Biology subjects

Stefanis, L.

Publications and source records attributed to Stefanis, L..

6 recordsLinked to original sources

bioRxiv2025

In vivo validation of novel non-invasive PHP.eB AAVs as a potential therapeutic approach for alpha-Synucleinopathies

Parkinsons disease (PD) is characterized by the accumulation of alpha-synuclein (aSyn) aggregates in specific brain regions, which are likely to be the disease-causing entities. Herein, we employed novel, systemically administered, brain-penetrating viral vectors (PHP.eB AAVs) in order to evaluate the potential therapeutic utility of lowering the endogenous aSyn protein burden in the aSyn pre-formed fibril (PFF)-mouse model. Such vectors expressing short hairpin RNAs or micro RNAs targeting the mouse Snca transcript (or respective scrambled control sequences) were intravenously administered in adult wild-type mice and two weeks later human aSyn PFFs were injected into the dorsal striatum. Following the administration of the Snca-targeting PHP.eB AAVs, a successful widespread viral transduction was achieved throughout the brain, accompanied by an efficient reduction of endogenous aSyn protein levels within transduced dopaminergic neurons. Intrastriatal injection of human aSyn PFFs led to the formation of pSer129-aSyn-rich cytoplasmic inclusions in brain regions connected to the PFF-injection site, nigrostriatal degeneration and relevant behavioral motor deficits, at 2.5 months post PFF-injection. Importantly, PHP.eB AAV-mediated down-regulation of endogenous aSyn reduced the accumulation of pSer129-aSyn+ inclusions, mitigated nigrostriatal degeneration and alleviated motor impairments. Spread of pathology to other brain regions was also attenuated. Overall, such data highlight further the contribution of the intracellular aSyn protein load to the spread of pathology and suggest that this non-invasive delivery strategy holds promise in the research avenues for treating neurodegenerative diseases with widespread pathology, such as Synucleinopathies.

neuroscience↗

bioRxiv2023

Flame (v2.0): advanced integration and interpretation of functional enrichment results from multiple sources

Functional enrichment is the process of identifying implicated functional terms from a given input list of genes or proteins. In this article, we present Flame (v2.0), a web tool which offers a combinatorial approach through merging and visualizing results from widely-used functional enrichment applications while also allowing various flexible input options. In this version, Flame utilizes the aGOtool, g:Profiler, WebGestalt and Enrichr pipelines and presents their outputs separately or in combination following a visual analytics approach. For intuitive representations and easier interpretation, it uses interactive plots such as parameterizable networks, heatmaps, barcharts and scatter plots. Users can also: (i) handle multiple protein/gene lists and analyze union and intersection sets simultaneously through interactive UpSet plots, (ii) automatically extract genes and proteins from free text through text-mining and Named Entity Recognition (NER) techniques, (iii) upload single nucleotide polymorphisms (SNPs) and extract their relative genes or (iv) analyze multiple lists of differentially-expressed proteins/genes after selecting them interactively from a parameterizable volcano plot. Compared to the previous version of 197 supported organisms, Flame (v2.0) currently allows enrichment for 14,436 organisms.

bioinformatics↗

bioRxiv2022

Microglia and astrocytes differentially endocytose exosomes facilitating alpha-Synuclein endolysosomal sorting

Exosomes have emerged as key players in cell-to-cell communication in both physiological and pathological processes in the Central Nervous System (CNS). Thus far, the intracellular pathways involved in uptake and trafficking of exosomes within different cell types of the brain (microglia and astrocytes) are poorly understood. In our study, the endocytic processes and subcellular sorting of exosomes were investigated in primary glial cells, particularly linked with the exosome-associated -synuclein (-syn) transmission. Mouse microglia and astrocytic primary cultures were incubated with DiI-stained mouse brain-derived exosomes. The internalization and trafficking pathways were analysed in cells treated with pharmacological reagents that block the major endocytic pathways. Brain-derived exosomes were internalized by both glial cell types; however, uptake was more efficient in microglia than in astrocytes. Colocalization of exosomes with early and late endocytic markers (Rab5, Lamp1) indicated that exosomes are sorted to endolysosomes for subsequent processing. Treatment with Cytochalasin D, that blocks actin-dependent phagocytosis and/or macropinocytosis, inhibited exosome entry into glial cells, whereas treatment with inhibitors that strip off cholesterol from the plasma membrane, induced uptake, however differentially altered endosomal sorting. Exosome-associated fibrillar -Syn was efficiently internalized and detected in Rab5- and Lamp1-positive compartments within microglia. Our study strongly suggests that exosomes enter glial cells through an actin network-dependent endocytic pathway and are sorted to endolysosomes for subsequent processing. Further, brain-derived exosomes are capable of mediating cell-to-glia transmission of pathological -Syn that is also targeted to the endosomal pathway, suggesting a possible beneficial role in microglia-mediated clearance of toxic protein aggregates, present in numerous neurodegenerative diseases.

neuroscience↗

bioRxiv2021

Distinct profiles of LRRK2 activation and Rab GTPase phosphorylation in clinical samples from different PD cohorts

Despite several advances in the field, pharmacodynamic outcome measures reflective of LRRK2 kinase activity in clinical biofluids remain urgently needed. A variety of targets and approaches have been utilized including assessments of LRRK2 itself (levels, phosphorylation), or its substrates (e.g. Rab10 or other Rab GTPases). We have previously shown that intrinsic kinase activity of LRRK2 isolated from PBMCs of G2019S carriers is elevated, irrespective of disease status. In the present study we find that phosphorylation of Rab10 is also elevated in G2019S carriers, but only those with PD. Additionally, phosphorylation of this substrate is also elevated in 2 separate idiopathic PD cohorts, but not in carriers of the A53T mutation in -synuclein. In contrast, Rab29 phosphorylation was specifically reduced in urinary exosomes from A53T and idiopathic PD patients. Taken together, our findings highlight the need for the assessment of multiple complimentary targets for a more comprehensive picture of the disease.

neuroscience↗

bioRxiv2021

A double-hit in vivo model of GBA1 viral microRNA-mediated downregulation and human alpha-synuclein overexpression demonstrates nigrostriatal degeneration

Preclinical and clinical studies support a strong association between mutations in the GBA1 gene that encodes {beta}-glucocerebrosidase (GCase) (EC 3.2.1.45; glucosylceramidase beta) and Parkinsons disease (PD). Alpha-synuclein (AS), a key player in PD pathogenesis, and GBA1 mutations may independently and synergistically cause lysosomal dysfunction and thus, embody clinically well-validated targets of the neurodegenerative disease process in PD. However, double-hit in vivo models, recapitulating pathological features of PD that can be used to dissect the nature of the complex relationship between GCase and AS on the nigrostriatal axis, the region particularly vulnerable in PD, are direly needed. To address this, we implemented a bidirectional approach in mice to examine the effects of: 1) GCase overexpression (wildtype and mutant N370S) on endogenous AS levels and 2) downregulation of endogenous GCase combined with AS overexpression. Striatal delivery of viral-mediated GCase overexpression revealed minimal effects on cortical and nigrostriatal AS tissue levels and no significant effect on dopaminergic system integrity. On the other hand, microRNA (miR)-mediated GBA1 downregulation (miR GBA), combined with virus-mediated human AS overexpression (+AS), yields decreased GCase activity in the cortex, mimicking levels seen in GBA1 heterozygous carriers (30-40%), increased astrogliosis and microgliosis, decreased striatal dopamine levels (50% compared to controls) and loss of nigral dopaminergic neurons (~33%)-effects that were all reversible with miR rescue. Most importantly, the synergistic neurodegeneration of miR GBA+AS correlated with augmented AS accumulation and extracellular release in the striatum. Collectively, our results suggest that GCase downregulation alone is not sufficient to recapitulate key pathological features of PD in vivo, but its synergistic interplay with AS, via increased AS levels and release, drives nigrostriatal neurodegeneration. Furthermore, we demonstrate a novel double-hit model that can be used to identify putative mechanisms driving PD pathophysiology and can be subsequently used to test novel therapeutic approaches.

neuroscience↗

bioRxiv2020

High Content Screening and Proteomic Analysis Identify the Kinase Inhibitor BX795 as a Potent Neuroprotective Compound in a Patient-Derived Model of Parkinson's Disease

Combining high throughput screening approaches with induced pluripotent stem cell (iPSC)-based disease modeling represents a promising unbiased strategy to identify therapies for neurodegenerative disorders. Here we applied high content imaging on iPSC-derived neurons from patients with familial Parkinsons disease bearing the G209A (p.A53T) -synuclein (Syn) mutation and launched a screening campaign on a small kinase inhibitor library. We thus identified the multi-kinase inhibitor BX795 that at a single dose effectively restores disease-associated neurodegenerative phenotypes. Proteomics profiling mapped the molecular pathways underlying the protective effects of BX795, comprising a cohort of 118 protein-mediators of the core biological processes of RNA metabolism, protein synthesis, modification and clearance, and stress response, all linked to the mTORC1 signaling hub. In agreement, expression of human p.A53T-Syn in neuronal cells affected key components of the mTORC1 pathway resulting in aberrant protein synthesis that was restored in the presence of BX795 with concurrent facilitation of autophagy. Taken together, we have identified a promising small molecule with neuroprotective actions as candidate therapeutic for PD and other protein conformational disorders.

neuroscience↗