Non-invasive systemic viral delivery of human alpha-synuclein mimics selective and progressive neuropathology of Parkinson's disease in rodent brains.

BACKGROUND: Alpha-synuclein (α-syn) aggregation into proteinaceous intraneuronal inclusions, called Lewy bodies (LBs), is the neuropathological hallmark of Parkinson's disease (PD) and related synucleinopathies. However, the exact role of α-syn inclusions in PD pathogenesis remains elusive. This lack of knowledge is mainly due to the absence of optimal α-syn-based animal models that recapitulate the different stages of neurodegeneration. METHODS: Here we describe a novel approach for a systemic delivery of viral particles carrying human α-syn allowing for a large-scale overexpression of this protein in the mouse brain. This approach is based on the use of a new generation of adeno-associated virus (AAV), AAV-PHP.eB, with an increased capacity to cross the blood-brain barrier, thus offering a viable tool for a non-invasive and large-scale gene delivery in the central nervous system. RESULTS: Using this model, we report that widespread overexpression of human α-syn induced selective degeneration of dopaminergic (DA) neurons, an exacerbated neuroinflammatory response in the substantia nigra and a progressive manifestation of PD-like motor impairments. Interestingly, biochemical analysis revealed the presence of insoluble α-syn oligomers in the midbrain. Together, our data demonstrate that a single non-invasive systemic delivery of viral particles overexpressing α-syn prompted selective and progressive neuropathology resembling the early stages of PD. CONCLUSIONS: Our new in vivo model represents a valuable tool to study the role of α-syn in PD pathogenesis and in the selective vulnerability of nigral DA neurons; and offers the opportunity to test new strategies targeting α-syn toxicity for the development of disease-modifying therapies for PD and related disorders.

Aggregation of alpha-synuclein disrupts mitochondrial metabolism and induce mitophagy via cardiolipin externalization.

Accumulation of α-synuclein aggregates in the substantia nigra pars compacta is central in the pathophysiology of Parkinson's disease, leading to the degeneration of dopaminergic neurons and the manifestation of motor symptoms. Although several PD models mimic the pathological accumulation of α-synuclein after overexpression, they do not allow for controlling and monitoring its aggregation. We recently generated a new optogenetic tool by which we can spatiotemporally control the aggregation of α-synuclein using a light-induced protein aggregation system. Using this innovative tool, we aimed to characterize the impact of α-synuclein clustering on mitochondria, whose activity is crucial to maintain neuronal survival. We observed that aggregates of α-synuclein transiently and dynamically interact with mitochondria, leading to mitochondrial depolarization, lower ATP production, mitochondrial fragmentation and degradation via cardiolipin externalization-dependent mitophagy. Aggregation of α-synuclein also leads to lower mitochondrial content in human dopaminergic neurons and in mouse midbrain. Interestingly, overexpression of α-synuclein alone did not induce mitochondrial degradation. This work is among the first to clearly discriminate between the impact of α-synuclein overexpression and aggregation on mitochondria. This study thus represents a new framework to characterize the role of mitochondria in PD.

Optogenetic-mediated induction and monitoring of α-synuclein aggregation in cellular models of Parkinson's disease.

Studying Parkinson's disease (PD) is complex due to a lack of cellular models mimicking key aspects of protein pathology. Here, we present a protocol for inducing and monitoring α-synuclein aggregation in living cells using optogenetics. We describe steps for plasmid transduction, biochemical validation, immunocytochemistry, and live-cell confocal imaging. These induced aggregates fulfill the cardinal features of authentic protein inclusions observed in PD-diseased brains and offer a tool to study the role of protein aggregation in neurodegeneration. For complete details on the use and execution of this protocol, please refer to Bérard et al.1.

Optimized protocol for the generation of functional human induced-pluripotent-stem-cell-derived dopaminergic neurons.

Generation of functional human dopaminergic (DA) neurons from human induced pluripotent stem cells (hiPSCs) is a crucial tool for modeling dopamine-related human diseases and cell replacement therapies. Here, we present a protocol to combine neuralizing transcription factor (NGN2) programming and DA patterning to differentiate hiPSCs into mature and functional induced DA (iDA) neurons. We describe steps from transduction of hiPSCs and neural induction through to differentiation and maturation of near-pure, fully functional iDA neurons within 3 weeks. For complete details on the use and execution of this protocol, please refer to Sheta et al. (2022).1.

Combining NGN2 programming and dopaminergic patterning for a rapid and efficient generation of hiPSC-derived midbrain neurons.

The use of human derived induced pluripotent stem cells (hiPSCs) differentiated to dopaminergic (DA) neurons offers a valuable experimental model to decorticate the cellular and molecular mechanisms of Parkinson's disease (PD) pathogenesis. However, the existing approaches present with several limitations, notably the lengthy time course of the protocols and the high variability in the yield of DA neurons. Here we report on the development of an improved approach that combines neurogenin-2 programming with the use of commercially available midbrain differentiation kits for a rapid, efficient, and reproducible directed differentiation of hiPSCs to mature and functional induced DA (iDA) neurons, with minimum contamination by other brain cell types. Gene expression analysis, associated with functional characterization examining neurotransmitter release and electrical recordings, support the functional identity of the iDA neurons to A9 midbrain neurons. iDA neurons showed selective vulnerability when exposed to 6-hydroxydopamine, thus providing a viable in vitro approach for modeling PD and for the screening of small molecules with neuroprotective proprieties.

Inhibition of PLK2 activity affects APP and tau pathology and improves synaptic content in a sex-dependent manner in a 3xTg mouse model of Alzheimer's disease.

Converging lines of evidence suggest that abnormal accumulation of the kinase Polo-like kinase 2 (PLK2) might play a role in the pathogenesis of Alzheimer's disease (AD), possibly through its role in regulating the amyloid ß (Aß) cascade. In the present study, we investigated the effect of inhibiting PLK2 kinase activity in in vitro and in vivo models of AD neuropathology. First, we confirmed that PLK2 overexpression modulated APP and Tau protein levels and phosphorylation in cell culture, in a kinase activity dependent manner. Furthermore, a transient treatment of triple transgenic mouse model of AD (3xTg-AD) with a potent and specific PLK2 pharmacological inhibitor (PLK2i #37) reduced some neuropathological aspects in a sex-dependent manner. In 3xTg-AD males, treatment with PLK2i #37 led to lower Tau burden, higher synaptic protein content, and prevented learning and memory deficits. In contrast, treated females showed an exacerbation of Tau pathology, associated with a reduction in amyloid plaque accumulation. Overall, our findings suggest that PLK2 inhibition alters key components of AD neuropathology in a sex-dependent manner and might display a therapeutic potential for the treatment for AD and related dementia.

A light-inducible protein clustering system for in vivo analysis of α-synuclein aggregation in Parkinson disease.

Neurodegenerative disorders refer to a group of diseases commonly associated with abnormal protein accumulation and aggregation in the central nervous system. However, the exact role of protein aggregation in the pathophysiology of these disorders remains unclear. This gap in knowledge is due to the lack of experimental models that allow for the spatiotemporal control of protein aggregation, and the investigation of early dynamic events associated with inclusion formation. Here, we report on the development of a light-inducible protein aggregation (LIPA) system that enables spatiotemporal control of α-synuclein (α-syn) aggregation into insoluble deposits called Lewy bodies (LBs), the pathological hallmark of Parkinson disease (PD) and other proteinopathies. We demonstrate that LIPA-α-syn inclusions mimic key biochemical, biophysical, and ultrastructural features of authentic LBs observed in PD-diseased brains. In vivo, LIPA-α-syn aggregates compromise nigrostriatal transmission, induce neurodegeneration and PD-like motor impairments. Collectively, our findings provide a new tool for the generation, visualization, and dissection of the role of α-syn aggregation in PD.

Alpha-Synuclein and the Endolysosomal System in Parkinson's Disease: Guilty by Association.

Abnormal accumulation of the protein α- synuclein (α-syn) into proteinaceous inclusions called Lewy bodies (LB) is the neuropathological hallmark of Parkinson's disease (PD) and related disorders. Interestingly, a growing body of evidence suggests that LB are also composed of other cellular components such as cellular membrane fragments and vesicular structures, suggesting that dysfunction of the endolysosomal system might also play a role in LB formation and neuronal degeneration. Yet the link between α-syn aggregation and the endolysosomal system disruption is not fully elucidated. In this review, we discuss the potential interaction between α-syn and the endolysosomal system and its impact on PD pathogenesis. We propose that the accumulation of monomeric and aggregated α-syn disrupt vesicles trafficking, docking, and recycling, leading to the impairment of the endolysosomal system, notably the autophagy-lysosomal degradation pathway. Reciprocally, PD-linked mutations in key endosomal/lysosomal machinery genes (LRRK2, GBA, ATP13A2) also contribute to increasing α-syn aggregation and LB formation. Altogether, these observations suggest a potential synergistic role of α-syn and the endolysosomal system in PD pathogenesis and represent a viable target for the development of disease-modifying treatment for PD and related disorders.

Development of a 3D functional assay and identification of biomarkers, predictive for response of high-grade serous ovarian cancer (HGSOC) patients to poly-ADP ribose polymerase inhibitors (PARPis): targeted therapy.

BACKGROUND: Poly(ADP-ribose) polymerase inhibitors (PARPis) specifically target homologous recombination deficiency (HRD) cells and display good therapeutic effect in women with advanced-stage BRCA1/2-mutated breast and epithelial ovarian cancer (EOC). However, about 50% of high grade serous ovarian cancers (HGSOC) present with HRD due to epigenetic BRCA1 inactivation, as well as genetic/epigenetic inactivation(s) of other HR genes, a feature known as "BRCAness". Therefore, there is a potential for extending the use of PARPis to these patients if HR status can be identified. METHODS: We have developed a 3D (spheroid) functional assay to assess the sensitivity of two PARPis (niraparib and olaparib) in ascites-derived primary cell cultures (AsPCs) from HGSOC patients. A method for AsPCs preparation was established based on a matrix (agarose), allowing for easy isolation and successive propagation of monolayer and 3D AsPCs. Based on this method, we performed cytotoxicity assays on 42 AsPCs grown both as monolayers and spheroids. RESULTS: The response to PARPis treatment in monolayer AsPCs, was significantly higher, compared to 3D AsPCs, as 88% and 52% of the monolayer AsPCs displayed sensitivity to niraparib and olaparib respectively, while 66% of the 3D AsPCs were sensitive to niraparib and 38% to olaparib, the latter being more consistent with previous estimates of HRD (40%-60%) in EOC. Moreover, niraparib displayed a significantly stronger cytotoxic effect in both in 3D and monolayer AsPCs, which was confirmed by consecutive analyses of the HR pathway activity (γH2AX foci formation) in PARPis-sensitive and resistant AsPCs. Global gene expression comparison of 6 PARPi-resistant and 6 PARPi-sensitive 3D AsPCs was indicative for the predominant downregulation of numerous genes and networks with previously demonstrated roles in EOC chemoresistance, suggesting that the PARPis-sensitive AsPCs could display enhanced sensitivity to other chemotherapeutic drugs, commonly applied in cancer management. Microarray data validation identified 24 potential gene biomarkers associated with PARPis sensitivity. The differential expression of 7 selected biomarkers was consecutively confirmed by immunohistochemistry in matched EOC tumor samples. CONCLUSION: The application of this assay and the potential biomarkers with possible predictive significance to PARPis therapy of EOC patients now need testing in the setting of a clinical trial.

The polypeptide GALNT6 Displays Redundant Functions upon Suppression of its Closest Homolog GALNT3 in Mediating Aberrant O-Glycosylation, Associated with Ovarian Cancer Progression.

Epithelial ovarian cancer (EOC) represents the most lethal gynecologic malignancy; a better understanding of the molecular mechanisms associated with EOC etiology could substantially improve EOC management. Aberrant O-glycosylation in cancer is attributed to alteration of N-acetylgalactosaminyltransferases (GalNAc-Ts). Reports suggest a genetic and functional redundancy between GalNAc-Ts, and our previous data are indicative of an induction of GALNT6 expression upon GALNT3 suppression in EOC cells. We performed single GALNT3 and double GALNT3/T6 suppression in EOC cells, using a combination of the CRISPR-Cas9 system and shRNA-mediated gene silencing. The effect of single GALNT3 and double GALNT3/T6 inhibition was monitored both in vitro (on EOC cells roliferation, migration, and invasion) and in vivo (on tumor formation and survival of experimental animals). We confirmed that GALNT3 gene ablation leads to strong and rather compensatory GALNT6 upregulation in EOC cells. Moreover, double GALNT3/T6 suppression was significantly associated with stronger inhibitory effects on EOC cell proliferation, migration, and invasion, and accordingly displayed a significant increase in animal survival rates compared with GALNT3-ablated and control (Ctrl) EOC cells. Our data suggest a possible functional redundancy of GalNAc-Ts (GALNT3 and T6) in EOC, with the perspective of using both these enzymes as novel EOC biomarkers and/or therapeutic targets.

Hic-5 regulates epithelial to mesenchymal transition in ovarian cancer cells in a TGFß1-independent manner.

The molecular basis of epithelial ovarian cancer (EOC) dissemination is still poorly understood. We have previously identified the hydrogen peroxide-inducible clone-5 (Hic-5) gene as hypomethylated in high-grade (HG) serous EOC tumors, compared to normal ovarian tissues. Hic-5 is a focal adhesion scaffold protein and has been primarily studied for its role as a key mediator of TGF-ß-induced epithelial-to-mesenchymal transition (EMT) in epithelial cells of both normal and malignant origin; however, its role in EOC has been never investigated. Here we demonstrate that Hic-5 is overexpressed in advanced EOC, and that Hic-5 is upregulated upon TGFß1 treatment in the EOC cell line with epithelial morphology (A2780s), associated with EMT induction. However, ectopic expression of Hic-5 in A2780s cells induces EMT independently of TGFß1, accompanied with enhancement of cellular proliferation rate and migratory/invasive capacity and increased resistance to chemotherapeutic drugs. Moreover, Hic-5 knockdown in the EOC cells with mesenchymal morphology (SKOV3) was accompanied by induction of mesenchymal-to-epithelial transition (MET), followed by a reduction of their proliferative, migratory/invasive capacity, and increased drugs sensitivity in vitro, as well as enhanced tumor cell colonization and metastatic growth in vivo. The modulation of Hic-5 expression in EOC cells resulted in altered regulation of numerous EMT-related canonical pathways and was indicative for a possible role of Hic-5 in controlling EMT through a RhoA/ROCK mediated mechanism. To our knowledge, this is the first report examining the role of Hic-5 in EOC, and its role in maintaining the mesenchymal phenotype of EOC cells independently of exogenous TGFß1 treatment.

Altered expression of different GalNAc­transferases is associated with disease progression and poor prognosis in women with high-grade serous ovarian cancer.

Protein glycosylation perturbations are implicated in a variety of diseases, including cancer. Aberrant glycosylation in cancer is frequently attributed to altered expression of polypeptide GalNAc transferases (GalNAc­Ts) - enzymes initiating mucin-type O-glycosylation. A previous study from our group demonstrated that one member of this family (GALNT3) is overexpressed in epithelial ovarian cancer (EOC), and GALNT3 expression correlated with shorter progression-free survival (PFS) in EOC patients with advanced disease. As considerable degree of redundancy between members of the GalNAc­Ts gene family has been frequently observed, we decided to investigate whether other members of this family are essential in EOC progression. In silico analysis based on publically available data was indicative for altered expression of five GalNAc­Ts (GALNT2, T4, T6, T9 and T14) in ovarian high-grade serous carcinoma (HGSC) samples compared to non-tumoral (control) ovarian tissue. We analyzed protein expression of these GalNAc­Ts in EOC cells and tumors by western blotting, followed by immunohistochemical (IHC) evaluation of their expression in EOC tumor and control samples using tissue microarrays (TMAs). Western blot analyses were indicative for low expression of GALNT2 and strong expression of GALNT6, T9 and T14 in both EOC cells and tumors. These observations were confirmed by IHC. GALNT2 displayed significantly lower expression, while GALNT6, GALNT9 and GALNT14 showed significantly higher expression in HGSC tumors compared to control tissue. Importantly, GALNT6 and GALNT14 expression correlated with poor prognosis of serous EOC patients. Moreover, our results suggest for overlapping functions of some GalNAc­Ts, more specifically GALNT3 and GALNT6, in directing EOC progression. Our results are indicative for a possible implication of different members of the GalNAc­T gene family in modulating EOC progression, and the potential use of GALNT6 and GALNT14 as novel prognostic EOC biomarkers. These data warrant future studies on the role of members of the GalNAc­Ts gene family in ovarian tumorigenesis.

Suppression of the grainyhead transcription factor 2 gene (GRHL2) inhibits the proliferation, migration, invasion and mediates cell cycle arrest of ovarian cancer cells.

Previously, we have identified the Grainyhead transcription factor 2 gene (GRHL2) as notably hypomethylated in high-grade (HG) serous epithelial ovarian tumors, compared with normal ovarian tissues. GRHL2 is known for its functions in normal tissue development and wound healing. In the context of cancer, the role of GRHL2 is still ambiguous as both tumorigenic and tumor suppressive functions have been reported for this gene, although a role of GRHL2 in maintaining the epithelial status of cancer cells has been suggested. In this study, we report that GRHL2 is strongly overexpressed in both low malignant potential (LMP) and HG serous epithelial ovarian tumors, which probably correlates with its hypomethylated status. Suppression of the GRHL2 expression led to a sharp decrease in cell proliferation, migration and invasion and induced G1 cell cycle arrest in epithelial ovarian cancer (EOC) cells displaying either epithelial (A2780s) or mesenchymal (SKOV3) phenotypes. However, no phenotypic alterations were observed in these EOC cell lines following GRHL2 silencing. Gene expression profiling and consecutive canonical pathway and network analyses confirmed these data, as in both these EOC cell lines, GRHL2 ablation was associated with the downregulation of various genes and pathways implicated in cell growth and proliferation, cell cycle control and cellular metabolism. Taken together, our data are indicative for a strong oncogenic potential of the GRHL2 gene in EOC progression and support recent findings on the role of GRHL2 as one of the major phenotypic stability factors (PSFs) that stabilize the highly aggressive/metastatic hybrid epithelial/mesenchymal (E/M) phenotype of cancer cells.

Proteomic dataset for altered glycoprotein expression upon GALNT3 knockdown in ovarian cancer cells.

This article contains raw and processed data related to research published in "Role of the polypeptide N-acetylgalactosaminyltransferase 3 in ovarian cancer progression: possible implications in abnormal mucin O-glycosylation" [1]. The data presented here was obtained with the application of a bioorthogonal chemical reporter strategy analyzing differential glycoprotein expression following the knock-down (KD) of the GALNT3 gene in the epithelial ovarian cancer (EOC) cell line A2780s. LC-MS/MS mass spectrometry analysis was then performed and the processed data related to the identified glycoproteins show that several hundred proteins are differentially expressed between control and GALNT3 KD A2780s cells. The obtained data also uncover numerous novel glycoproteins; some of which could represent new potential EOC biomarkers and/or therapeutic targets.

A metabolic labeling approach for glycoproteomic analysis reveals altered glycoprotein expression upon GALNT3 knockdown in ovarian cancer cells.

UNLABELLED: Epithelial ovarian cancer (EOC) is a disease responsible for more deaths among women in the Western world than all other gynecologic malignancies. There is urgent need for new therapeutic targets and a better understanding of EOC initiation and progression. We have previously identified the polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3) gene, a member of the GalNAc-transferases (GalNAc-Ts) gene family, as hypomethylated and overexpressed in high-grade serous EOC tumors, compared to low malignant potential EOC tumors and normal ovarian tissues. This data also suggested for a role of GALNT3 in aberrant EOC glycosylation, possibly implicated in disease progression. To evaluate differential glycosylation in EOC caused by modulations in GALNT3 expression, we used a metabolic labeling strategy for enrichment and mass spectrometry-based characterization of glycoproteins following GALNT3 gene knockdown (KD) in A2780s EOC cells. A total of 589 differentially expressed glycoproteins were identified upon GALNT3 KD. Most identified proteins were involved in mechanisms of cellular metabolic functions, post-translational modifications, and some have been reported to be implicated in EOC etiology. The GALNT3-dependent glycoproteins identified by this metabolic labeling approach support the oncogenic role of GALNT3 in EOC dissemination and may be pursued as novel EOC biomarkers and/or therapeutic targets. BIOLOGICAL SIGNIFICANCE: Knowledge of the O-glycoproteome has been relatively elusive, and the functions of the individual polypeptide GalNAc-Ts have been poorly characterized. Alterations in GalNAc-Ts expression were shown to provide huge variability in the O-glycoproteome in various pathologies, including cancer. The application of a chemical biology approach for the metabolic labeling and subsequent characterization of O-glycoproteins in EOC using the Ac4GalNAz metabolite has provided a strategy allowing for proteomic discovery of GalNAc-Ts specific functions. Our study supports an essential role of one of the GalNAc-Ts - GALNT3, in EOC dissemination, including its implication in modulating PTMs and EOC metabolism. Our approach validates the use of the applied metabolic strategy to identify important functions of GalNAc-Ts in normal and pathological conditions.

Functional importance of cardiac enhancer-associated noncoding RNAs in heart development and disease.

The key information processing units within gene regulatory networks are enhancers. Enhancer activity is associated with the production of tissue-specific noncoding RNAs, yet the existence of such transcripts during cardiac development has not been established. Using an integrated genomic approach, we demonstrate that fetal cardiac enhancers generate long noncoding RNAs (lncRNAs) during cardiac differentiation and morphogenesis. Enhancer expression correlates with the emergence of active enhancer chromatin states, the initiation of RNA polymerase II at enhancer loci and expression of target genes. Orthologous human sequences are also transcribed in fetal human hearts and cardiac progenitor cells. Through a systematic bioinformatic analysis, we identified and characterized, for the first time, a catalog of lncRNAs that are expressed during embryonic stem cell differentiation into cardiomyocytes and associated with active cardiac enhancer sequences. RNA-sequencing demonstrates that many of these transcripts are polyadenylated, multi-exonic long noncoding RNAs. Moreover, knockdown of two enhancer-associated lncRNAs resulted in the specific downregulation of their predicted target genes. Interestingly, the reactivation of the fetal gene program, a hallmark of the stress response in the adult heart, is accompanied by increased expression of fetal cardiac enhancer transcripts. Altogether, these findings demonstrate that the activity of cardiac enhancers and expression of their target genes are associated with the production of enhancer-derived lncRNAs.

Myosin-Va-interacting protein, RILPL2, controls cell shape and neuronal morphogenesis via Rac signaling.

Neuronal morphology plays an essential role in neuronal function. The establishment and maintenance of neuronal morphology is intimately linked to the actin cytoskeleton; however, the molecular mechanisms that regulate changes in neuronal morphology are poorly understood. Here we identify a novel myosin-Va (MyoVa)-interacting protein, RILPL2, which regulates cellular morphology. Overexpression of this protein in young or mature hippocampal neurons results in an increase in the number of spine-like protrusions. By contrast, knockdown of endogenous RILPL2 in neurons by short hairpin RNA (shRNA) interference results in reduced spine-like protrusions, a phenotype rescued by overexpression of an shRNA-insensitive RILPL2 mutant, suggesting a role for RILPL2 in both the establishment and maintenance of dendritic spines. Interestingly, we demonstrate that RILPL2 and the Rho GTPase Rac1 form a complex, and that RILPL2 is able to induce activation of Rac1 and its target, p21-activated kinase (Pak). Notably, both RILPL2-mediated morphological changes and activation of Rac1-Pak signaling were blocked by expression of a truncated tail form of MyoVa or MyoVa shRNA, demonstrating that MyoVa is crucial for proper RILPL2 function. This might represent a novel mechanism linking RILPL2, the motor protein MyoVa and Rac1 with neuronal structure and function.