The Two Cysteines of Tau Protein Are Functionally Distinct and Contribute Differentially to Its Pathogenicity in Vivo

Research Articles: Neurobiology of Disease The two Cysteines of Tau protein are functionally distinct and contribute differentially to its pathogenicity in vivo https://doi.org/10.1523/JNEUROSCI.1920-20.2020 Cite as: J. Neurosci 2020; 10.1523/JNEUROSCI.1920-20.2020 Received: 23 July 2020 Revised: 21 October 2020 Accepted: 25 November 2020 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2020 the authors 1 The two Cysteines of Tau protein are functionally distinct and contribute 2 differentially to its pathogenicity in vivo. 3 Engie Prifti1*, Eleni N. Tsakiri1*, Ergina Vourkou1, George Stamatakis2, Martina 4 Samiotaki2 and Katerina Papanikolopoulou1,§. 5 1Institute for Fundamental Biomedical Research and 2Institute for Bio- 6 innovation, Biomedical Sciences Research Centre “Alexander Fleming”, 34 Fleming 7 Street, Vari 16672, Greece 8 *Equal contribution to this work. 9 § To whom correspondence should be addressed. 10 e-mail: [email protected] 11 12 13 - Number of Figures: 7 14 - Number of Tables: 1 15 - Multimedia: 0 16 - Extended data figures/tables: 0 17 18 Number of words for: 19 - Abstract: 164 20 - Significance statement: 99 21 - Introduction: 473 22 - Discussion: 1465 23 24 25 1 26 Author Contributions: 27 K.P. wrote the manuscript with input from all authors. 28 E.P., E.T, E.V. and K.P. designed research. 29 E.P., E.T, E.V., G.S., M.S. and K.P. performed research. 30 E.P., E.T, E.V., G.S., M.S. and K.P. analyzed data. 31 32 Acknowledgments 33 The authors acknowledge support for their research by a grant from the Stavros 34 Niarchos Foundation to the Biomedical Sciences Research Center “Alexander 35 Fleming”, as part of the Foundation’s initiative to support the Greek research center 36 ecosystem, Fondation Sante and The Hellenic Foundation for Research and Innovation. 37 We thank Dr. Efthimios Skoulakis for fly strains and for critical reading of the 38 manuscript. Finally, we thank Dr. Jean Y Gouzi for performing pilot LTM experiments. 39 Conflict of interest 40 The authors declare no conflict of interest. 41 42 43 44 45 46 47 48 49 2 50 ABSTRACT 51 Although Tau accumulation is clearly linked to pathogenesis in Alzheimer’s 52 disease (AD) and other Tauopathies, the mechanism that initiates the aggregation of 53 this highly soluble protein in vivo remains largely unanswered. Interestingly, in vitro 54 Tau can be induced to form fibrillar filaments by oxidation of its two cysteine 55 residues, generating an intermolecular disulfide bond that promotes dimerization and 56 fibrillization. The recently solved structures of Tau filaments revealed that the two 57 cysteine residues are not structurally equivalent since Cys-322 is incorporated into the 58 core of the fibril whereas Cys-291 projects away from the core to form the fuzzy coat. 59 Here, we examined whether mutation of these cysteines to alanine affects 60 differentially Tau mediated toxicity and dysfunction in the well-established 61 Drosophila Tauopathy model. Experiments were conducted with both sexes, or with 62 either sex. Each cysteine residue contributes differentially to Tau stability, 63 phosphorylation status, aggregation propensity, resistance to stress, learning and 64 memory. Importantly, our work uncovers a critical role of Cys-322 in determining 65 Tau toxicity and dysfunction. 66 SIGNIFICANCE STATEMENT 67 Cysteine-291 and Cysteine-322, the only two cysteine residues of Tau present in 68 only 4-Repeat or all isoforms respectively, have competing functions: as the key 69 residues in the catalytic center, they enable Tau auto-acetylation, and as residues 70 within the microtubule-binding repeat region are important not only for Tau function 71 but also instrumental in the initiation of Tau aggregation. In this study, we present the 72 first in vivo evidence that their substitution leads to differential consequences on 73 Tau’s physiological and pathophysiological functions. These differences raise the 3 74 possibility that cysteine residues play a potential role in determining the functional 75 diversity between isoforms. 76 INTRODUCTION 77 Tau is a microtubule-associated protein that occurs mainly in neurons, crucial 78 for the elongation of microtubule labile domains (Qiang et al., 2018) among several 79 other physiological functions at the synapse and in the nucleus, as well as interactions 80 with the plasma membrane and the actin cytoskeleton (Sotiropoulos et al., 2017). In 81 the adult human brain, alternative splicing of a single-copy gene generates six Tau 82 isoforms that differ by the number of N-terminal insertions and microtubule-binding 83 repeats (MBRs). Tau has 85 potential phosphorylation sites that regulate its affinity 84 for microtubules, with excess steady state phosphorylation being associated with 85 disease (Wang and Mandelkow, 2015). 86 Intracellular Tau aggregates are the defining feature of a group of 87 neurodegenerative dementias called Tauopathies, presenting varying clinical 88 symptoms (Arendt et al., 2016). In addition to serving as markers for differential 89 diagnosis, Tau aggregates can mediate disease propagation (Peng et al., 2020) and 90 serve as direct drivers of toxicity (Ballatore et al., 2007). In recent years, studies in 91 animal models have shown that Tau oligomers correlate with synapse loss and 92 behavioral deficits much better than the appearance of mature fibrils and pre-fibrillar 93 events have gained considerable attention with respect to their toxic potential 94 (Santacruz et al., 2005; Berger et al., 2007). 95 Tau presents conformational flexibility rendering it highly soluble and it is 96 therefore surprising that it assembles into filaments (Wang and Mandelkow, 2015). It 97 has been proposed that Tau fibrillization may be associated with folding events which 98 allow Tau monomers to acquire partially folded, β-sheet- enriched structures that 4 99 further self-assemble to form highly ordered fibrils (Jeganathan et al., 2008), 100 potentially mediated by two hexapeptide motifs in the MBR (von Bergen et al., 2005). 101 In addition, cysteines appear capable of forming disulfide linked dimers that 102 accelerate the conformational conversion of Tau into fibrillary aggregates in vitro 103 (Schweers et al., 1995; Bhattacharya et al., 2001) and compounds that target these 104 residues prevent Tau aggregation (Soeda et al., 2015). However, the specific events 105 leading in Tau aggregation in vivo are not yet established. 106 Of the two Tau cysteines, Cys-322 is present in all isoforms, whereas Cys-291 107 is found only in 4R species. A recent study revealed the ability of Tau to perform 108 thiol/disulfide exchanges with tubulin and brought new insights into the role of these 109 two residues in the correct localization of Tau on microtubules (Martinho et al., 110 2018). In the two C-shaped protofilaments constituting the Tau filaments from AD 111 brains, Cys-322 is incorporated into the core of the fibril whereas Cys-291 is in the 112 disordered portion of the protein forming the “fuzzy coat” (Fitzpatrick et al., 2017). 113 This was evidence that two Tau cysteines are not structurally equivalent. Based on 114 this distinction we aimed to determine whether they were equivalent or contributed 115 differentially to Tau mediated neuronal toxicity and dysfunction in vivo using the 116 established Drosophila melanogaster Tauopathy model (Papanikolopoulou and 117 Skoulakis, 2011). 118 . 119 120 121 122 123 5 124 125 126 127 MATERIALS AND METHODS 128 Drosophila culture and strains. 129 Flies were cultured in standard sugar-wheat flour food supplemented with soy 130 flour and CaCl2 (Acevedo et al., 2007). Pan-neuronal transgene expression was 131 achieved using the ElavC155-Gal4;Ras2-Gal4 driver (Robinow and White, 1988; 132 Walker et al., 2006; Keramidis et al., 2020). The double driver was constructed using 133 standard genetic crosses. The gstD-ARE:GFP (ARE of the gstD gene) reporter 134 transgenic line was a kind gift from Prof. D. Bohmann (University of Rochester, 135 NY,USA). Experiments were performed at 25°C unless noted otherwise. To generate 136 the new equivalently-expressing transgenes within the same attp site we used the 137 UAS-htauFLAG-2N4R which has been described previously (Kosmidis et al., 2010). The 138 same cDNA was subcloned into pUAS.attB (Bischof et al., 2007) as EcoRI/XbaI 139 fragment. The Cys mutants were generated by replacing Cys-291 and Cys-322 with 140 Ala using the QuickChange XL site-directed mutagenesis kit (Agilent) according to 141 the manufacturer’s instructions. The mutagenic oligonucleotides for each mutant are 142 shown below. The silent restriction site BamHI introduced for effective screening of 143 positive clones appears underlined in italics whereas the amino acid substitution is 144 shown in bold. The mutagenic oligonucleotides for C291A 145 5’-GCAACGTCCAGTCCAAGGCTGGATCCAAGGATAATATC-3’ 146 5’- GATATTATCCTTGGATCCAGCCTTGGACTGGACGTTGC-3’ and for C322A 147 5’-CCTGAGCAAGGTGACCTCCAAGGCTGGATCCTTAGGCAACATCC-3’ 6 148 5’- GGATGTTGCCTAAGGATCCAGCCTTGGAGGTCACCTTGCTCAGG-3’ 149 were annealed onto the pUAS.attB UAS-htauFLAG-2N4R plasmid template. For the 150 generation of the double Cys mutant the pUAS.attB UAS-htauC291A FLAG-2N4R served as 151 a template for cloning with the C322A primers. The sequence of the mutants was 152 confirmed by dsDNA sequencing (VBC-biotech). Transgenic flies were generated by 153 phiC31-mediated transgenesis by BestGene Inc. (Chino Hills, CA, USA). DNAs were 154 injected into genomic landing site attP2 on the third chromosome (BDSC #8622). 155 RNA extraction and RT-PCR. 156 Total RNA was extracted from Drosophila heads using TRI Reagent® (Sigma- 157 Aldrich) following the manufacturer’s instructions.

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