PEOPLEAnne Picard

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Anne Picard

Technician
Institute for Biomedicine

T +39 0471 055 498

anne.picard@eurac.edu

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About

Set-up and adaptation of new protocols, laboratory safety

Technical Skills:

RT-PCR and qPCR, ddPCR,

NGS library preparations (miRNA, WES, 16S amplicon, Covid-19 variants sequencing), NGS library runs on the Illumina MiSeq platform

Cell culture techniques (Hybridoma cell lines, primary culture of skeletal and cardiac muscle, immortal cell lines such as HEK293 and Hela, IPSc), in vitro transient transfection, RNA interference, immunofluorescence microscopy, immunohistochemistry, in situ hybridization

Molecular cloning, cDNA library construction,

biochemical and enzymatical protein analysis, Western Blot, Northern Blot, Southern blot, Sanger sequencing, DNA footprinting, epitope mapping, EMSA, ChIP, calcium signaling by bioenergetic assessment of cells (aequorin measurements and confocal microscopy), drug library screening, TMRE (mitochondrial membrane potential measurements).

Scientific Publications

Journal article2023

Increased Levels of the Parkinson's Disease-Associated Gene ITPKB Correlate with Higher Expression Levels of α-Synuclein, Independent of Mutation Status

Di Leva F, Filosi M, Silvestri E, Picard A, Lavdas AA, Pramstaller PP, Hicks AA, Corti C

Erica Silvestri, Andrew Hicks, Alexandros Lavdas, Corrado Corti, Peter P. Pramstaller, Francesca Di Leva, Anne Picard

Autosomal dominant mutations in the gene encoding α-synuclein (SNCA) were the first to be linked with hereditary Parkinson's disease (PD). Duplication and triplication of SNCA has been observed in PD patients, together with mutations at the N-terminal of the protein, among which A30P and A53T influence the formation of fibrils. By overexpressing human α-synuclein in the neuronal system of Drosophila, we functionally validated the ability of IP3K2, an ortholog of the GWAS identified risk gene, Inositol-trisphosphate 3-kinase B (ITPKB), to modulate α-synuclein toxicity in vivo. ITPKB mRNA and protein levels were also increased in SK-N-SH cells overexpressing wild-type α-synuclein, A53T or A30P mutants. Kinase overexpression was detected in the cytoplasmatic and in the nuclear compartments in all α-synuclein cell types. By quantifying mRNAs in the cortex of PD patients, we observed higher levels of ITPKB mRNA when SNCA was expressed more (p < 0.05), compared to controls. A positive correlation was also observed between SNCA and ITPKB expression in the cortex of patients, which was not seen in the controls. We replicated this observation in a public dataset. Our data, generated in SK-N-SH cells and in cortex from PD patients, show that the expression of α-synuclein and ITPKB is correlated in pathological situations.

DOI

DOI: 10.3390/ijms24031984

Journal article2022

GCN5 contributes to intracellular lipid accumulation in human primary cardiac stromal cells from patients affected by Arrhythmogenic cardiomyopathy

Volani C, Pagliaro A, Rainer J, Paglia G, Porro B, Stadiotti I, Foco L, Cogliati E,

Alexandros Lavdas, Marzia De Bortoli, Anne Picard, Alessandra Rossini, Johannes Rainer

Projekt: Mitochondriale Dysfunktion bei der Pathogenese der ACM

Arrhythmogenic cardiomyopathy (ACM) is a genetic disease associated with sudden cardiac death and cardiac fibro-fatty replacement. Over the last years, several works have demonstrated that different epigenetic enzymes can affect not only gene expression changes in cardiac diseases but also cellular metabolism. Specifically, the histone acetyltransferase GCN5 is known to facilitate adipogenesis and modulate cardiac metabolism in heart failure. Our group previously demonstrated that human primary cardiac stromal cells (CStCs) contribute to adipogenesis in the ACM pathology. Thus, this study aims to evaluate the role of GCN5 in ACM intracellular lipid accumulation. To do so, CStCs were obtained from right ventricle biopsies of ACM patients and from samples of healthy cadaveric donors (CTR). GCN5 expression was increased both in ex vivo and in vitro ACM samples compared to CTR. When GCN5 expression was silenced or pharmacologically inhibited by the administration of MB-3, we observed a reduction in lipid accumulation and a mitigation of reactive oxygen species (ROS) production in ACM CStCs. In agreement, transcriptome analysis revealed that the presence of MB-3 modified the expression of pathways related to cellular redox balance. Altogether, our findings suggest that GCN5 inhibition reduces fat accumulation in ACM CStCs, partially by modulating intracellular redox balance pathways.

DOI Quelle

DOI: 10.1111/jcmm.17396

Journal article2021

Exome-wide association study of levodopa-induced dyskinesia in Parkinson's disease

König E, Nicoletti A, Pattaro C, Annesi G, Melotti R, Gialluisi A, Schwienbacher C, Picard A,

Andrew Hicks, Cristian Pattaro, Roberto Melotti, Irene Pichler, Peter P. Pramstaller, Anne Picard, Hagen Blankenburg, Eva König

Levodopa is the standard long-term dopamine replacement therapy to treat Parkinson’s disease (PD) symptoms. With time, levodopa may induce debilitating dyskinesias (LID), the treatment of which represents a large clinically unmet need. However, time-to-LID onset varies between patients, reflecting a possible genetic component. We performed an hypothesis-free whole-exome sequencing (WES)-based screening of time-to-LID onset and attempted replication of previously published candidate gene studies. A WES association analysis was carried out in 134 PD patients in a meta-analytical framework. Replication was attempted in an independent study of 97 PD patients. Variants from previously reported candidate genes (OPRM1, COMT, BDNF) were also specifically examined. We significantly replicated, for the first time, an association of variant rs1799971 in the OPRM1 gene with time-to-LID onset. Furthermore, we identified two novel potentially functional variants, in the MAD2L2 (rs2233019) and MAP7 (rs35350783) genes, which were significantly associated at the discovery stage. In the replication study, the two variants showed direction-consistent effects but did not achieve the replication significance threshold. Our study provides the first WES results for time-to-LID onset, where we replicate association at OPRM1, and suggest new variants in MAD2L2 and MAP7 genes that are significant in discovery, but require larger datasets for replication. The results are being made publicly available to allow for independent external validation.

DOI Quelle

DOI: 10.1038/s41598-021-99393-8

Journal article2020

Kinase inhibition of G2019S-LRRK2 enhances autolysosome formation and function to reduce endogenous alpha-synuclein intracellular inclusions

Obergasteiger J, Frapporti G, Lamonaca G, Pizzi S, Picard A, Lavdas AA, Pischedda F, Piccoli G,

Andrew Hicks, Alexandros Lavdas, Mattia Volta, Corrado Corti, Peter P. Pramstaller, Anne Picard

The Parkinson’s disease (PD)-associated kinase Leucine-Rich Repeat Kinase 2 (LRRK2) is a crucial modulator of the autophagy-lysosome pathway, but unclarity exists on the precise mechanics of its role and the direction of this modulation. In particular, LRRK2 is involved in the degradation of pathological alpha-synuclein, with pathogenic mutations precipitating neuropathology in cellular and animal models of PD, and a significant proportion of LRRK2 patients presenting Lewy neuropathology. Defects in autophagic processing and lysosomal degradation of alpha-synuclein have been postulated to underlie its accumulation and onset of neuropathology. Thus, it is critical to obtain a comprehensive knowledge on LRRK2-associated pathology. Here, we investigated a G2019S-LRRK2 recombinant cell line exhibiting accumulation of endogenous, phosphorylated alpha-synuclein. We found that G2019S-LRRK2 leads to accumulation of LC3 and abnormalities in lysosome morphology and proteolytic activity in a kinase-dependent fashion, but independent from constitutively active Rab10. Notably, LRRK2 inhibition was ineffective upon upstream blockade of autophagosome-lysosome fusion events, highlighting this step as critical for alpha-synuclein clearance

DOI

DOI: 10.1038/s41420-020-0279-y

Journal article2019

Application of CRISPR/Cas9 editing and digital droplet PCR in human iPSCs to generate novel knock-in reporter lines to visualize dopaminergic neurons

Überbacher C, Obergasteiger J, Volta M, Venezia S, Müller S, Pesce I, Pizzi S, Lamonaca G,

Andrew Hicks, Mattia Volta, Corrado Corti, Peter P. Pramstaller, Anne Picard, Giada Cattelan

Human induced pluripotent stem cells (hiPSCs) have become indispensable for disease modelling. They are an important resource to access patient cells harbouring disease-causing mutations. Derivation of midbrain dopaminergic (DAergic) neurons from hiPSCs of PD patients represents the only option to model physiological processes in a cell type that is not otherwise accessible from human patients. However, differentiation does not produce a homogenous population of DA neurons and contaminant cell types may interfere with the readout of the in vitro system. Here, we use CRISPR/Cas9 to generate novel knock-in reporter lines for DA neurons, engineered with an endogenous fluorescent tyrosine hydroxylase - enhanced green fluorescent protein (TH-eGFP) reporter. We present a reproducible knock-in strategy combined with a highly specific homologous directed repair (HDR) screening approach using digital droplet PCR (ddPCR). The knock-in cell lines that we created show a functioning fluorescent reporter system for DA neurons that are identifiable by flow cytometry.

DOI

DOI: 10.1016/j.scr.2019.101656

Journal article2019

Compound heterozygous SZT2 mutations in two siblings with early-onset epilepsy, intellectual disability and macrocephaly

Domingues FS, König E, Schwienbacher C, Volpato CB, Picard A, Cantaloni C, Mascalzoni D, Lackner P,

Andrew Hicks, Deborah Mascalzoni, Peter P. Pramstaller, Anne Picard, Claudia Volpato, Eva König

PURPOSE:

Mutations in SZT2 have been previously reported in several cases of early onset epilepsy and intellectual disability. In this study we investigate potential causal mutations in two male siblings affected by early onset epilepsy, intellectual disability and macrocephaly.

METHODS:

We use family-based whole-exome sequencing to identify candidate variants.

RESULTS:

We report the identification of two potential causal SZT2 mutations in compound heterozygous state. We observe considerable differences in the clinical phenotype severity of the two affected individuals. The cerebral MRI revealed no abnormalities in the older affected brother, while in the youngest one it revealed a right frontal polymicrogiria. Moreover, while good seizure control was achieved in the older affected individual the younger brother is affected by pharmacoresistant epilepsy, progressive spastic paraplegia, cortical myoclonus and a more severe intellectual disability. We also analyzed the relative location of the reported pathogenic mutations in the SZT2 protein.

CONCLUSION:

Variable phenotypic expressivity is observed for this condition, while the location and type of mutations in SZT2 also has a potential impact on epilepsy severity. These findings extend our knowledge of epileptogenic conditions related to SZT2 and mTOR signaling.

DOI

DOI: 10.1016/j.seizure.2018.12.021

Journal article2019

Generation of an induced pluripotent stem cell line (EURACi005-A) from a Parkinson's disease patient carrying a homozygous exon 3 deletion in the PRKN gene.

Zanon A, Riekschnitz D, von Troyer M, Volpato C, Picard A, Cantaloni C, Di Segni M, Silipigni R,

Andrew Hicks, Irene Pichler, Peter P. Pramstaller, Anne Picard, Diana Riekschnitz, Claudia Volpato

Mutations in the PRKN gene, encoding parkin, are the most frequent known cause of recessive Parkinson's disease (PD). We report the generation of an induced pluripotent stem cell (iPSC) line of a patient carrying a homozygous deletion of exon 3 in the PRKN gene. Skin fibroblasts were reprogrammed using non-integrating episomal plasmids. The generated cell line (EURACi005-A; iPS-2011) exhibits expression of pluripotency markers, the potential to differentiate into all three germ layers, and a stable karyotype. This iPSC line provides a valuable resource for further research on the pathomechanism and drug testing for PRKN-linked PD.

DOI

DOI: 10.1016/j.scr.2019.101624

Journal article2019

Microbiota, type 2 diabetes and non-alcoholic fatty liver disease: protocol of an observational study

Motta BM, Grander C, Gögele M, Foco L, Vukovic V, Melotti R, Fuchsberger C, De Grandi A,

Cristian Pattaro, Martin Gögele, Roberto Melotti, Deborah Mascalzoni, Luisa Foco, Peter P. Pramstaller, Alessandro De Grandi, Christian Fuchsberger, Anne Picard, Alessandra Rossini

BACKGROUND:

Non-alcoholic fatty liver disease (NAFLD) is characterized by triglyceride accumulation in the hepatocytes in the absence of alcohol overconsumption, commonly associated with insulin resistance and obesity. Both NAFLD and type 2 diabetes (T2D) are characterized by an altered microbiota composition, however the role of the microbiota in NAFLD and T2D is not well understood. To assess the relationship between alteration in the microbiota and NAFLD while dissecting the role of T2D, we established a nested study on T2D and non-T2D individuals within the Cooperative Health Research In South Tyrol (CHRIS) study, called the CHRIS-NAFLD study. Here, we present the study protocol along with baseline and follow-up characteristics of study participants.

METHODS:

Among the first 4979 CHRIS study participants, 227 individuals with T2D were identified and recalled, along with 227 age- and sex-matched non-T2D individuals. Participants underwent ultrasound and transient elastography examination to evaluate the presence of hepatic steatosis and liver stiffness. Additionally, sampling of saliva and faeces, biochemical measurements and clinical interviews were carried out.

RESULTS:

We recruited 173 T2D and 183 non-T2D participants (78% overall response rate). Hepatic steatosis was more common in T2D (63.7%) than non-T2D (36.3%) participants. T2D participants also had higher levels of liver stiffness (median 4.8 kPa, interquartile range (IQR) 3.7, 5.9) than non-T2D participants (median 3.9 kPa, IQR 3.3, 5.1). The non-invasive scoring systems like the NAFLD fibrosis score (NFS) suggests an increased liver fibrosis in T2D (mean - 0.55, standard deviation, SD, 1.30) than non-T2D participants (mean - 1.30, SD, 1.17).

DISCUSSION:

Given the comprehensive biochemical and clinical characterization of study participants, once the bioinformatics classification of the microbiota will be completed, the CHRIS-NAFLD study will become a useful resource to further our understanding of the relationship between microbiota, T2D and NAFLD.

DOI

DOI: 10.1186/s12967-019-02130-z

Journal article2017

Elevated levels of alpha-synuclein blunt cellular signal transduction downstream of Gq protein-coupled receptors

Volta M, Lavdas AA, Obergasteiger J, Überbacher C, Picard A, Pramstaller PP, Hicks AA, Corti C

Andrew Hicks, Alexandros Lavdas, Mattia Volta, Corrado Corti, Peter P. Pramstaller, Anne Picard

Alpha-synuclein is central to Parkinson's disease pathogenesis and pathology, however its precise functions are still unclear. It has been shown to bind both PLCβ1 and MAPKs, but how this property influences the downstream signaling of Gq protein-coupled receptors has not been elucidated. Here we show that recombinant expression of alpha-synuclein in human neuroblastoma cells enhances cellular levels of PLCβ1 but blunts its signaling pathway, preventing the agonist-dependent rise of cytoplasmic Ca2+. In addition, overexpressing alpha-synuclein abolishes the activation of ERK1/2 upon agonist stimulation, indicating an upstream action in the signal transduction pathway. This data demonstrates that alpha-synuclein, when recombinantly expressed, interferes with the normal signaling of Gq-protein coupled receptors, which are then dysfunctional. Since many neurotransmitter systems utilize these receptor signaling pathways to mediate different abilities affected in Parkinson's disease, we argue this novel perspective might be helpful in designing treatment strategies for some of the non-motor symptoms in Parkinson's disease and synucleinopathies.

DOI

DOI: 10.1016/j.cellsig.2016.11.012

Journal article2017

Exploring digenic inheritance in arrhythmogenic cardiomyopathy

König E, Volpato CB, Motta BM, Blankenburg H, Picard A, Pramstaller PP, Casella M, Rauhe W,

Alessandra Rossini, Peter P. Pramstaller, Anne Picard, Hagen Blankenburg, Claudia Volpato, Eva König

BACKGROUND:
Arrhythmogenic cardiomyopathy (ACM) is an inherited genetic disorder, characterized by the substitution of heart muscle with fibro-fatty tissue and severe ventricular arrhythmias, often leading to heart failure and sudden cardiac death. ACM is considered a monogenic disorder, but the low penetrance of mutations identified in patients suggests the involvement of additional genetic or environmental factors.

METHODS:
We used whole exome sequencing to investigate digenic inheritance in two ACM families where previous diagnostic tests have revealed a PKP2 mutation in all affected and some healthy individuals. In family members with PKP2 mutations we determined all genes that harbor variants in affected but not in healthy carriers or vice versa. We computationally prioritized the most likely candidates, focusing on known ACM genes and genes related to PKP2 through protein interactions, functional relationships, or shared biological processes.

RESULTS:
We identified four candidate genes in family 1, namely DAG1, DAB2IP, CTBP2 and TCF25, and eleven candidate genes in family 2. The most promising gene in the second family is TTN, a gene previously associated with ACM, in which the affected individual harbors two rare deleterious-predicted missense variants, one of which is located in the protein's only serine kinase domain.

CONCLUSIONS:
In this study we report genes that might act as digenic players in ACM pathogenesis, on the basis of co-segregation with PKP2 mutations. Validation in larger cohorts is still required to prove the utility of this model.

DOI

DOI: 10.1186/s12881-017-0503-7

Journal article2017

Plasma and White Blood Cells Show Different miRNA Expression Profiles in Parkinson's Disease

Schwienbacher C, Foco L, Picard A, Corradi E, Serafin A, Panzer J, Zanigni S, Blankenburg H,

Andrew Hicks, Luisa Foco, Peter P. Pramstaller, Anne Picard, Hagen Blankenburg

Parkinson's disease (PD) diagnosis is based on the assessment of motor symptoms, which manifest when more than 50% of dopaminergic neurons are degenerated. To date, no validated biomarkers are available for the diagnosis of PD. The aims of the present study are to evaluate whether plasma and white blood cells (WBCs) are interchangeable biomarker sources and to identify circulating plasma-based microRNA (miRNA) biomarkers for an early detection of PD. We profiled plasma miRNA levels in 99 L-dopa-treated PD patients from two independent data collections, in ten drug-naïve PD patients, and in unaffected controls matched by sex and age. We evaluated expression levels by reverse transcription and quantitative real-time PCR (RT-qPCR) and combined the results from treated PD patients using a fixed effect inverse-variance weighted meta-analysis. We revealed different expression profiles comparing plasma and WBCs and drug-naïve and L-dopa-treated PD patients. We observed an upregulation trend for miR-30a-5p in L-dopa-treated PD patients and investigated candidate target genes by integrated in silico analyses. We could not analyse miR-29b-3p, normally expressed in WBCs, due to the very low expression in plasma. We observed different expression profiles in WBCs and plasma, suggesting that they are both suitable but not interchangeable peripheral sources for biomarkers. We revealed miR-30a-5p as a potential biomarker for PD in plasma. In silico analyses suggest that miR-30a-5p might have a regulatory role in mitochondrial dynamics and autophagy. Further investigations are needed to confirm miR-30a-5p deregulation and targets and to investigate the influence of L-dopa treatment on miRNA expression levels.

DOI Quelle

DOI: 10.1007/s12031-017-0926-9

Journal article2015

Overexpression of blood microRNAs 103a, 30b, and 29a in L-dopa-treated patients with PD

Serafin A, Foco L, Zanigni S, Blankenburg H, Picard A, Zanon A, Giannini G, Pichler I,

Andrew Hicks, Irene Pichler, Luisa Foco, Peter P. Pramstaller, Anne Picard, Hagen Blankenburg

OBJECTIVE:

The aims of the present study were to profile the expression of several candidate microRNAs (miRNAs) in blood from L-dopa-treated and drug-naive patients with Parkinson disease (PD) vs unaffected controls and to interpret the miRNA expression data in a biological context.

METHODS:

We analyzed RNAs from peripheral blood of 36 L-dopa-treated, 10 drug-naive patients with PD and unaffected controls matched 1:1 by sex and age. We evaluated expression by reverse transcription-quantitative real-time PCR, and we analyzed data using a 2-tailed paired t test. To detect miRNA targets, several miRNA resources were combined to generate an overall score for each candidate gene using weighted rank aggregation.

RESULTS:

Significant overexpression of miR-103a-3p (p < 0.0001), miR-30b-5p (p = 0.002), and miR-29a-3p (p = 0.005) in treated patients with PD was observed, and promising candidate target genes for these were revealed by an integrated in silico analysis.

CONCLUSIONS:

We revealed 3 candidate biomarkers for PD. miRNAs 30b-5p and 29a-3p replicated a documented deregulation in PD albeit opposite to published data, while for miR-103a-3p, we demonstrated for the first time an overexpression in treated patients with PD. Expression studies in patients and/or in isolated peripheral blood mononuclear cells before and after L-dopa administration are necessary to define the involvement of L-dopa treatment in the observed overexpression. Our in silico analysis to prioritize targets of deregulated miRNAs identified candidate target genes, including genes related to neurodegeneration and PD. Despite the preliminary character of our study, the results provide a rationale for further clarifying the role of the identified miRNAs in the pathogenesis of PD and for validating their diagnostic potential.

DOI

DOI: 10.1212/WNL.0000000000001258

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Journal article2019

Microbiota, type 2 diabetes and non-alcoholic fatty liver disease: protocol of an observational study

Motta BM, Grander C, Gögele M, Foco L, Vukovic V, Melotti R, Fuchsberger C, De Grandi A,

Cristian Pattaro, Martin Gögele, Roberto Melotti, Deborah Mascalzoni, Luisa Foco, Peter P. Pramstaller, Alessandro De Grandi, Christian Fuchsberger, Anne Picard, Alessandra Rossini

BACKGROUND:

METHODS:

RESULTS:

DISCUSSION:

DOI

DOI: 10.1186/s12967-019-02130-z