Attenuation of epigenetic regulator SMARCA4 and ERK‐ETS signaling suppresses aging‐related dopaminergic degeneration

Abstract How complex interactions of genetic, environmental factors and aging jointly contribute to dopaminergic degeneration in Parkinson's disease (PD) is largely unclear. Here, we applied frequent gene co‐expression analysis on human patient substantia nigra‐specific microarray datasets to identify potential novel disease‐related genes. In vivo Drosophila studies validated two of 32 candidate genes, a chromatin‐remodeling factor SMARCA4 and a biliverdin reductase BLVRA. Inhibition of SMARCA4 was able to prevent aging‐dependent dopaminergic degeneration not only caused by overexpression of BLVRA but also in four most common Drosophila PD models. Furthermore, down‐regulation of SMARCA4 specifically in the dopaminergic neurons prevented shortening of life span caused by α‐synuclein and LRRK2. Mechanistically, aberrant SMARCA4 and BLVRA converged on elevated ERK‐ETS activity, attenuation of which by either genetic or pharmacological manipulation effectively suppressed dopaminergic degeneration in Drosophila in vivo. Down‐regulation of SMARCA4 or drug inhibition of MEK/ERK also mitigated mitochondrial defects in PINK1 (a PD‐associated gene)‐deficient human cells. Our findings underscore the important role of epigenetic regulators and implicate a common signaling axis for therapeutic intervention in normal aging and a broad range of age‐related disorders including PD.


| INTRODUC TI ON
Among age-related diseases, Parkinson's disease (PD) is the most common neurodegenerative movement disorder, with an incidence rate above 1% among individuals over 65 years of age (Nalls et al., 2014). The pathologic manifestations of PD include age-dependent progressive dopaminergic (DA) neuronal deterioration in basal ganglia and substantia nigra, with reduction of dopamine release.
Remarkable similarities at the molecular and cellular levels exist between PD and normal aging. Current treatments for PD are only symptomatic, ameliorating disease symptoms for a limited period of time, without retarding or halting disease progression.
Parkinson's disease is a complex disease with high heterogeneity.
Typically, these known PD genes participate in diverse cellular processes; however, common themes in PD pathogenesis have been proposed, such as aberrant proteostasis and vesicle trafficking, mitochondrial dysfunction, altered epigenetic regulation, and inflammation (Coppedè, 2013). Notably, all these involved pathogenic agents are similar to those in normal aging. Therefore, the insights involved in PD pathogenesis may be critical for understanding and modifying aging, and vice versa. Nevertheless, the majority of PD heritable components remain elusive (Lill et al., 2012;Nalls et al., 2014). How to extract contributing factors from limited human brain specimens is the main challenge to solve this heterogeneous disease, because only postmortem human brain specimens can be available.
More challengingly, most postulated novel genetic associations or risk factors await further validation.
Gene co-expression analysis allows identifying genes with similar expression patterns across a set of samples, which has facilitated identifying genes involved in certain disease pathways, new gene functions, and potential biomarkers (Zhang et al., 2012). In this study, we used the known PD genes as "anchors" in order to identify new PD candidate genes that are highly co-expressed with known PD genes with multiple human brain microarray datasets. Among the predicted 32 candidate genes, SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 4 (SMARCA4) and biliverdin reductase A (BLVRA) were further studied in vivo using Drosophila melanogaster, and their potential involvement in PD pathogenesis was confirmed. Furthermore, we revealed a potential common aging-related pathogenic signaling pathway consisting of the chromatin-remodeling factor SMARCA4 and the ERK-ETS signaling axis, suggesting new therapeutic targets for PD and other aging-related disorders, as three ERK-ETS inhibitors were tested for their efficacy in multiple Drosophila PD models. Our work also illustrates the high efficiency of combining bioinformatics analysis of large-scale human transcriptomic data and small-scale genetic screening using model organisms to interrogate highly heterogeneous diseases including age-related disorders.

| Gene co-expression network analysis identified 32 novel PD-associated candidate genes
We chose seven of the most commonly known PD genes as anchor genes, namely SCNA, LRRK2, PARKIN, DJ1, PINK1, ATP13A2, and HTR2A. Eleven datasets from NCBI Gene Expression Omnibus (GEO) were used, which contained samples from human brain tissues, especially the substantia nigra region. Our workflow is illustrated in Figure 1a. A total of 32 genes were identified to have high Pearson's correlation coefficients (PCC) with at least three anchor genes in at least five datasets (Table 1 and Table S1). According to the gene ontology (GO) enrichment analysis, the identified candidate PD genes were highly enriched with the genes associated with age-dependent metabolic reprogramming and neural disorders (Blalock et al., 2004).

| Inhibition of Brahma rescued progressive DA degeneration caused by overexpression of BVR in Drosophila
Among the 32 candidate genes, we chose SMARCA4 and BLVRA (biliverdin reductase A) for further studies based on the rationales  (Table S2). First, according to the Human Brain Transcriptome (HBT: https://hbatl as.org/pages/ hbtd) and the Allen Brain Atlas (http://human.brain-map.org/), both SMARCA4 and BLVRA are expressed widely in the human brain, including the substantia nigra.
Second, mutations of SMARCA4/Brm, a subunit of the SWI/SNF chromatin-remodeling complex that regulates higher order chromatin structure and gene expression, have been linked to multiple neurological and psychiatric disorders including autism spectrum disorders and schizophrenia (De Rubeis et al., 2014;Koga et al., 2009). Although expression of SMARCA4/Brm has been reported in both murine and human DA neurons by recent single-cell RNA-seq profiling (Hook et al., 2018;Sandor et al., 2017), there has been no functional reports of its role for DA neurons yet. Biliverdin reductases (BLVRs), together with hemeoxygenases (HOs), constitute the evolutionarily conserved enzymes in the heme metabolism, exert multiple physiological functions, and have been considered as a potential biomarker for AD and mild cognitive impairment (Barone, Di Domenico, Mancuso, & Butterfield, 2014). We then queried with gene symbol "SMARCA4" and "BLVRA" in PD gene database PDGene (http://www.pdgene.org), which incorporates all available SNP data pertaining to the discovery phase of the GWAS meta-analysis (Nalls et al., 2014). We found that both SMARCA4 (Table S3) and BLVRA (Table S4) harbor SNPs with meta-analysis p value between 1E-4 and 0.05, which can be regarded as the potential PD risk SNPs albeit not in the top 10,000 most significant GWAS results. Furthermore, both SMARCA4 and BLVRA are highly conserved among vertebrate and invertebrate species (Brahma or Brm for Drosophila SMARCA4 homologue and dBVR (CG9471) for Drosophila BLVRA homologue).

F I G U R E 1 Bioinformatics analyses. (a)
Overview of frequent gene co-expression analysis in identifying novel Parkinson's diseaseassociated genes. Eleven NCBI GEO DataSets were identified with the microarray datasets of the substantia nigra brain region of human PD patients. Seven genes were used as the anchor PD genes. See Section 4 for more details. To study the in vivo roles of candidate genes in PD pathogenesis, we took advantage of the Drosophila melanogaster model organism.
The age-dependent progressive DA neuronal loss in the lateral protocerebral posterior 1 (PPL1) cluster was used as the neurodegenerative index (Figure 2a-b). We then used an available Brm::GFP reporter fly strain to examine whether Brahma is expressed in fly DA neurons (Venken et al., 2011). Brm::GFP was first verified by its nuclear localization in Drosophila larval tissues ( Figure S1a-c).
Whole-mount immunostaining then confirmed the expression of Brm in the fly DA neurons (Figure 2c), consistent with a previous report (Abruzzi et al., 2017).
Drosophila genetic manipulations were then carried out to dissect the roles of Brm and dBVR. When Brm RNAi-mediated down-regulation (Herr et al., 2010)

PD models
We then examined how Brm and dBVR genetically interact with known PD genes. Four previously reported Drosophila PD models were successfully reproduced in our laboratory (Clark et al., 2006;Feany & Bender, 2000;Greene et al., 2003;Imai et al., 2008;Park et al., 2006;Yang et al., 2006). Given that the homozygous Parkin null alleles were found to be unhealthy in our laboratory, we used Parkin RNA interference (RNAi) flies instead. These four Drosophila PD models were abbreviated as αSyn (A30P), LRRK2 (I1915T), Parkin, Pink1 (Pink1 mut or Pink1 RNAi) PD models, respectively (see Section 4 & fly genotypes listed in the Appendix S2). Consistent with previous findings, age-dependent progressive degeneration was mild but statistically significant, as evidenced by the decreased number of PPL1 DA neurons in 30-day-old flies compared with agematched controls. In contrast, young 2-day-old PD flies displayed no DA neuronal loss ( Figure S4).
By labeling DA neurons with red fluorescent protein (RFP) in parallel, we found that Brm::GFP level exhibited an age-dependent progressive elevation in the PD fly brains compared with controls ( Figure 2f). We next addressed how Brm is upregulated in the degenerative dopaminergic neurons. One possibility is there could be a link between Brm activity and oxidative stress, which has been widely believed to be a common pathogenic factor in PD. To this end,

PD models
When Brm was downregulated or upregulated specifically in the Drosophila DA neurons, only slight perturbances in the life span were observed ( Figure 3a,b, Table S4). In line with previous reports (Imai et al., 2008;Todd & Staveley, 2008), overexpression of αSyn (A30P)

| Genetic manipulations of Brm or BVR modulated DA degeneration in multiple Drosophila PD models
When

PD models
We next examined whether directly reducing MEK-ERK-ETS activation was sufficient to prevent those common forms of DA degeneration. MEK RNAi fully rescued PPL1 DA neuronal loss in three 30-day-old PD model flies (Figure 5f-i, Figure S11a,b), and the rescue was not owing to titration of UAS-mediated overexpression ( Figure S3). In contrast, Drosophila ERK (rolled or rl) RNAi (Slack et al., 2015) exerted no apparent rescuing effects, suggesting that it was the activated fraction of ERK (pERK), not the abundance of the ERK protein, that caused the neurotoxicity. In parallel, when MEK RNAi or ERK RNAi was induced alone specifically in DA neurons, no changes in the PPL1 DA neurons were observed when compared with age-matched controls, suggesting that the rescuing effects of MEK RNAi were epistatic, but not due to simple addition ( Figure   S11a). Overexpression of constitutively active ERK led to early larval lethality before eclosion. We further found that the DA neuron- to early lethality before eclosion, and overexpression of Aop [wt] or Aop RNAi alone resulted in mild DA neuronal loss ( Figure S11a).
Based on all the above evidences, we conclude that there might be a delicate range of the ERK-ETS signaling strength that is beneficial for the maintenance of DA neurons, while deviation from that range such as prolonged over-activation could be rather detrimental.

PD models
To examine the MEK/ERK signaling pathway as a drug target for intervening in DA degeneration, we started with the MEK1 inhibitor, U0126. The effective inhibitory dose was first determined in flies subjected to 7 days of drug feeding ( Figure S12a inhibitor, PD0325901 (10 μg/ml), also completely blocked PPL1 DA neuronal loss (Figure 5o). We further tested Trametinib, another potent and highly specific MEK1 inhibitor, an FDA-approved drug for the treatment of melanoma (Yamaguchi, Kakefuda, Tajima, Sowa, & Sakai, 2011). With the optimal feeding concentration of 16 μM of Trametinib ( Figure S12c,d) (Slack et al., 2015), PPL1 DA neurons were fully protected from degeneration ( Figure 5p). No global brain or motor behavioral abnormalities were detected with all these drug treatments. Taken together, our data demonstrated that the MEK-ERK pathway could be a valid drug target to revert DA degeneration and illustrated that oral administration could be a promising pharmacological intervention.

| Knockdown of SMARCA4 or drug inhibition of ERK activity ameliorated mitochondrial defects in PINK1-deficient human cells
In humans, PINK1-deficiency leads to mitochondrial defects (Bueno et al., 2015). To determine whether our findings were relevant to human pathology, we first used human SH-SY5Y cells to examined the potential impact of SMARCA4 on PINK1 depletion. Lower mitochondrial contents and more fragmentated mitochondrial network were observed in the PINK1 RNAi-treated SH-SY5Y cells. While SMARCA4 RNAi alone did not significantly perturb the mitochondrial content and morphology, the genetic manipulation ameliorated the mitochondrial abnormality caused by PINK1 RNAi (Figure 6a shown to reverse PD-associated phenotypes induced by pathological LRRK2 alleles in cultured human iPS derived neurons (Reinhardt et al., 2013).

| D ISCUSS I ON
In summary, we identified a subset of 32 novel PD-associated genes, which were highly enriched in aging and neural disorders. The roles of two candidate genes SMARCA4/Brm and BLVRA/BVR were validated in vivo. The activity of ERK-ETS signaling, as a common effector for SMARCA4/Brm and BVR, was found to be also elevated in different genetic forms of Drosophila PD models. Thus, we have discovered a potential convergent PD pathogenesis pathway. Our Brm was found here to be progressively upregulated in the aging brains of PD fly models. One possibility is through a genetic imprinting response to elevated calcium level triggered by aberrant neuronal activities or calcium metabolism (Zhang et al., 2016). Alternatively, Brm could be activated through NF-kB mediated inflammatory responses that are well recognized in the development of neurodegenerative diseases (Bonnay et al., 2014). Besides, there is a clue that Brm functions downstream of Hippo pathway and plays an important role in a feedback loop between Crumb and Yorkie in this pathway (Zhu et al., 2015). On the other hand, in our unpublished data, we did observe the differential expression of other oxidoreductases (e.g., P450, sulfiredoxin, and phenoloxidase) at the early-middle stage of PD. Therefore, it remains possible that Brm might directly interact with Keap1/Nrf to elicit a program of sequential cellular responses to oxidative stress in DA neurons.
Inactivation of Brm was shown here to prevent dopaminergic neurons from degeneration. One putative route was through modulating the MAPK/ERK signally activity. Accordingly, Brm was shown to directly interact with Dsor1 (MEK1) and promotes EGFR-Ras-MAPK signaling activity (Friedman et al., 2011;Herr et al., 2010).
Nevertheless, the possibility could not be ruled out that direct interactions exist between Brm with transcriptional factors downstream of MAPK/ERK signaling, such as Pnt or Aop, constituting a positive auto-regulation feedback loop. Alternative mechanisms await further investigation to elucidate the effects of Brm.
Our finding that Brm inactivation protects DA degeneration seems to be at odds with the positive roles of Brm in the neural development (De Rubeis et al., 2014;Koga et al., 2009;Zhang et al., 2016 Previously, targeted inhibition of ERK signaling was demonstrated to mitigate spinocerebellar ataxia type 1 in Drosophila and mice models (Park et al., 2013). In addition, direct attenuation of MAPK/ERK was shown to be sufficient to extend life span of Drosophila (Slack et al., 2015). Indeed, three compounds that inhibited MEK-ERK signaling also ameliorated DA degeneration in all PD fly models in current studies. Among them, Trametinib, an FDAapproved drug for the treatment of melanoma, has been shown to extend life span (Slack et al., 2015). Our finding thus adds another piece of evidence that MAPK/ERK signaling could be a crucial intervention for aging and age-related disorders. Nevertheless, different tissues or cellular contexts might differentially respond to distinct level of MAPK/ERK, and the understanding of the quantitative relationship between ERK signaling and outcomes would benefit future efficacy study of ERK inhibitors upon life span and age-related disorders.
To our knowledge, this manuscript represents the first report of compounds that are effective in preventing DA degeneration in vivo in the four most common genetic forms of PD, compared to a previous report of Rapamycin (Tain et al., 2009 extend the life span in humans (Figure 6g).

| Drosophila Stocks and nomenclature
Fly genotypes for each experiment were listed in the Appendix S2.
Detail information of fly strains and husbandry was described in the Appendix S1.

| Drosophila PD models and pathologic phenotype evaluation
At least 20 hemisphere brains were quantified via double-blinded fashion for each data point (n > 20), details in the Appendix S1.

| Semi-quantitative RT-PCR and Quantitative Real-time PCR
Details in the Appendix S1.

| Whole-mount Brain Immunostaining, live imaging, and Microscopy
All images were taken by a confocal microscopy (Leica TCS SP5) with identical instrument parameters for any given individual experimental series, details in the Appendix S1.

| Western Blot Analysis
Details in the Appendix S1.

| Generation of PINK1 knockout HeLa cell lines using CRISPR/Cas9 gene editing
See Appendix S1 for details.

| Assessment of mitochondrial membrane potential (MMP)
Mitochondrial membrane potential was assessed with the probe JC-1 (Invitrogen). See Appendix S1 for details.

| Assessment of mitochondrial content and morphology
The mitochondrial content and morphology were assessed with Mito-Morphology Macro in ImageJ as previously described (Dagda et al., 2009;Schneider, Rasband, & Eliceiri, 2012).

| Genes knockdown in SH-SY5Y cells
Three pairs of siRNAs target to human PINK1 or SMARCA4 were designed and synthetized. SH-SY5Y cells were transfected with siRNAs to perform mitochondrial content and morphology analysis.

| Quantification and statistical analysis
Mann-Whitney test or log-rank test was used, respectively. See details in the Appendix S1.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are openly available in Mendeley Data at http://dx.doi.org/10.17632/ 6587k sptsf.1.