Biology subjects

Bonifati, V.

Publications and source records attributed to Bonifati, V..

7 recordsLinked to original sources

bioRxiv2024

DJ-1 mediates regulation of metabolism and immune response in Parkinsons disease astrocytes and Glioblastoma cells

An inverse correlation for the expression of Parkinsons disease (PD)- and cancer-associated genes has been previously reported. Genes that are upregulated in cancer are frequently downregulated in PD and vice versa. PARK7, encoding DJ-1, was initially identified as an oncogene, but loss of DJ-1 causes early-onset PD. However, it remains elusive how differential DJ-1 levels contribute to opposite cell fates in cancer and PD. Here, we demonstrate specific effects of differential DJ-1 protein levels on the energy metabolism and cell growth in patient-derived cellular models of PD and glioblastoma (GBM) cell lines. Impaired energy metabolism was associated with an increased immune response upon IL-1{beta} stimulation and increased apoptosis and decreased cell growth in models of PD, whereas in GBM cells increased metabolic activity translated into a reduced immune response and increased cell growth. Furthermore, we found decreased glutathione (GSH) synthesis and therefore increased levels of reactive oxygen species (ROS) and oxidized glutathione (GSSG) in models of DJ-1 deficiency and decreased ROS levels in GBM cell lines. Thus, the mechanism by which DJ-1 modulates these phenotypes is the same in both diseases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/621212v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@4eccb4org.highwire.dtl.DTLVardef@161669corg.highwire.dtl.DTLVardef@197bfc3org.highwire.dtl.DTLVardef@1aecb36_HPS_FORMAT_FIGEXP M_FIG C_FIG DJ-1 levels modulate GSSG/GSH ratio and ROS levels, which results in divergent effects on cell growth and immune response in DJ-1-dependent glial pathologies in glioblastoma and PD. In models of PD, DJ-1 level dependent phenotypes can be rescued by antioxidant treatment that reduces the GSSG/GSH ratio and ROS levels.

neuroscience↗

bioRxiv2023

Interactome mapping reveals a role for LRP10 in autophagy and NDFIP1-mediated alpha-synuclein secretion

Variants in the LRP10 gene have been found in a spectrum of neurodegenerative disorders, including Lewy body diseases (LBDs). In brains of LBD patients, LRP10 is found in neuronal -synuclein-containing Lewy bodies, astrocytes, and vasculature, but not in inclusion-free neurons. Furthermore, recent work suggests that LRP10 is involved in -synuclein processing and transmission, which is disrupted by the LBD-associated LRP10:c.1424+5G>A variant (LRP10-Splice). In spite of the cumulating genetic and functional evidence for a role of LRP10 in neurodegenerative disorders, our knowledge about the biological processes in which LRP10 is involved is incomplete. In this work, we provide a list of LRP10 interactors identified via LRP10 co-immunoprecipitation and mass spectrometry in LRP10-overexpressing cells and induced pluripotent stem cells (iPSC)-derived astrocytes. In addition to interactors and biological processes previously associated with LRP10, we identified novel interactors and pathways that may provide new insights into LRP10 function. Based on these findings, we focused on the involvement of LRP10 in the autophagy and unconventional secretion pathways via its interaction with the autophagy receptor SQSTM1/p62 and the ubiquitin-proteasome adaptor protein NDFIP1, respectively. We demonstrate that changes in LRP10 levels, either via knock-out or overexpression, affect p62 levels and autophagy in HuTu-80 cells and iPSC-derived astrocytes. Furthermore, we found that both LRP10 and NDFIP1 stimulate -synuclein secretion and synergistically affect intracellular -synuclein levels. Next, we studied the LRP10 interactome and related biological processes in iPSC-derived astrocytes carrying the LRP10-Splice variant. Although various interactors and biological processes were shared between wild-type LRP10 (LRP10-WT) and LRP10-Splice, others were only found in either LRP10-WT or LRP10-Splice. Interestingly, we found that LRP10-Splice responded differently to autophagy-modulating drugs in comparison to LRP10-WT. Furthermore, we show that LRP10-Splice interferes with the LRP10-WT:NDFIP1 interaction and NDFIP1-mediated -synuclein secretion. Finally, we investigated the interactome of a secreted LRP10 species only found in conditioned media from LRP10-Splice carrier cells, and identify biological processes that might be impacted by the secreted LRP10-Splice specific protein. In summary, this study enhances our understanding of LRP10 biology, describes LRP10 functions in autophagy and NDFIP1-mediated -synuclein secretion, and reveals potentially interesting differences between LRP10-WT and LRP10-Splice carrier cells that might be relevant to better understand the role of LRP10 in LBDs pathogenesis.

neuroscience↗

bioRxiv2023

Altered TFEB subcellular localization in nigral dopaminergic neurons of subjects with prodromal, sporadic and GBA-related Parkinson's disease and Dementia with Lewy bodies

Transcription factor EB is a master regulator of genes involved in the maintenance of autophagic and lysosomal homeostasis, processes which have been implicated in the pathogenesis of GBA-related and sporadic Parkinsons disease (PD) and dementia with Lewy bodies (DLB). TFEB activation at the lysosomal level results in its translocation from the cytosol to the nucleus. Here, we aimed at investigating whether TFEB subcellular localization is altered in post-mortem human brain of aged individuals with either prodromal PD/DLB (incidental Lewy body disease, iLBD, N=3), GBA-related PD/DLB (N=9) or sPD/DLB (N=9), compared to control subjects (N=12). We scanned nigral dopaminergic neurons using high-resolution confocal and stimulated emission depletion (STED) microscopy and semi-quantitatively scored the observed TFEB subcellular localization patterns. In line with previous studies, we observed reduced nuclear TFEB immunoreactivity in PD/DLB patients compared to controls, both sporadic and GBA-related cases, as well as in iLBD cases. Nuclear depletion of TFEB was more pronounced in neurons with Ser129-phosphorylated (pSer129) aSyn cytopathology and in cases carrying pathogenic GBA variants. Interestingly, we further observed previously unidentified TFEB-immunopositive somatic clusters in human brain dopaminergic neurons and in human embryonic stem cell (hESC)-derived neurons, which localized at the Golgi apparatus. The TFEB clustering was more frequently observed and more severe in iLBD, sPD/DLB and GBA-PD/DLB compared to controls, particularly in pSer129 aSyn-positive neurons but also in neurons without apparent cytopathology. Notably, increased frequency of cytoplasmic TFEB clusters in aSyn-negative cells correlated with reduced total GBA enzymatic activity and higher Braak LB stage. In the studied patient population, altered TFEB distribution was accompanied by a reduction in overall mRNA expression levels of selected CLEAR genes, indicating a possible early dysfunction of lysosomal regulation. Overall, these findings suggest the early cytoplasmic TFEB retention and accumulation at the Golgi prior pSer129 aSyn accumulation in incidental, GBA-related and sporadic PD/DLB and indicate TFEB as potential as early therapeutic target for synucleinopathies

neuroscience↗

bioRxiv2023

LRP10 as a novel α-synuclein regulator in Lewy body diseases

Autosomal dominant variants in LRP10 have been identified in patients with Lewy body diseases (LBDs), including Parkinsons disease (PD), Parkinsons disease-dementia (PDD), and dementia with Lewy bodies (DLB). Nevertheless, there is little mechanistic insight into the role of LRP10 in disease pathogenesis. In the brains of non-demented individuals, LRP10 is typically expressed in non-neuronal cells like astrocytes and neurovasculature, but in idiopathic and genetic cases of PD, PDD, and DLB it is also present in -synuclein-positive neuronal Lewy bodies. These observations raise the questions of what leads to the accumulation of LRP10 in Lewy bodies and whether a possible interaction between LRP10 and -synuclein plays a role in disease pathogenesis. Here, we demonstrate that wild-type LRP10 is secreted via extracellular vesicles (EVs) and can be internalised via clathrin-dependent endocytosis. Additionally, we show that LRP10 secretion is highly sensitive to autophagy inhibition, which induces the formation of atypical LRP10 vesicular structures in neurons in human induced pluripotent stem cells (iPSC)-derived midbrain-like organoids (hMLOs). Furthermore, we show that LRP10 overexpression leads to a strong induction of monomeric -synuclein secretion, together with time-dependent, stress-sensitive changes in intracellular -synuclein levels. Interestingly, patient-derived astrocytes carrying the c.1424+5G>A LRP10 variant secrete aberrant high-molecular-weight species of LRP10 in EV-free media fractions. Finally, we show that the truncated LRP10splice protein binds to wild-type LRP10, reduces LRP10 wild-type levels, and antagonises the regulatory effect of LRP10 on -synuclein levels and distribution. Together, this work provides initial evidence for a functional role of LRP10 in LBDs by regulating intra- and extracellular -synuclein levels, and pathogenic mechanisms linked to the disease-associated c.1424+5G>A LRP10 variant, pointing towards potentially important disease mechanisms in LBDs.

neuroscience↗

bioRxiv2022

deCLUTTER2+ pipeline to analyze calcium traces in a novel stem cell model for ventral midbrain patterned astrocytes

Astrocytes are the most populous cell type of the human central nervous system and are essential for physiological brain function. Increasing evidence suggests multiple roles for astrocytes in Parkinsons disease (PD), nudging a shift in the research focus, which historically pivoted around the ventral midbrain dopaminergic neurons (vmDANs). Studying human astrocytes and other cell types in vivo remains technically and ethically challenging. However, in vitro reprogrammed human stem cell-based models provide a promising alternative. Here, we describe a novel protocol for astrocyte differentiation from human stem cell-derived vmDANs-generating progenitors. This protocol simulates the regionalization, gliogenic switch, radial migration, and final differentiation that occur in the developing human brain. We have characterized the morphological, molecular, and functional features of these ventral midbrain astrocytes with a broad palette of techniques. In addition, we have developed a new pipeline for calcium imaging data analysis called deCLUTTER2+ (deconvolution of Ca2+ fluorescent patterns) that can be used to discover spontaneous or cue-dependent patterns of Ca2+ transients. Altogether, our protocol enables the characterization of the functional properties of human ventral midbrain astrocytes under physiological conditions and in PD.

neuroscience↗

bioRxiv2021

Correcting differential gene expression analysis for cyto-architectural alterations in substantia nigra of Parkinson's disease patients reveals known and potential novel disease-associated genes and pathways

Several studies have analyzed gene expression profiles in the substantia nigra to better understand the pathological mechanisms causing Parkinsons disease (PD). However, the concordance between the identified gene signatures in these individual studies was generally low. This might be caused by a change in cell type composition as loss of dopaminergic neurons in the substantia nigra pars compacta is a hallmark of PD. Through an extensive meta-analysis of nine previously published microarray studies, we demonstrated that a big proportion of the detected differentially expressed genes was indeed caused by cyto-architectural alterations due to the heterogeneity in the neurodegenerative stage and/or technical artifacts. After correcting for cell composition, we identified a common signature that deregulated the previously unreported ammonium transport, as well as known biological processes including bioenergetic pathways, response to proteotoxic stress, and immune response. By integrating with protein-interaction data, we shortlisted a set of key genes, such as LRRK2, PINK1, and PRKN known to be related to PD; others with compelling evidence for their role in neurodegeneration, such as GSK3{beta}, WWOX, and VPC; as well as novel potential players in the PD pathogenesis, including NTRK1, TRIM25, ELAVL1. Together, these data showed the importance of accounting for cyto-architecture in these analyses and highlight the contribution of multiple cell types and novel processes to PD pathology providing potential new targets for drug development. Significance StatementThe exploration of the transcriptomic landscape in PD is pivotal for the understanding of the pathological mechanisms of this disease. Nonetheless, little attention has been paid to the influence of cell composition on the transcriptome even though it is known that cyto-architecture undergoes major alterations in neurodegenerative diseases such as PD. Our study signifies that changes in cellular architecture of human substantia nigra in PD have a strong effect on the set of detected differentially expressed genes. By reanalyzing the data and accounting for cell composition, we provide an updated description of deregulated biological processes in PD and nominate a shortlist of PD-associated genes for further investigations.

bioinformatics↗

bioRxiv2017

Lewy pathology in Parkinson’s disease consists of a crowded organellar membranous medley

Parkinsons disease, the most common age-related movement disorder, is a progressive neurodegenerative disease with unclear etiology. Key neuropathological hallmarks are Lewy bodies and Lewy neurites, which are neuronal inclusions that are immunopositive for the protein -synuclein. In-depth ultrastructural analysis of this Lewy pathology is crucial to understanding pathogenesis and progression of the disease. Using correlative light and electron microscopy/tomography on brain tissue from five Parkinsons disease brain donors, we identified -synuclein immunopositive Lewy pathology and could show that the majority of these features including Lewy bodies and Lewy neurites primarily consists of a crowded membranous medley of vesicular structures and dysmorphic organelles. Only a small fraction of observed Lewy bodies contained predominant proteinaceous filaments, as previously described. The crowding of organellar components was confirmed by STED- based super-resolution microscopy, and high lipid content within the -synuclein immunopositive inclusions was corroborated by confocal imaging, CARS/FTIR imaging and lipidomics. Applying this correlative high-resolution imaging and biophysical approach, we discovered in the postmortem brain of Parkinsons patients a subcellular protein-lipid compartmentalization not previously described in Lewy pathology.

neuroscience↗