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Expression of the Parkinson’s Disease-Associated Gene Alpha-Synuclein is Regulated by the Neuronal Cell Fate Determinant TRIM32

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Abstract

Alpha-synuclein is an abundant neuronal protein which has been associated with physiological processes like synaptic function, neurogenesis, and neuronal differentiation but also with pathological neurodegeneration. Indeed, alpha-synuclein (snca) is one of the major genes implicated in Parkinson’s disease (PD). However, little is known about the regulation of alpha-synuclein expression. Unveiling the mechanisms that control its regulation is of high importance, as it will enable to further investigate and comprehend the physiological role of alpha-synuclein as well as its potential contribution in the aetiology of PD. Previously, we have shown that the protein TRIM32 regulates fate specification of neural stem cells. Here, we investigated the impact of TRIM32 on snca expression regulation in vitro and in vivo in neural stem cells and neurons. We demonstrated that TRIM32 is positively influencing snca expression in a neuronal cell line, while the absence of TRIM32 is causing deregulated levels of snca transcripts. Finally, we provided evidence that TRIM32 binds to the promoter region of snca, suggesting a novel mechanism of its transcriptional regulation. On the one hand, the presented data link the PD-associated gene alpha-synuclein to the neuronal cell fate determinant TRIM32 and thereby support the concept that PD is a neurodevelopmental disorder. On the other hand, they imply that defects in olfactory bulb adult neurogenesis might contribute to early PD-associated non-motor symptoms like hyposmia.

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Acknowledgments

The authors would like to thank Dr. Leonidas Stefanis and Dr. Daniel Haber for plasmids as well as Thea van Wuellen and Inga Werthschulte for excellent technical assistance.

Author Contributions

MASP did the conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing and final approval of the manuscript. NC and JG did the model design and interpretation. SFA, LGC and MCM did the collection and assembly of data, data analysis and interpretation. SN did the generation of mNSCs lines. JCS did the conception and design, financial support, data analysis and interpretation, manuscript writing and final approval of the manuscript.

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Authors and Affiliations

  1. Developmental and Cellular Biology Group, Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, 6 avenue du Swing, 4367, Belvaux, Luxembourg

    Maria Angeliki S. Pavlou, Sarah Nicklas, Laura Gonzalez Cano & Jens C. Schwamborn

  2. Systems control group, Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, 6 avenue du Swing, 4367, Belvaux, Luxembourg

    Nicoló Colombo & Jorge Goncalves

  3. Instituto de Biomedicina (IBIOMED), Department of Molecular Biology, Universidad de León, Campus de Vegazana, León, 24071, Spain

    Sandra Fuertes-Alvarez & Maria C. Marín

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  1. Maria Angeliki S. Pavlou
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  4. Sarah Nicklas
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Corresponding author

Correspondence to Jens C. Schwamborn.

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Funding

The J. C. S.’s lab is supported by the Boehringer Ingelheim Foundation and the fund “Innovative Medical Research” of the University of Münster Medical School, Schram-Stiftung (T287/21795/2011) by the Fonds National de la Recherche (FNR) Luxembourg (CORE, C13/BM/5791363), a University Luxembourg Internal Research Project (MidNSCs) and the EU Joint Programme - Neurodegenerative Disease Research (JPND) project (supported by the FNR). L.G.C. was supported by a fellowship from the FNR (AFR, Aides à la Formation-Recherche). M.C.M ’s lab is supported by Grant SAF2012-36143 from Spanish Ministerio de Ciencia e and LE310U14 from the Junta de Castilla y Leon. S.F.A holds a predoctoral contract (PIRTU) from Junta de Castilla y Leon.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Online Resource 1

TRIM32 is localised in the nucleus of neurons of the mouse olfactory bulb and in differentiated mouse neuronal cells. a Immunostainings of mouse brain sections labelled with the indicated antibodies. Images were taken in the olfactory bulb (OB). Scale bars = 20 μm. b Immunostainings of mouse neural stem cells submitted to 5-day differentiation, labelled with the indicated antibodies. Scale bars = 20 μm (PDF 6935 kb)

Online Resource 2

Investigation of possible links in the regulatory network involving trim32 and snca The plots show the difference between experimental data (coloured stars) and simulations (black dots). For each combination of input/output genes a model has been trained using the experimental data and then used to simulate the gene response. The FIT between simulation and data can be used as a measure of the strength of the corresponding causal link. a, b Analysis of other possible regulatory effects of trim32 on a neuronal differentiation-associated genes (map2, neuroD, rbfox3) and b the regulatory effects of the same genes on snca. c, d Analysis of other possible regulatory effects of trim32 on c housekeeping genes (chmpa2, gpi1, psmb2, rab7) and d the regulatory effects of the same genes on snca. (PDF 445 kb)

Online Resource 3

TRIM32 is inducing or inhibiting the transcriptional activity of snca in a concentration-dependent manner, in HEK293T cells Diagram showing the normalised luciferase activity, in the presence of TRIM32 overexpression; 1.33 μg of TRIM32 were coexpressed together with 1.33 μg of an empty vector and 1.33 μg of the luciferase constructs (basic pGL3 vector or pGL3/snca intron 1) in HEK293T cells (mean ± SD; n = 6 independent experiments; Mann-Whitney U test, *p ≤ 0.05); 1.75 μg of TRIM32 were coexpressed together with 0.75 μg of an empty vector and 1.75 μg of the luciferase constructs (basic pGL3 vector or pGL3/snca intron 1) in HEK293T cells (mean ± SD; n = 6 independent experiments; Mann-Whitney U test, *p ≤ 0.05); 2.0 μg of TRIM32 were coexpressed together with 2.0 μg of the luciferase constructs (basic pGL3 vector or pGL3/snca intron 1) in HEK293T cells (mean ± SD; n = 5 independent experiments; t test, *p ≤ 0.05) (PDF 338 kb)

Online Resource 4

ChIP results indicating interaction of TRIM32 with the snca promoter region. Original agarose gel electrophoresis showing PCR results from ChIP-eluted DNA, with representative primer pairs of snca promoter region. A 100-bp DNA ruler was used on the first lane of both upper and lower images and the samples are indicated. (PDF 537 kb)

Online Resource 5

TRIM32 interaction with the snca promoter region. RT-qPCR results showing the DNA fold enrichment under different immunoprecipitations. RT-qPCR was performed on the eluted DNA derived from chromatin immunoprecipitations either with the IgG (negative control), histone 3 (H3, positive control), or TRIM32 antibodies. Results from three primer pairs are shown, depicting the possible interaction of H3 or TRIM32 relative to IgG, (mean ± SD; n = 3 independent experiments; t test, *p ≤ 0.05, **p ≤ 0.001). Fold enrichment was calculated by using the formula 2-DDCt, where DDCt is (Ct H3 or TRIM32) – (Ct IgG)) (PDF 326 kb)

Online Resource 6

Interaction with the snca promoter region is abolished when TRIM32 is knocked down. a, b RT-qPCR results of eluted DNA derived from three independent ChIP experiments, where a shRNA scrambled (a) or a shRNA TRIM32 (b) constructs were used. Five primer pairs are depicting the fold enrichment of TRIM32 compared to IgG (mean ± SD; n = 3 independent experiments; t test, *p ≤ 0.05). Fold enrichment was calculated by using the formula 2−DDCt, where DDCt is (Ct TRIM32) – (Ct IgG) (PDF 177 kb)

Online Resource 7

mRNA levels of trim32 on wt and p73 ko mNSCs are significantly different on the transition from maintenance towards differentiation conditions. a RT-qPCR measuring the relative trim32 mRNA expression levels in wt and TRIM32 ko mNSCs. Values were normalised to GAPDH levels, (mean ± SD; n = 8 independent experiments with n = 4 different cell lines; Mann-Whitney U test, *p ≤ 0.05, **p ≤ 0.001). b RT-qPCR measuring the relative trim32 mRNA expression levels in wt mNSCs under maintenance conditions or after 5 days induction of neuronal differentiation. Note how trim32 mRNA levels are significantly increased on the transition from maintenance towards differentiation. Values were normalised to GAPDH levels, (mean ± SD; n = 4 independent experiments with n = 4 different cell lines; Mann-Whitney U test, *p ≤ 0.05). c RT-qPCR measuring the relative trim32 mRNA expression levels in wt or p73 ko mNSCs, under maintenance conditions and when cells were submitted for 1, 3 or 5 days of neuronal differentiation. Note how trim32 mRNA levels were increased in the mNSCs of both genotypes, though the rate of increase is significantly lower in the p73 ko cells. Values were normalised to 18S levels, (mean ± SD; n ≥ 3 independent experiments; t test, *p ≤ 0.05, **p ≤ 0.001), maint maintenance. (PDF 160 kb)

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mRNA levels of snca between wt and TRIM32 ko or p73 ko MEFs reveal dissimilarities. a RT-qPCR measuring the relative trim32 mRNA expression levels in wt and TRIM32 ko MEFs. Values were normalised to GAPDH levels, (mean ± SD; n = 3 independent experiments with n = 3 different cell lines; Mann-Whitney U test, *p ≤ 0.05, **p ≤ 0.001). b RT-qPCR measuring the relative snca mRNA expression levels in wt and TRIM32 ko MEFs. The levels of snca mRNA do not seem to change between the two different genotypes, supporting the hypothesis of a cell type-specific effect of snca. Values were normalised to GAPDH levels, (mean ± SD; n = 3 independent experiments with n = 3 different cell lines; Mann-Whitney U test, *p ≤ 0.05). c, d RT-qPCR measuring the relative trim32 and scna mRNA expression levels in wt and p73 ko MEFs. Absence of p73 influences the mRNA levels of both genes validating for the former already published results [22] and revealing a novel effect on the latter possibly attributed to the downregulation of trim32. Values were normalised to 18S levels, (mean ± SD; n ≥3 independent experiments with n = 4 different clones; t test, *p ≤ 0.05, **p ≤ 0.001). (PDF 154 kb)

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Pavlou, M.A.S., Colombo, N., Fuertes-Alvarez, S. et al. Expression of the Parkinson’s Disease-Associated Gene Alpha-Synuclein is Regulated by the Neuronal Cell Fate Determinant TRIM32. Mol Neurobiol 54, 4257–4270 (2017). https://doi.org/10.1007/s12035-016-9989-9

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