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Targeted Downregulation of dMyc Suppresses Pathogenesis of Human Neuronal Tauopathies in Drosophila by Limiting Heterochromatin Relaxation and Tau Hyperphosphorylation

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Abstract

Human tauopathies such as Alzheimer’s Disease (AD), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), Pick’s disease etc., are a group of neurodegenerative diseases which are characterized by abnormal hyperphosphorylation of tau that leads to formation of neurofibrillary tangles. Recapitulating several features of human neurodegenerative disorders, the Drosophila tauopathy model displays compromised lifespan, locomotor function impairment, and brain vacuolization in adult brain which is progressive and age dependent. Here, we demonstrate that tissue-specific downregulation of the Drosophila homolog of human c-myc proto-oncogene (dMyc) suppresses tau-mediated morphological and functional deficits by reducing abnormal tau hyperphosphorylation and restoring the heterochromatin loss. Our studies show for the first time that the inherent chromatin remodeling ability of myc proto-oncogenes could be exploited to limit the pathogenesis of human neuronal tauopathies in the Drosophila disease model. Interestingly, recent reports on successful uses of some anti-cancer drugs against Alzheimer's and Parkinson's diseases in clinical trials and animal models strongly support our findings and proposed possibility.

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Acknowledgments

We are thankful to Prof. Mel Feany (Harvard Medical School, USA) and the Bloomington Stock Center for providing different fly stocks used in this study. We gratefully thank Prof. Bob Eisenman (Fred Hutchinson Cancer Research Center, USA) for anti-dMyc and Prof. T. Lilja (Stockholm University, USA) for anti-CBP antibodies. This work was supported by research grants from the Department of Biotechnology (DBT), Government of India, New Delhi, India, to S.S. SIC is supported by the Senior Research Fellowship (SRF) from the University Grant Commission (UGC), New Delhi, India. We also thank Delhi University for financial support under R&D scheme, DST-FIST(L2) support to the Department, and Central Instrument Facility (CIF) at South Campus. We are grateful to Ms. Nabanita Sarkar and Ms. Nisha for technical support.

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Authors and Affiliations

  1. Department of Genetics, University of Delhi South Campus, Benito Juarez Road, Dhaula Kuan, New Delhi, 110 021, India

    Soram Idiyasan Chanu & Surajit Sarkar

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  1. Soram Idiyasan Chanu
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  2. Surajit Sarkar
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Correspondence to Surajit Sarkar.

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Fig. S1

Targeted downregulation of dMyc utilizing second independent RNAi line (UAS-dmyc JF01762) also suppresses human tau mediated rough eye phenotypes and degeneration of retinal tissues (a, b). Induced expression of human c-myc isoform (c-myc2) significantly aggravates tau mediated neurodegenerative phenotype in Drosophila as revealed by bright field and scanning electron microscopy (c, d). Targeted downregulation of dMyc suppresses neurodegenerative phenotypes induced by mutant human tauV337M or tauR406W isoforms (e-n). Compared to GMR-Gal4/+ flies (e, f), eye specific expression of tauV337M (g, h) and tauR406W (k, l) resulted in mild and severe degeneration respectively, which was suppressed upon targeted downregulation of dMyc in tauV337M (i, j) or tauR406W (m, n) expressing cells. In contrast, increased expression of dMyc in tauV337M expressing eye cells resulted in notable aggravation in neurodegenerative phenotype (o, p). (Scale bars: b, d, f, h, j, l, n, p = 100 μm). (GIF 299 kb)

High Resolution Image (TIF 15321 kb)

Fig. S2

Expression of tauWT induces neurodegenerative phenotypes in Drosophila pupal eyes. 55 h old pupal eye disc stained with anti-armadillo (a-c’) and anti-Disc large (d-f’) antibodies and counterstained with DAPI. With normal development and arrangement of primary, secondary, tertiary and cone cells in control pupae (a, a’; d, d’), targeted expression of tauWT deteriorates the gross morphology of the pupal eyes associated with fusion of the surrounding primary, secondary and tertiary cells (b, b’; e, e’) and these morphological impairments were dominantly suppressed upon downregulation of dMyc (c, c’; f, f’). (Scale bars: a-f’ = 10 μm). (GIF 215 kb)

High Resolution Image (TIF 4418 kb)

Fig. S3

Human tauWT mediated neurodegeneration and vacuolization was dominantly suppressed by tissue specific downregulation of dMyc. Paraffin sections of adult head across the midbrain stained with DAPI (a-d) revealed normal internal cellular architecture in GMR-Gal4/+ (a) and degeneration of optic lobes and retina with prominent vacuoles in tauWT expressing neurons (b). Targeted downregulation of dMyc in tauWT expressing photoreceptors suppressed neuronal degeneration in rescue flies (c) or aggravated further upon upregulation of dMyc (d). (Scale bar: a-d = 100 μm). (GIF 191 kb)

High Resolution Image (TIF 3129 kb)

Fig. S4

Graph representing relative level of dMyc expression (±SD) in eye imaginal discs of various genotypes as quantified by calculating total fluorescence intensity (a). Altered expression of dMyc did not make any negative impact on cell cycle as almost equal BrdU incorporation efficiency was evident at the eye morphogenetic furrows (arrow in b-e) of eye discs in controls (b), tauWT expressing (c), tauWT expressing with downregulated dMyc (d) and upregulated dMyc larvae (e). Assessment of the size of adult eye (f) and ommatidium (f’) of GMR-Gal4/+ and GMR-Gal4/+;UAS-dmycRNAi flies (g, g’) did not show any significant difference. (Scale bars: b-e = 100 μm; f’-g’ = 50 μm). (GIF 150 kb)

High Resolution Image (TIF 9289 kb)

Fig. S5

Downregulation of dMyc reduces the abnormal phosphorylation of tau during the early phase of disease pathogenesis. Larval eye imaginal disc stained with anti-human PHF-tauSer202/Thr205 (AT8) antibody showed relatively high level of tau phosphorylation in differentiated neurons of tauWT expressing larval eye disc (a, a’, c) which was reduced following downregulation of dMyc (d, d’, f) and enhanced significantly after dMyc overexpression (g, g’, i). DAPI counterstained discs are shown in (b, e, h). (Scale bars: a-i = 100 μm; a',d',g' = 10 μm). (GIF 181 kb)

High Resolution Image (TIF 5720 kb)

Three dimensional view of a normally developed mushroom body (MB) in adult brain of control Elav-Gal4/+ flies. (MP4 2518 kb)

Three dimensional view of a severely degenerated mushroom body (MB) and neuropil structures in tauWT expressing adult brain. (MP4 2658 kb)

Three dimensional view of a completely restored and normally developed mushroom body (MB) in tauWT expressing adult brain with reduced expression of dMyc. (MP4 2858 kb)

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Chanu, S.I., Sarkar, S. Targeted Downregulation of dMyc Suppresses Pathogenesis of Human Neuronal Tauopathies in Drosophila by Limiting Heterochromatin Relaxation and Tau Hyperphosphorylation. Mol Neurobiol 54, 2706–2719 (2017). https://doi.org/10.1007/s12035-016-9858-6

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