried out either in cell cultures or very thin tissue sections, while there are only a few examples of super-resolution imaging in thick (> ~50 m) biological samples. In that context, the mammalian whole-mount retina has rarely been studied with super-resolution microscopy. Here, we aimed at establishing a stimulated-emission-depletion (STED) microscopy protocol for imaging whole-mount retina. To this end, we developed sample preparation including horizontal slicing of retinal tissue, an immunolabeling protocol with STED-compatible fluorophores and optimised the STED microscopes settings. We labelled subcellular structures in somata, dendrites, and axons of retinal ganglion cells in the inner mouse retina. Under optimal conditions, we achieved a mean lateral spatial resolution of ~120 nm (using the full width of half-maximum as a proxy) for the thinnest filamentous structures in our preparation and a resolution enhancement of two or higher compared to conventional confocal images. When combined with horizontal slicing of the retina, these settings allowed us visualisation of putative GABAergic horizontal cell synapses in the outer retina with a similar resolution. Taken together, we successfully established a STED protocol for reliable super-resolution imaging in the whole-mount mouse retina, which enables investigating, for instance, protein complex composition and cytoskeletal ultrastructure at retinal synapses in health and disease.

neuroscience↗

bioRxiv2022

The kinesin motor KIF1C is a putative transporter of the exon junction complex in neuronal cells

Neurons critically depend on regulated RNA localization and tight control of spatio-temporal gene expression to maintain their morphological and functional integrity. Mutations in the kinesin motor protein gene KIF1C cause Hereditary Spastic Paraplegia, an autosomal recessive disease leading to predominant degeneration of the long axons of central motoneurons. In this study we aimed to gain insight into the molecular function of KIF1C and understand how KIF1C dysfunction contributes to motoneuron degeneration. We used affinity proteomics in neuronally differentiated neuroblastoma cells (SH-SY5Y) to identify the protein complex associated with KIF1C in neuronal cells; candidate interactions were then validated by immunoprecipitation and mislocalization of putative KIF1C cargoes was studied by immunostainings. We found KIF1C to interact with all core components of the exon junction complex (EJC); expression of mutant KIF1C in neuronal cells leads to loss of the typical localization distally in neurites. Instead, EJC core components accumulate in the pericentrosomal region, here co-localizing with mutant KIF1C. These findings suggest KIF1C as a neuronal transporter of the EJC. Interestingly, the binding of KIF1C to the EJC is RNA-mediated, as treatment with RNAse prior to immunoprecipitation almost completely abolishes the interaction. Silica-based solid-phase extraction of UV-crosslinked RNA-protein complexes furthermore supports direct interaction of KIF1C with RNA, as recently also demonstrated for kinesin heavy chain. Taken together, our findings are consistent with a model where KIF1C transports mRNA in an EJC-bound and therefore transcriptionally silenced state along neurites, thus providing the missing link between the EJC and mRNA localization in neurons.

neuroscience↗

bioRxiv2022

Increased H3K27 trimethylation contributes to cone survival in a mouse model of cone dystrophy

Inherited retinal diseases (IRDs) are a heterogeneous group of blinding disorders, which result in dysfunction or death of the light-sensing cone and rod photoreceptors. Despite individual IRDs being rare, collectively, they affect up to 1:2000 people worldwide, causing a significant socioeconomic burden, especially when cone-mediated central vision is affected. This study uses the Pde6ccpfl1 mouse model of achromatopsia, a cone-specific vision loss IRD, to investigate the potential gene-independent therapeutic benefits of a histone demethylase inhibitor GSK-J4 on cone cell survival. We investigated the effects of GSK-J4 treatment on cone cell survival in vivo and ex vivo and changes in cone-specific gene expression via single-cell RNA sequencing. A single intravitreal GSK-J4 injection led to transcriptional changes in pathways involved in mitochondrial dysfunction, endoplasmic reticulum stress, among other key epigenetic pathways, highlighting the complex interplay between methylation and acetylation in healthy and diseased cones. Furthermore, continuous administration of GSK-J4 in retinal explants increased cone survival. Our results suggest that IRD-affected cones respond positively to epigenetic modulation of histones, indicating the potential of this approach in the development of a broad class of novel therapies to slow cone degeneration.

neuroscience↗

bioRxiv2021

mTOR inhibition via Rapamycin treatment partially reverts the deficit in energy metabolism caused by FH loss in RPE cells

Age-related macular degeneration (AMD) is a complex degenerative disease of the retina with multiple risk-modifying factors, including ageing, genetics and lifestyle choices. The combination of these factors leads to oxidative stress, inflammation and metabolic failure in the retinal pigment epithelium (RPE) with subsequent degeneration of photoreceptors in the retina. The alternative complement pathway is tightly linked to AMD. In particular, the genetic variant in the complement factor H gene (CFH), that leads to the Y402H polymorphism in the factor H protein (FH), confers the second highest risk for the development and progression of AMD. While the association between the FH Y402H variant and increased complement system activation is known, recent studies have uncovered novel FH functions not tied to this activity and highlighted functional relevance for intracellular FH. In our previous studies, we show that loss of CFH expression in RPE cells causes profound disturbances in cellular metabolism, increases the vulnerability towards oxidative stress and modulates the activation of pro-inflammatory signaling pathways, most importantly the NF-kB pathway. Here, we silenced CFH in hTERT-RPE1 cells to investigate the mechanism by which intracellular FH regulates RPE cell homeostasis. We found that silencing of CFH results in hyperactivation of mTOR signaling along with decreased mitochondrial respiration and that mTOR inhibition via rapamycin can partially rescue these metabolic defects. To obtain mechanistic insight into the function of intracellular FH in hTERT-RPE1 cells, we analyzed the interactome of FH via immunoprecipitation followed by Mass spectrometry-based analysis. We found that FH interacts with essential components of the ubiquitin-proteasomal pathway (UPS) as well as with factors associated with RB1/E2F signalling in a complement-pathway independent manner. Moreover, we found, that FH silencing affects mRNA levels of the E3 Ubiquitin-Protein Ligase Parkin and PTEN induced putative kinase (Pink1), both of them are associated with UPS. As inhibition of mTORC1 has been previoulsy shown to result in increased overall protein degradation via UPS and as FH mRNA and protein levels were shown to be affected by inhibition of UPS, our data stress a potential regulatory link between endogenous FH activity and the UPS.

molecular biology↗

bioRxiv2021

Synaptotagmin 13 orchestrates pancreatic endocrine cell egression and islet morphogenesis

Epithelial cell egression is important for organ development, but also drives cancer metastasis. Better understandings of pancreatic epithelial morphogenetic programs generating islets of Langerhans aid to diabetes therapy. Here we identify the Ca2+-independent atypical Synaptotagmin 13 (Syt13) as a key driver of endocrine cell egression and islet formation. We detected upregulation of Syt13 in endocrine precursors that correlates with increased expression of unique cytoskeletal components. High-resolution imaging reveals a previously unidentified apical-basal to front-rear repolarization during endocrine cell egression. Strikingly, Syt13 interacts with acetylated tubulin and phosphatidylinositol phospholipids and localizes to the leading-edge of egressing cells. Knockout of Syt13 impairs endocrine cell egression and skews the - to-{beta}-cell ratio. Mechanistically, Syt13 regulates endocytosis to remodel the basement membrane and cell-matrix adhesion at the leading-edge of egressing endocrine cells. Altogether, these findings implicate an unexpected role of Syt13 in regulating cell polarity to orchestrate endocrine cell egression and islet morphogenesis.

developmental biology↗

bioRxiv2021

FH loss in RPE cells causes retinal degeneration in a human RPE-porcine retinal explant co-culture model

Age-related Macular degeneration (AMD) is a degenerative disease of the macula affecting the elderly population. Treatment options are limited, partly due to the lack of understanding of AMD pathology and the sparse availability of research models, that replicate the complexity of the human macula and the intricate interplay of the genetic, aging and life-style risk factors contributing to AMD. One of the main genetic risks associated with AMD is located on Complement Factor H (CFH) gene, leading to an amino acid substitution in the FH protein (Y402H). However, the mechanism of how this FH variant promotes the onset of AMD remains unclear. Previously, we have shown that FH deprivation in RPE cells, via CFH silencing, leads to increased inflammation, metabolic impairment and vulnerability towards oxidative stress. In this study, we established a novel co-culture model comprised of CFH silenced RPE cells and porcine retinal explants derived from the visual streak of the porcine eyes, closely resembling the human macula. We show that retinae exposed to FH-deprived RPE cells show signs of retinal degeneration, with rod cells being the first cells to undergo degeneration. Moreover, via Raman analyses, we observe that the main changes involve the mitochondria and lipid composition of the co-cultured retinae upon FH loss. Interestingly, the detrimental effects of FH loss in RPE cells on the neuroretina were independent of glial cell activation and external complement sources. Moreover, we show that the co-culture model is also suitable for human retinal explants, and we observed a similar trend when RPE cells deprived of FH were co-cultured with human retinal explants from a single donor eye. Our findings highlight the importance of RPE derived FH for retinal homeostasis and provide a valuable model for AMD research.

cell biology↗

bioRxiv2021

Pharmacological inhibition of the VCP/proteasome axis rescues photoreceptor degeneration in RHOP23H rat retinal explants

1.Rhodopsin (RHO) misfolding mutations are a common cause of the blinding disease autosomal dominant retinitis pigmentosa (adRP). The most prevalent mutation, RHOP23H, results in its misfolding and retention in the Endoplasmic Reticulum (ER). Under homeostatic conditions, misfolded proteins are selectively identified, retained at the ER, and cleared via ER-associated degradation (ERAD) and/or autophagy. Overload of these degradation processes for a prolonged period leads to imbalanced proteostasis and may eventually result in cell death. ERAD of misfolded proteins like RHOP23H includes the subsequent steps of protein recognition, targeting for ERAD, retrotranslocation, and proteasomal degradation. In the present study, we investigated and compared pharmacological modulation of ERAD at these four different major steps. We show that inhibition of the VCP/proteasome activity favors cell survival and suppresses P23H-mediated retinal degeneration in RHOP23H rat retinal explants. We suggest targeting this activity as a therapeutic approach for patients with currently untreatable adRP.

cell biology↗

bioRxiv2021

WDR31 is a novel ciliopathy protein displaying functional redundancy with GTPase-activating proteins ELMOD and RP2 in recruiting BBSome to cilium

The correct intraflagellar transport (IFT) assembly at the ciliary base and the IFT turnaround at the ciliary tip are key for the IFT to perform its function, but we still have poor understanding about how these processes are regulated. Here, we identify WDR31 as a new ciliary protein, and analysis from zebrafish and Caenorhabditis elegans reveals the role of WDR31 in regulating the cilia morphology. We find that loss of WDR-31 together with RP-2 and ELMD-1 (the sole ortholog ELMOD1-3) results in ciliary accumulations of IFT Complex B components and KIF17 kinesin, with fewer IFT/BBSome particles traveling along cilia in both anterograde and retrograde directions, suggesting that the IFT/BBSome entry into cilia and exit from cilia are impacted. Furthermore, anterograde IFT in the middle segment travel at increased speed in wdr-31;rpi-2;elmd-1. Remarkably, a non-ciliary protein leaks into cilia of wdr-31;rpi-2;elmd-1 possible due to IFT defects. This work reveals WDR31-RP-2-ELMD-1 as IFT and BBSome trafficking regulators.

genetics↗

bioRxiv2021

Self-assembled nanoparticles enable delivery of VCP inhibitor, resulting in enhanced photoreceptor protection in retinal explants

1.Mutations in rhodopsin lead to its misfolding resulting in autosomal dominant retinitis pigmentosa (adRP). Pharmacological inhibition of the ATP-driven chaperone valosin- containing protein (VCP), a molecular checkpoint for protein quality control, slows down retinal degeneration in animal models. However, poor water-solubility of VCP inhibitors poses a challenge to their clinical translation as intravitreal injections for retinal treatment. In order to enable the delivery of VCP inhibitors, we have developed and investigated two formulations for the VCP inhibitor ML240. Nanoformulations of ML240 were obtained by using amphiphilic polymers methoxy-poly (ethylene glycol)5kDa-cholane (mPEG5kDa-cholane) and methoxy-poly (ethylene glycol)5kDa- cholesterol (mPEG5kDa-cholesterol). Both formulations increased the water-solubility of ML240 by two orders of magnitude and prolonged the drug released over ten days. Encapsulation of ML240 in mPEG5kDa-cholane showed superior photoreceptor protection at lower drug concentrations, normalized rhodopsin localization, and alleviated inflammatory microglial responses in an ex vivo rat model of retinal degeneration. The study demonstrates the potential of VCP inhibitor nanoformulations to treat adRP, a pharmacologically orphan disease.

pharmacology and toxicology↗