Biliverdin Reductase A is a major determinant of neuroprotective Nrf2 signaling

Biliverdin reductase A, oxidative stress, Nrf2 signaling, neuroprotection, gene 53 regulation 54 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 8, 2025. ; https://doi.org/10.1101/2025.06.04.657936doi: bioRxiv preprint

1 Biliverdin reductase A (BVRA), the terminal enzyme in heme catabolism, generates the 2 neuroprotective and lipophilic antioxidant bilirubin. Here, we identify a novel non-enzymatic role for 3 BVRA in redox regulation. We show that BVRA directly interacts with nuclear factor erythroid -4 derived factor-like 2 (Nrf2), the master regulator of redox homeostasis, to modulate target signaling 5 pathways. ChIP-seq and RNA-seq analyses reveal that this interaction coordinates the expression 6 of neuroprotective genes that are typically dysregulated in Alzheimer’s disease and other 7 neurodegenerative conditions. Thus, this previously unknown BVRA-Nrf2 axis controls an essential 8 pathway of redox signaling in neuroprotection. Our findings establish BVRA as a dual -function 9 integrator of antioxidant defenses in both the lipophilic and hyd rophilic subcellular compartments, 10 bridging these two distinct and critical cellular protection mechanisms in the brain. This 11 advancement in understanding the endogenous antioxidant system of the brain positions the 12 BVRA-Nrf2 axis as a promising therapeutic target for neurodegenerative disease. 13 Significance Statement 14 We show a non -canonical role for biliverdin reductase A (BVRA), classically known as the 15 biosynthetic enzyme for bilirubin, in nonenzymatic modulation of antioxidant neuroprotective 16 nuclear factor erythroid -derived factor -like 2 (Nrf2) signaling in the brain. Both BVRA and Nrf2 17 signaling are compromised in neurodegenerative diseases such as Alzheimer’s disease, and the 18 BVRA-Nrf2 axis offers a new direction for developing neuroprotective therapies. 19 20 Main Text 21 22

23 24 Biliverdin reductase A (BVRA), encoded by the Blvra gene, and the terminal enzyme in heme 25 catabolism and the major biosynthetic enzyme for bilirubin in adults ( Fig. 1A) (1, 2). Bilirubin is a 26 potent lipophilic antioxidant with protective efficacy in advanced cardiovascular disease associated 27 with hyperbilirubinemia (3-9), as well as in malaria (10) and metabolic disorders (11, 12). Beyond 28 its canonical role, BVRA exhibits additional diverse enzymatic and non-enzymatic functions, 29 modulating key signaling pathways such as focal adhesion kinase signaling in the brain, insulin 30 signaling, phosphoinositide 3-kinase (PI3K)/Protein kinase B (Akt) signaling, and mitogen-activated 31 protein kinase (MAPK) cascades (11, 13-21). Interestingly, at near-neutral pH BVRA utilizes NADH 32 as a cofactor, whereas it uses NADPH at alkaline pH, enabling flexibility of function under different 33 cellular conditions (22-24). Importantly, while BVRA’s roles in peripheral tissues are well -34 documented, its functions in the central nervous system are not as well understood, despite its 35 significant expression in brain (2). 36 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 8, 2025. ; https://doi.org/10.1101/2025.06.04.657936doi: bioRxiv preprint We previously showed that both bilirubin and BVRA are highly abundant in the lipid -rich 37 brain (2). Bilirubin, being lipophilic, functions in a manner complementary to glutathione (GSH), the 38 abundant hydrophilic antioxidant to confer beneficial effects (2, 7-9, 25). Bilirubin more effectively 39 prevents lipid peroxidation than soluble cellular components, such as protecting cytosolic proteins 40 from oxidation (2, 8). Bilirubin is also a potent s cavenger of superoxide (O 2) radicals, with mice 41 lacking BVRA displaying elevated susceptibility to oxidative stress (2, 9) . We have shown that 42 bilirubin is a potent scavenger of O2 and cannot neutralize other forms of reactive oxygen species 43 (ROS) such as H2O2 and the hydroxyl radical (OH )(2). 44 Despite bilirubin’s apparent lack of reactivity towards these ROS, Blvra-/- mice are still 45 hypersensitive to H 2O2 and complementing cells with BVRA is protective, suggesting that these 46 mice may be deficient in a mechanism dependent on BVRA, but not bilirubin to counteract oxidative 47 stress (2). Here, we reveal a redox-regulatory role of BVRA that entails a non-enzymatic interaction 48 with the nuclear factor erythroid-derived factor-like 2 (Nrf2) transcription factor, which is well known 49 as the master regulator of antioxidant responses (26). We demonstrate that direct binding of BVRA 50 to Nrf2 enhances transcriptional regulation of neuroprotective gene networks implicated in 51 Alzheimer’s disease (AD) and neurodegeneration. This BVRA -Nrf2 axis establishes a novel link 52 between heme metabolism and physiologic redox homeostasis, revealing opportunities to 53 therapeutically bolster endogenous neuroprotection. 54 55 56 57

58 59 Heme-independent expression patterns and functional roles of brain BVRA 60 The first enzyme in heme catabolism in the brain, heme oxygenase 2 (HO -2) accounts for 61 most of the HO activity in the nervous system and, like HO -1, regulates redox balance (27, 28). 62 HO-2 deletion aggravates oxidative stress induced by seizures, glutamate, and inflammation, and 63 also causes cerebral vascular injury (29). To determine whether BVRA operates independently of 64 the heme oxygenase (HO) system, we first identified brain cell types in which heme metabolism is 65 crucial. Using single -cell RNA sequencing (scRNA -seq), we compared Blvra (which encodes 66 BVRA) and Hmox2 (which encodes HO -2) expression profiles in whole -brain lysates. Both Blvra 67 and Hmox2 were detected in numerous cell types in the brain (Fig. 1B). On the other hand, Hmox1 68 mRNA (which encodes HO -1) is inducible (30) and not expressed at baseline, rendering it 69 undetectable in most cells analyzed. Notably, Blvra exhibited more restricted cellular expression 70 than Hmox2, with HO -2 mRNA levels nearly tripling those of Blvra transcripts. Strikingly, 23% of 71 brain cells expressed Blvra mRNA without detectable Hmox2 mRNA and only 10% expressed both 72 Blvra and Hmox2 (Fig. 1C), a pattern observed broadly across cell types and brain regions ( Fig. 73 1D). This surprisingly broad presence of BVRA in the absence of HO -2, is puzzling and suggests 74 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 8, 2025. ; https://doi.org/10.1101/2025.06.04.657936doi: bioRxiv preprint a potential function independent of heme metabolism. This dissociation, corroborated by 75 independent datasets (Allen Brain Atlas, Protein Atlas) (31), implies that BVRA operates in contexts 76 unrelated to bilirubin generation. 77 We next examined the evolutionary histories of these principal enzymes involved in heme 78 degradation: the BVRs (BVRA, BVRB), and HO. BVRA and BVRB are distinct enzymes with BVRA 79 being the main bilirubin producing enzyme in adults and BVRB in the fetal stage s (32). BVRA 80 belongs to the expansive glyceraldehyde 3 -phosphate dehydrogenase (GAPDH) -like clade of 81 redox enzymes, whose me mbers feature an N -terminal NADPH/NADH -binding Rossmann fold 82 catalytic domain and a C -terminal GADC domain responsible for substrate selection and binding. 83 Structural studies of the related Synechocystis BVRA revealed that its C -terminal domain 84 accommodates two biliverdin molecules via π-π stacking, both of which are believed to participate 85 in the redox reaction. Based on the Synechocystis BVRA structure and its interactions with NADPH 86 and biliverdin, and the sequence conservation pattern, we identified sev eral residues for further 87 mutational studies (G17, E97, R172, R46, R227, K219). We also noticed a cysteine residue (C292) 88 that is conserved across vertebrates and some cyanobacteria, which may play a role in BVRA -89 mediated redox homeostasis (Fig. S1 ). In co ntrast, BVRB lacks the GADC domain and is 90 comprised of only the NADPH -binding Rossmann fold catalytic domain, which is also present in 91 BVRA (Fig. 1F). Consistent with the absence of a dedicated substrate -binding domain, it exhibits 92 greater substrate promiscuity. HO, on the other hand, is a heme-dependent all–α-helical oxygenase 93 that is structurally unrelated to BVRA and BVRB. 94 Given that homologs of these enzymes are reported in prokaryotes, we explored their 95 phylogenetic affinities and genomic contexts to gain insights into the evolution of this pathway. 96 Phylogenetic analyses show that both BVRA and HO are ultimately of cyanobacterial origin. 97 However, their patterns of acquisition by eukaryotes differ, with the gene for BVRA appearing to 98 have been transferr ed from cyanobacteria to an early metazoan ( Fig. 1E) and HO having been 99 acquired earlier in eukaryotic evolution, as evidenced by its presence in diverse eukaryotic lineages 100 such as oomycetes and haptophytes. BVRB, by contrast, appears to represent a lateral transfer 101 from a firmicute lineage again into an early metazoan. Notably, homologs related to all three 102 enzymes were transferred on multiple independent occasions from prokaryotes to eukaryotes. For 103 example, one such paralog of BVRA in eukaryotes, the diphenol detoxifying enzyme dihydrodiol 104 dehydrogenase (DHDH), was independently acquired from bacteria. 105 Furthermore, even within metazoans, the phyletic patterns of BVRA and BVRB enzymes 106 differ considerably: for example, BVRB is widely present in insects, where HO and BVRA are largely 107 absent. However, HO and BVRA, display a degree of concordance in their distribution among other 108 metazoans. These patterns support the hypothesis that the heme catabolic pathway combining the 109 roles of BVRA and HO likely evolved near the base of the animal lineage, whereas BVRB, given 110 its broader substrate range, including flavins and ferric ions, may have been secondarily 111 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 8, 2025. ; https://doi.org/10.1101/2025.06.04.657936doi: bioRxiv preprint incorporated into heme catabolism in vertebrates, perhaps as an extension of its more generalized 112 role in redox modification of exogenous toxic small molecules. The cyanobacterial versions of HO 113 and BVRA likely functioned originally in the metabolism of phycobilins associated with proteins of 114 their light-harvesting systems. Their early incorporation and shared distribution in animals imply 115 that they were co-opted for the emerging importance of heme metabolism in the context of oxygen 116 transport in multicellular animals. 117 To further characterize the potentially novel function of BVRA decoupled from HO, we 118 utilized Blvra-/- mice lacking exon 3 (encoding the NAD(P)H -binding domain) abolishing bilirubin 119 production (2). Despite originally creating this model to interrogate bilirubin’s antioxidant effects, 120 our earliest observations implicated BVRA itself in redox regulation, including protection agains t 121 the hydrophilic H 2O2 oxidant. We therefore generated a series of mutants in the NADPH and 122 biliverdin binding domains of BVRA, including the G17A mutant described earlier (21, 33) to assess 123 their roles in BVRA f unction ( Fig. S1 ). As previously reported, a glycine to alanine mutation of 124 residue 17 (G17A) disrupted NADPH binding and significantly diminished BVRA-mediated bilirubin 125 production ( Fig. 1 G). Specifically, G17A conferred a 5 -fold reduction in Vmax and a 3 00-fold 126 increase in Km (Fig. 1H) of the enzyme. Furthermore, several other mutants derived from residues 127 that are predicted to bind NADPH/NADH or biliverdin also showed diminished activities ( Fig. 1G). 128 Next, we transfected BVRA-/- mouse embryonic fibroblasts (MEFs) with WT or G17A-BVRA 129 to test whether this enzymatically dead mutant could rescue cells exposed to H 2O2 despite its 130 inability to produce bilirubin ( Fig. 1I). G17A-BVRA rescues Blvra-/- cells to a similar extent as WT -131 BVRA, hinting further at a redox function of BVRA independent of bilirubin production and heme 132 catabolism (Fig. 1J). 133 134 Depleting BVRA blunts Nrf2 signaling 135 136 To assess how BVR may exert antioxidant activity independent of its role in bilirubin production, 137 we monitored the gene expression of several antioxidant genes via unbiased whole transcriptome 138 sequencing of WT and BVRA -/- hippocampal neurons at baseline and under oxidative stress 139 induced by H2O2. After this toxic exposure, several genes were differentially expressed in both cell 140 types (Fig. 2A-C, (SI Data Table 1). We focused on genes that show a fold difference of at least 2 141 in upregulation or downregulation between the WT and Blvra-/- genotypes. Among the genes 142 upregulated, the cholesterol biosynthetic pa thway was most prominently represented, with 143 upregulation in BVRA-/- cells under stress BVRA-/- cells. Most significant among the downregulated 144 pathways was the nuclear factor erythroid -derived factor-like 2 (Nrf2) signaling pathway ( Fig. 2D, 145 right panel), with Nrf2 ranking significantly high amongst the transcription factors implicated in the 146 differential gene expression between the WT and BVRA -/- cells (Fig. 2E). We therefore chose to 147 focus on Nrf2 signaling for the remainder of our study to determine the specific role of BVRA in the 148 response to oxidative stress. 149 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 8, 2025. ; https://doi.org/10.1101/2025.06.04.657936doi: bioRxiv preprint Nrf2 is a transcription factor that regulates the expression of several antioxidant response 150 proteins. Under basal conditions, Nrf2 is sequestered by the Kelch -like ECH-associated protein 1 151 (Keap1) in the cytosol and ubiquitinated for targeted degradation by the 26S proteasome (34). In 152 response to oxidative stress, Keap1 is modified on its reactive cysteine residues and dissociates 153 from N rf2, preventing its ubiquitination and proteasomal degradation (35-39). Nrf2 then 154 translocates to the nucleus, where it binds the antioxidant response element (ARE) to orchestrate 155 transcription of a battery of vital antioxidant genes necessary for cytoprotection. To evaluate 156 whether Nrf2 signaling is disrupted in BVRA-/- cells, we analyzed the transcript levels of Nrf2 target 157 genes such as Hmox1, Gclc, Gsta2 and Cxcl2 (Fig. 2F-I). The expression of Hmox1, Gclc (which 158 encodes the catalytic subunit of glutamate –cysteine ligase, a key enzyme in glutathione 159 biosynthesis) and Gsta2 (which encodes glutathione S -transferase A2, an enzyme involved in 160 detoxification of electrophilic compounds) were blunted in the BVRA -/- cells, whereas the proteins 161 involved in the immune response and inflammation such as Cxcl2 (encoding the chemokine (C-X-162 C motif) ligand 2) were upregulated. 163 Next, we compared the activity of the Nrf2 promoter in WT and BVRA -/- cells using a 164 luciferase reporter assay. In this dual -luciferase firefly reporter system, where the luciferase gene 165 is regulated by the promoter for glutathione S-transferase A4 (GSTA4), a target of Nrf2. Thus, Nrf2 166 activity at the Gsta4 promoter initiates luciferase transcription, which can be measured via luciferin 167 luminescence. We quantified the activation of the Gsta4 promoter in WT and BVRA -/- MEFs in 168 response to three Nrf2 activators: H2O2, sulforaphane (SFN), and tert-butyl hydroquinone (t-BHQ). 169 SFN is a compound known to modify the reactive cysteines on Keap1, thus releasing Nrf2 before 170 ubiquitination and increasing overall expression of intr acellular Nrf2 (40). t-BHQ is also known to 171 activate Nrf2, although its mechanism not well-established (41). H2O2 treatment increases luciferin 172 reporter detection in WT cells, indicating Nrf2 activation by proxy of GST gene transcription. 173 However, this increase is not seen in BVRA-/- cells, which are unable to elicit Nrf2 activity and GST 174 luciferase expression. The same pattern was also noted in the SFN and tBHQ treatment conditions 175 (Fig. 2J). 176 To determine whether enzymatically dead BVRA also potentiates Nr f2 transcriptional 177 activity, we introduced WT and G17A BVRA in a HEK293 cell line harboring an Nrf2 regulated 178 luciferase reporter. In this HEK293 cell line, the promoter controlling luciferase expression is directly 179 downstream of the antioxidant response e lement promoter that Nrf2 binds, allowing us to use 180 luciferase expression as a readout for Nrf2 transcriptional activity in general and not limited to 181 GSTA4 expression as in the previous experiment. WT - and G17A-BVRA both increase luciferase 182 activity significantly and to the same degree in each treatment condition ( Fig. 2K). Enzymatically 183 dead BVRA was equally efficacious as WT BVRA in activating Nrf2 in vitro, underscoring BVRA’s 184 novel non-catalytic Nrf2-inducing role. We next analyzed the effect of erasti n, an inhibitor of the 185 cystine transporter, one of whose subunits is encoded by Slc7a11, a target of Nrf2, on the viability 186 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 8, 2025. ; https://doi.org/10.1101/2025.06.04.657936doi: bioRxiv preprint of these cells. BVRA -/- MEFs were more susceptible to increasing concentrations of erastin as 187 compared to WT cells, further confirming that depletion of BVRA compromises Nrf2 signaling (Fig. 188 2L). 189 190 BVRA physically and genetically interacts with Nrf2 191 To characterize the relationship between BVRA and Nrf2, we aimed to breed BVRA and Nrf2 192 double-knockout mice. In our attempts to create such mice, no Blvra-/-/ Nfe2l2-/- pups were ever 193 obtained, indicating that this interaction is crucial for survival and function. In the crosses 194 conducted, we expected roughly 9% of the pups to present with the double-null genotype but were 195 unable to obtain double knockout pups ( Fig. 3A, B). All other possible genotypes were generated 196 at near their expected frequencies. This finding suggests that BVRA and Nrf2 are genetically linked 197 and that deleting both genes is embryonic lethal, confirming a genetic interaction between Blvra 198 and Nfe2l2 that is crucial for survival. 199 To determine whether BVRA interacts with Nrf2, we condu cted a GST pull down assay in 200 HEK293 transfected with constructs harboring GFP -tagged Nrf2 and either GST vector alone or a 201 construct encoding GST-BVRA. GST-BVRA robustly bound Nrf2 unlike GST alone, as revealed by 202 western blot analysis ( Fig. 3 C). Given th at BVRA and BVRB are the two enzymes involved in 203 bilirubin production, but have completely different evolutionary histories and are only remotely 204 related, we analyzed whether BVRB has an Nrf2 -interacting role. We simultaneously co -205 expressing GFP -Nrf2 with either GST -BVRB, GST -BVRA or GST alone in HEK293 cells and 206 conducted GST pulldown assays. We observed that BVRB does not associate with Nrf2 and this 207 function of BVR remains specific to BVRA ( Fig. 3 D). To determine whether this interaction is 208 dependent on the BVRA’s bilirubin -producing activity, we transfected cells with Nrf2 and 209 enzymatically dead G17A BVRA, which cannot bind the cofactors NADPH or NADH. G17A BVRA 210 not only pulls down Nrf2 but does so more robustly than WT BVRA, indicating that this intera ction 211 is independent of its catalytic function (Fig. 3E). 212 As both BVRA and Nrf2 modulate redox homeostasis, we also analyzed whether the 213 formation of the BVRA -Nrf2 complex increases in response to oxidative stress. We therefore 214 simulated pro-oxidant condi tions to monitor changes in the BVR -Nrf2 interaction. HEK293 cells 215 exposed to H 2O2 and pyrogallol, a superoxide generator (42), exhibit increased Nrf2 expression 216 compared to vehicle conditions, which w as associated with increased BVRA -Nrf2 complex 217 formation via co -immunoprecipitation. ( Fig. 3 F). Notably, there was no affinity difference in 218 interaction under different redox conditions. 219 We also analyzed whether BVRA influences stability of Nrf2. As Nrf2 stability and levels 220 are regulated by ubiquitination, we first examined whether BVRA increases Nrf2 expression by 221 binding and hindering ubiquitin association. Through co -immunoprecipitation, we find that BVRA 222 does not bind or associate with ubiquitin (Fig. S2A). Additionally, we observed no changes in levels 223 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 8, 2025. ; https://doi.org/10.1101/2025.06.04.657936doi: bioRxiv preprint of Nrf2 in the presence or absence of BVRA both under basal conditions or in response to H 2O2, 224 sulforaphane or t-BHQ exposure (Fig. S2B). Next, we monitored Nrf2 levels in the nucleus of WT 225 and BVRA-/- cells upon induction of oxidative stress or with treatment with Nrf2 activators such as 226 sulforaphane. While whole cell levels of Nrf2 are higher in WT cells with oxidative stress compared 227 to cells lacking BVRA, the levels of Nrf2 in the nucleus are simila r in both WT and BVRA -/- MEFs 228 under the conditions analyzed (Fig. S3C). 229 Lastly, we investigated whether binding of BVRA to Nrf2 is modulated by its substrate, 230 biliverdin. We transfected HEK293 cells with GFP -Nrf2 and either GST -BVRA or GST vector, 231 treated cells with biliverdin and conducted a pulldown assay. In the presence of biliverdin, 232 interaction of BVRA with Nrf2 decreased as compared to cells which were not treated ( Fig. 3G). 233 Thus it appears that the presence of substrates for BVRA can influence its non-canonical functions. 234 235 BVRA is a component of the transcriptional program of Nrf2-regulated promoters in vivo 236 237 BVRA has been reported to be a transcription factor regulating the expression of activating 238 transcription factor 2 (ATF2) and HO -1 by binding to the AP -1 sites in their promoters (43, 44). 239 Considering the physical and genetic interaction of Nrf2 and BVRA, we examined whether BVRA 240 might potentially coregulate Nrf2 target genes. We performed a genome -wide ChIP-seq analysis 241 of MEFs generated from prev iously described FLAG -BVRA transgenic mouse line (21) using 242 FLAG and Nrf2 antibodies ( Fig. 4A-D). The peak distribution demonstrated that approximately 10 243 % of the peaks were located in promoter regions with majority (approximately 40%) harbored in 244 distal intergenic regions (10 to 50kb; Fig. 4B). Motif analysis of both FLAG -BVRA and Nrf2 ChIP-245 seq peaks identified bZIP motifs such as Bach1::Mafk motif and Nrf2 motif respectively ( Fig. 4B). 246 ChIP-seq peaks were annotated by nearest neighbor method to identify genes that both Nrf2 and 247 BVRA potentially regulate. Approximately 43% of the genes in the annot ated ChIP-seq data had 248 both BVRA and Nrf2 peaks ( SI Data Table 2 ). We further overlaid the BVRA -Nrf2 gene set with 249 RNAseq-data from WT and BVRA-/- cells. K-means clustering of the Nrf2 -BVRA gene expression 250 identified two clusters that are predominantly en riched for pathways that are dysregulated in 251 neurodegenerative diseases (Fig. S3). Further, Gene Set Enrichment Analysis (GSEA) analysis of 252 BVR-Nrf2 associated genes revealed inflammatory pathways, heme metabolism and redox 253 signaling (Fig. 4E). 254 255 Thus, interaction of BVRA with Nrf2 is physiologically relevant and fits in well with the observation 256 that this association is increased in response to oxidative stress to increase the transcription of 257 Nrf2 targets to maintain redox balance in cells 258 259

260 261 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 8, 2025. ; https://doi.org/10.1101/2025.06.04.657936doi: bioRxiv preprint In this study, we show that BVRA, the biosynthetic enzyme for bilirubin, is an essential 262 regulator of neuroprotective signaling by the master regulator of redox homeostasis, Nrf2. We 263 showed previously that bilirubin is a potent antioxidant capable of sca venging superoxide (2). 264 However, the role of BVRA in oxidative stress independent of its catalytic activity has not been 265 explored (2). Using advanced genomic techniques, as well as evolutionary, mutational, genetic, 266 and biochemical studies, we examined the non -canonical role of BVRA in the antioxidant stress 267 response. sc -RNA-seq analysis of the brain revealed th at a significant proportion of brain cell 268 populations expressed BVRA but not HO-2, the precursor to BVRA in heme catabolism in the brain, 269 indicating additional functions for BVRA distinct from its classical role in bilirubin production. 270 Phylogenetic analysis revealed that although HO -2 and BVRA originated in the cyanobacteria in 271 the context of phycobilisome synthesis in the light -harvesting complex, they were acquired at 272 different points in time in the eukaryotes. Thus, they were co -opted and combined in a common 273 heme catabolism pathway only in Metazoa. Using sequence conservation information from a 274 multiple sequence alignment, we mutated key residues in BVRA and tested these mutants for 275 BVRA activity. Further, the presence of a single exposed conserved cyst eine within a CC motif at 276 the extreme C-terminus in vertebrates suggests that this residue might be involved in the sensing 277 of the redox status of the cell. 278 Amongst the mutants generated, the G17A mutant was severely compromised in the ability 279 to produce bilirubin as this mutation affects its ability to bind the cofactor, NAD(P)H as reported 280 earlier (21, 33) . Interestingly, G17A BVRA rescu ed H 2O2-mediated cell death, demonstrating a 281 protective role of BVRA, distinct from its catalytic activity. Furthermore, unbiased RNA-seq analysis 282 of hippocampal neurons derived from BVRA-/- mice, identified Nrf2 signaling as a top downregulated 283 pathway. I n support of these findings, both WT and G17A BVRA bind Nrf2 and stimulate 284 transcription of target genes. We also noted that in the presence of biliverdin, the substrate for 285 production of bilirubin, the binding of BVRA to Nrf2 is diminished. This suggests a dual yet distinct 286 role of BVRA with its additional ability to regulate redox metabolism via Nrf2. Although BVRA does 287 not influence the nuclear translocation of Nrf2, it is essential for optimal function of Nrf2 as revealed 288 by our RNA -seq and ChIP -seq da ta. This role of BVRA as a regulator that interacts with a 289 transcription factor is closely paralleled by another catalytically inactive Rossman fold 290 oxidoreductase harboring a from the same clade as BVRA, Gal80, which has been co -opted as a 291 regulator that binds transcription factors (45). 292 Our studies revealed that multiple pathways are co -regulated by the BVRA -Nrf2-bound 293 genes, which encompass a range of functions, including mitochondrial function, heme metabolism 294 to cytokine signaling and oxidative stress response. Delineating this function of BVRA allows us to 295 understand heme metabolism in the context of oxidant -induced aging and disease. While heme 296 oxygenases, which catalyze the first step of heme degradation, are known targets for redox 297 regulation, their overexpression leads to deleterious off -target effects including inflammation and 298 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 8, 2025. ; https://doi.org/10.1101/2025.06.04.657936doi: bioRxiv preprint cognitive decline (46) reducing its therapeutic efficacy (47). Likewise, while bilirubin itself acts as 299 an endogenous and exogenous ROS suppressor, its effects are limited to distinct forms of ROS 300 (2). While exogenous bilirubin may only be effective in curtailing excess O2, activators of BVRA 301 expression may be able to regulate ROS in a more all -encompassing manner. The BVRA/Nrf2 302 interaction is relevant to a wide array of ROS -mediated pathologies. Suboptimal Nrf2 activity has 303 been observed in neurodegenerative diseases such as PD and AD (48, 49). However, currently 304 known activators of Nrf2 are predominantly electrophilic compounds that exert adverse side effects. 305 In such a scenario, molecules that activate BVRA expression or stimulate its transcriptional activity 306 are of significant therapeutic value. BVRA’s role in activating Nrf2 provides a new therapeutic 307 signaling pathway that mitiga tes a broader array of oxidative species via multiple downstream 308 effectors. Augmenting BVRA therefore promises to a more reliable and efficacious approach for 309 diseases involving imbalanced Nrf2 activity. Additionally, modulating the Nrf2 pathway through 310 BVRA may also mitigate ferroptosis, an iron -mediated form of cell death, which is observed in 311 several neurodegenerative diseases as both BVRA and Nrf2 regulate iron disposition, through its 312 metabolism or transport, and loss of either of these proteins result in accumulation of excess iron 313 and vulnerability to oxidative stress (50-53). 314 315 Collectively, our findings reveal a previously unrecognized and crucial function of BVRA, 316 which bridges two distinct and critical pathways, namely heme metabolism and Nrf2 signaling, to 317 afford cytoprotective effects. Furthermore, BVRA orchestrates antioxidant stress responses in both 318 the lipophilic and hydrophilic subcellular compartments of the cell through not only the production 319 of bilirubin which directly scavenges O2radicals, but also through its role as a transcriptional 320 regulator, respectively ( Fig. 4F). Augmenting this latter pathway may offer therapeutic benefits in 321 diseases characterized by redox imbalance. Both the enzymatic and non-enzymatic roles of BVRA 322 participate in diverse aspects of normal physiology, and characterizing these activities a nd their 323 cell-type-specific roles will likely reveal new paradigms for treating neurodegenerative diseases. 324 325 326

327 328 Plasmids/cDNA 329 Plasmids encoding GST -BVR (GST -BVRA) and GST -BVRB were generated by cloning cDNA 330 encoding human BVRA and BVRB into pCMV-GST vector (Clontech/TaKaRa). Likewise, the myc-331 BVRA (myc-BVRA) plasmid was generated by cloning cDNA of human BLVRA into pCMV -myc 332 vector. The GFP-Nrf2 plasmid was generated by cloning cDNA of Nrf2 into GFP-vector. 333 334 Cell Culture 335 Mouse Embryonic Fibroblasts (MEFs) 336 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 8, 2025. ; https://doi.org/10.1101/2025.06.04.657936doi: bioRxiv preprint WT, BVRA-/-, and Flag BVRA MEFs were isolated as previously described (2). In brief, embryonic 337 day 14.5 (E14.5) embryos were obtained from timed BVRA+/− matings. The pups were decapitated 338 and eviscerated, after which the remaining portion was trypsinized and sheared. Isolated MEFs 339 were plated in 2 wells of a 6-well plate and cultured overnight in DMEM (Gibco) supplemented with 340 10% FBS, 100 U/mL penicillin and streptomycin, and 2 mM glutamine at 37°C with 5% CO2. MEFs 341 were then expanded to 6-well plates and transiently transfected with SV40T antigen (Addgene) and 342 maintained until stably proliferative. DNA was isolated from the heads of the pups and genotyped 343 to confirm the genotype of the corresponding MEF cell line. Data for WT and BVRA -/- cells were 344 drawn from experiments on at least two independent cell lines of each genotype. 345 346 Human Embryonic Kidney (HEK) 293 Cells 347 HEK293 cells were obtained from the American Type Culture Collection. HEK293 cells were 348 cultured in Dulbecco's Modified Eagle Medium (DMEM), 10% fetal bovine serum, 349 penicillin/streptomycin (100 U/ml), and glutamine (2 mM) in an atmosphere of 5% CO 2 at 37°C. 350 351 Primary neurons 352 Primary WT and BVRA -/- neurons were isolated as previously described (2). In brief, hippocampi 353 from embryonic day 18 (E18) embryos were isolated and washed with HBSS. Tissue was then 354 incubated with 0.25% trypsin in HBSS for 18 min at 37°C in a conical tube, after which trypsin was 355 inactivated with the addition of FBS. Tissue were then pelleted at 2000 g for 5 min at 25°C, after 356 which the supernatant trypsin/FBS was aspirated off. Tissue were then rinsed twice with HBSS, 357 resuspended in HBSS, and then gently triturated through a fire -polished glass pipette to promo te 358 additional dissociation. Tissue was then filtered through a 70 μm nylon cell strainer to remove 359 debris. The dissociated cells were pelleted at 2000 g for 5 min at 25°C and then resuspended in 360 neuronal culture media (composed of Neurobasal (Gibco) supplemented with 2% B-27 supplement 361 and 2 mM glutamine). Cells were filtered once more through a 70 μm nylon cell strainer. Dissociated 362 cells were then plated onto rinsed, poly -D-lysine-coated dishes. Four days later, the conditioned 363 media was supplemented with fresh neuronal culture media. The day before dissections, plates 364 were prepared and coated in 0.25 mg/mL poly -D-lysine overnight at 37°C. Just prior to plating, 365 plates were rinsed three times with sterile water and once with neuronal culture media. 366 367 Transfection 368 HEK293 cells were transfected with Lipofectamine 3000 (ThermoFisher) as per the manufacturer’s 369 instructions. Briefly, HEK293 cells were seeded in either 10 cm dishes (3x10 6 cells) or in 60 mm 370 dishes (1x106 cells). The next day, transfection complexes were added dropwise to the plated cells. 371 For cells in 10 cm dishes, 25 g of DNA, 23.7 μL of Lipofectamine, and 50 μL of P3000 were 372 brought to a volume of 1 mL in serum-free Optimem media (Thermo). For cells in 60 mm dishes, 5 373 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 8, 2025. ; https://doi.org/10.1101/2025.06.04.657936doi: bioRxiv preprint ug of DNA, 8.25 μL Lipofectamine, and 10 μL P300 were brought to a volume of 500 μL in Optimem. 374 Cells were harvested 48 hours after transfection. 375 376 Additional details of reagents and methods are available in the Supplementary Section. 377 378 Acknowledgments 379 380 BDP, SHS and AAP were supported by the American Heart Association and Paul Allen Foundation 381 Initiative in Brain Health and Cognitive Impairment (19PABH134580006). BDP and AAP by 382 NIH/NIA 1R01AG071512. BDP also acknowledges support from NIH/NIA 1R21AG073684, NIH 383 NIDA, grant P50 DA044123, funding from the Solve -ME foundation and the Catalyst Award from 384 Johns Hopkins University. B.T. was supported by NIH AG077396, NS101967, NS133688, and the 385 Department of Defense HT94252310443. M.C.O. and S.M.S. were supported by NIH 386 P01CA236778. AAP was supported by The Valour Foundation, as the Rebecca E. Barchas, MD, 387 Professor in Translational Psychiatry of Case Western Reserve University and the Morley -Mather 388 Chair in Neuropsychiatry of University Hospitals of Cleveland Medi cal Center, and by the Wick 389 Foundation, Department of Veterans Affairs Merit Award I01BX005976, NIH/NIA RO1AGs066707, 390 NIH/NIA 1 U01 AG073323, the Louis Stokes VA Medical Center resources and facilities, the Lincoln 391 Neurotherapeutics Research Fund, the Leon ard Krieger Fund of the Cleveland Foundation, the 392 Meisel & Pesses Family Foundation, and an anonymous donor. LMI and LA are supported by the 393 funds of the Division of Intramural Research of the National Library of Medicine at the National 394 Institutes of Hea lth. We also acknowledge biorender.com, a service we used to design our 395 schematic figures and graphical abstract, and Intelligenomica LLC for genomics support. 396 397

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Mezzanotte M & Stanga S (2024) Brain Iron Dyshomeostasis and Ferroptosis in 521 Alzheimer's Disease Pathophysiology: Two Faces of the Same Coin. Aging Dis. 522 52. Berndt C, et al. (2024) Ferroptosis in health and disease. Redox Biol 75:103211. 523 53. Wang ZL, Yuan L, Li W, & Li JY (2022) Ferroptosis in Parkinson's disease: glia -neuron 524 crosstalk. Trends Mol Med 28(4):258-269. 525 54. Pereira PJ , et al. (2001) Structure of human biliverdin IXbeta reductase, an early fetal 526 bilirubin IXbeta producing enzyme. Nat Struct Biol 8(3):215-220. 527 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 8, 2025. ; https://doi.org/10.1101/2025.06.04.657936doi: bioRxiv preprint 528 529 Figure Legends 530 531 Fig. 1. BVRA exhibits expression profiles and redox homeostatic activity unlinked to heme 532 catabolism. (A) Schematic representation of the heme catabolic pathway. Heme derived 533 from hemoglobin from senescent red blood cells is metabolized by heme oxygenases (HO) 534 to produce the green pigment, biliverdin. Biliverdin reductase A (BVRA) then reduces 535 biliverdin to produce the yellow pigment, bilirubin, which is a potent antio xidant. ( B) T -536 distributed stochastic neighbor embedding (t -SNE) analysis of single -cell RNA expression in 537 primary neuronal cultures from WT C57BL/6J mice. Each point represents a single cell. Left 538 panel: Cells are clustered by gene expression into populat ions with similar cell type and 539 function. Right panel: Expression distribution of Blvra, Hmox2, both individually and together, 540 across cell population clusters. (C) Quantified percentage of total cells expressing Blvra, 541 Hmox2, and a combination of both gen es. (D) Scaled Pearson residuals of Blvra and Hmox2 542 gene expression in different cell populations. A residual >1 indicates a higher observed to 543 expected frequency ratio. Each point represents a single cell. ( E) Phylogenetic trees of BVRA, 544 HO, and BVRB respectively. (F) Protein structures of BVRA and BVRB obtained from Kavanagh 545 et. al. (PDB Entry - 2H63 (pdb_00002h63) and Periera et. al., respectively (54). Structural 546 comparison of the two BVR proteins aligned at the Rossmann fold of the NADPH-binding domain. 547 (G) Activity of WT BVRA and BVRA mutants measured at varying concentrations of NADPH and 548 10 M biliverdin. Activity measured by absorbance reading of bilirubin production at 442 nM with 549 Vo calculated according to the method by Michaelis and Menten. n = 3 in triplicate. (H) Quantified 550 Vmax and Km of WT BVRA and BVRA mutants. ( I) Viability of WT and BVRA MEFs transfected 551 with empty vector, WT BVRA, and G17A- BVRA after 8 h of exposure to serial dosing of H 2O2. 552 Data is normalized to viability of vehicle condition. ( J) Quantified LD50s (I) of H 2O2 exposure in 553 WT and BVRA MEFS. n = 3 in triplicate. 554 555 Fig.2. Nrf2 activity is disrupted in BVRA-/- neurons and fibroblasts 556 557 (A) Volcano plot analysis of genes from WT and Blvra-/- primary hippocampal neurons. Each plot 558 displays the fold change of gene expression on a log scale with their corresponding P values plotted 559 on a log scale. Each dot represents a gene of interest. Change in gene expression between WT 560 from vehicle to H 2O2 condition are shown ( B) Change in gene expression in Blvra-/- neurons from 561 vehicle to H 2O2 condition. (C) Change in gene expression between WT and Blvra-/- neurons after 562 H2O2 treatment. Red dots indicate genes that are upregulated with H2O2 condition in Blvra-/- neurons. 563 Blue dots indicate genes that are downregulated with H 2O2 condition in Blvra-/- neurons. ( D) 564 Logarithmically-scaled p values of molecular pathways that are upregulated (left panel) and 565 downregulated (right panel) in Blvra-/- neurons compared to WT controls under the H 2O2 condition. 566 (E) Transcription factors ranked by log -scaled p values. A higher p value indicates transcription 567 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 8, 2025. ; https://doi.org/10.1101/2025.06.04.657936doi: bioRxiv preprint factors that are more significantly dysregulated in BVRA-/- compared to WT neurons in the H 2O2 568 condition. Transcription factors ranked from 1, most significant, to 1279, least significant. ( F-I) 569 Validation of expression of genes regulated by Nrf2 in WT and BVRA-/- neurons. Wild -type (WT) 570 and BVRA-/- primary hippocampal neurons were treated with or without 200 M H 2O2 for 6 h and 571 expression of Nrf2 targets were analyzed by qPCR. n=3, SEM, p <0.01, Student’s t test. ( J) 572 Luciferase reporter assays in WT and BVRA-/- MEFs transfected with Gsta4 luciferase reporter 573 construct after exposure to vehicle, 50 M H2O2, 10 M SFN, and 25 M TBHQ. n = 4 in triplicate. 574 Sidak’s multiple comparison (K) Luciferase reporter assays in stable cell HEK293 cell lines harboring 575 a 5xARE reporter constructs transfected with either WT BVRA or G17A- BVRA and after exposure 576 to either vehicle, 200 M H2O2, 10 M SFN, and 25 M TBHQ. n = 4 in triplicate. Tukey’s multiple 577 comparison. (L) BVRA-/- MEFs are more sensitive to erastin, an inhibitor of the XcT transporter. WT 578 and BVRA-/- MEFs were treated overnight for 18 hours with increasing concentrations of erastin, a 579 xCT inhibitor and ferroptosis inducer, and cell viability was assessed by MTT assay. n=7, SEM, ∗p 580 < 0.05, **p <0.01, ***p <0.001, Student’s t test. 581 582 Fig. 3. BVRA genetically and physically interacts with Nrf2 583 (A) Expected versus observed frequency of pups from the Blvra-/- and Nfe2l2-/- (Nrf2-/-) cross. (B) 584 Number of observed pups of each genotype. n = 114 pups total. (C) GST pulldown with glutathione 585 sepharose beads of GST-BVRA and GFP-Nrf2 transfected HEK293 cells. GST-only vector control 586 was used as a negative control. Blots were probed for GFP -Nrf2 and GST -BVRA with their 587 respective tags. (D) Co-immunoprecipitation of GFP-Nrf2 with GST- BVRA and GST-BVRB. Inputs 588 at the right indicate samples taken before incubation in beads to monitor whole cell expression of 589 transfected proteins. Blots were probed for GFP -NRF2 and GST-BVRA with their respective tags. 590 (E) GFP -Nrf2 co -immunoprecipitation with GST -WT BVRA and GST -G17A-BVRA. Shorter 591 exposure was used for G17A IP lanes and longer exposure was used for the remaining lanes. Blots 592 were probed for GFP-Nrf2 and GST-BVR with their respective tags. (F) Co-immunoprecipitation of 593 GST-BVRA and GFP-Nrf2 with varying oxidative stress conditions, including vehicle, 200 M H2O2, 594 and 100 M pyrogallol. Blots were probed for GFP-Nrf2 and GST-BVRA with their respective tags. 595 (G) Interaction of GST -BVRA and GFP-Nrf2 in the presence of 10 M biliverdin in HEK293 cells. 596 Blots were probed for GFP-Nrf2 and GST-BVRA with their respective tags. 597 598 599 Fig. 4. ChIP -Seq analysis reveals genes regulated by both BVRA and Nrf2. (A) Schematic 600 representation of methods used for genomic analysis. (The data obtained from ChIP-seq was 601 overlaid onto the RNA -seq data from the primary hippocampal neurons in vehicle treated (V) or 602 H2O2-treated conditions (T) (B) Global representation of Flag-BVRA and Nrf2 peak distribution and 603 Motifs. (C) Venn diagram showing overlap of genes a ssociated with both BVRA and Nrf2 peaks. 604 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 8, 2025. ; https://doi.org/10.1101/2025.06.04.657936doi: bioRxiv preprint (D) MA plot showing differentially expressed readcounts between WT and BVRA -/-. (E) GSEA 605 analysis of BVRA -Nrf2 associated genes from C. ( F) Summary of canonical and non -canonical 606 roles of BVRA. BVRA acts on heme -derived biliverdin to produce the antioxidant -cytoprotectant 607 bilirubin, which scavenges superoxide generated from mitochondrial activity and other processes 608 to maintain redox balance. In addition to this role in heme catabolism, BVR also functions as a 609 serine-threonine and tyrosine kinase that modulates several signaling pathways. We show here 610 that BVRA also regulates Nrf2 signaling, independent of its enzymatic roles. 611 612 613 614 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 8, 2025. ; https://doi.org/10.1101/2025.06.04.657936doi: bioRxiv preprint A B C D E F G I J H Figure 1 was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 8, 2025. ; https://doi.org/10.1101/2025.06.04.657936doi: bioRxiv preprint A B C D E G HF Figure 2 I J K was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 8, 2025. ; https://doi.org/10.1101/2025.06.04.657936doi: bioRxiv preprint A B C D E F Figure 3 GST alone GST- BVRA GST alone GST- BVRA input pull down input pull down BVRA BVRA input pull down G was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 8, 2025. ; https://doi.org/10.1101/2025.06.04.657936doi: bioRxiv preprint E Figure 4 A B C D BVRA ChIP-seq Nrf2 ChIP-seq BVRA ChIP-seq genes Nrf2 ChIP-seq genes Co-regulated genes F was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 8, 2025. ; https://doi.org/10.1101/2025.06.04.657936doi: bioRxiv preprint Supplementary Information Biliverdin Reductase A is a major determinant of neuroprotective Nrf2 signaling Chirag Vasavda a,1, Ruchita Kothari a,1, Navneet Ammal Kaidery b,c, Suwarna Chakraborty d, Sunil Jamuna Tripathi d, Ryan S. Dhindsa e,f,g, Samaneh Saberi h, Julia E. Lefler h, Priyanka Kothari i, Kalyani Chaubey j,k,l,m, Adele M. Snowman a, Michael C. Ostrowski h, Eugenio Barone n, Lakshminarayan M. Iyer o, L. Aravind o, Sudarshana M. Sharma h, Andrew A. Pieper j,k,l,m,p,q, Bobby Thomasb,c,r,s, Solomon H. Snydera,d,t,2, Bindu D. Paula,d,t,u,2 *Solomon H. Snyder; *Bindu D. Paul Email: [email protected] [email protected] was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 8, 2025. ; https://doi.org/10.1101/2025.06.04.657936doi: bioRxiv preprint Supporting Information Text

Western Blotting For western blot analysis, tissues were dounce homogenized at 4°C in lysis buffer (pH 7.4 solution of 50 mM Tris -HCl, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, and 1% Triton X-100) supplemented with protease inhibitors (Sigma). Lysates were then pulse sonicated and centrifuged at 16,000 g for 15 min at 4°C. Fifteen micrograms of cleared lysate were run on a 4 -12% polyacrylamide Bis-Tris gradient gel in running buffer (pH 7.3 solution of 50 mM MES, 50 mM Tris Base, 0.1% SDS, 1 mM EDTA) and then transferred to a PVDF membrane. Membranes were blocked with 5% milk in TBS -T (pH 7.6 solution of 16 mM Tris -HCl, 140 mM NaCl, 0.1% Tween - 20) and incubated with primary antibodies in 3% bovine serum albumin (BSA) (w/v) overnight at 4°C. The following day, membranes were washed with TBS-T, and then incubated with secondary antibodies in 3% BSA for 1 h at 25°C. The following primary antibodies were used: rabbit anti - BVRA (Abcam ab180208 ; 1:1,000), rabbit anti -Nrf2 (Abcam ab62352; 1:1,000), rabbit anti -Nrf2 (Santa Cruz sc -722; 1:1,000), rabbit anti -GFP (Cell Signaling 2956; 1:1,000), mouse anti -GST (Abcam ab3416 1:20,000), mouse anti -myc (Abcam ab9106; 1:10,000), and mouse anti -β-actin (Santa Cruz sc-47778 HRP; 1:10,000). The following secondary antibodies were used: sheep anti- mouse IgG (GE Healthcare NA931; 1:10,000), and donkey anti-rabbit IgG (GE Healthcare NA934; 1:10,000). Subcellular Fractionation Cells were lysed in membrane prep buffer (10 mM Hepes pH 7.4, containing 1 mM EGTA, 1 mM dithiothreitol, 10% sucrose) supplemented with protease inhibitors (Sigma). After lysis cells were snap-frozen in liquid nitrogen and then thawed briefly in a cold -water bath, and then kept on ice thereafter. A 30G needle was then used to lyse cells 3 -4 times. The cell homogenate was then filtered through a nylon mesh (pore size 75 μm) to remove any intact cells. 100μL of this sample was collected and set aside for the whole cell fraction. The remainder of the sample was centrifuged at 1,000 g for 10 minutes at 4°C. The supernatant was collected and set aside as the cytosolic fraction. The pellet was then re -homogenized in membrane buffer and spun down once again at 1,000 g for 10 minutes at 4°C. The pe llet was then washed once more in membrane buffer by pipetting buffer over the pellet without homogenizing it. The cells were then centrifuged one last time at 1,000 g for 10 minutes at 4°C, and the supernatant was discarded. The pellet was re - homogenized as the nuclear fraction. The previously collected cytosolic fraction was then centrifuged at 11,000g for 20 minutes at 4°C and the pellet was discarded. All whole cell, cytosolic, and nuclear fractions were then pulse -sonicated to shear DNA and spun down a t 16,000 g for 15 minutes at 4°C. The fractions were then processed for immunoblot analysis. was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 8, 2025. ; https://doi.org/10.1101/2025.06.04.657936doi: bioRxiv preprint BVRA enzymatic activity assays BVRA activity was assayed at pH 8.5 at 37°C. 250 nM of purified WT and mutant BVRA were separately incubated for 5 min at room temp erature in a pH 8.5 solution of 50 mM Tris, NADP+, glucose-6-phosphate, and glucose -6-phosphate dehydrogenase (NADPH Regeneration System, Promega v9510). The samples were then placed at 37°C and spiked with varying concentrations of biliverdin IXα (250 nM to 20 uM). Reaction rates were determined by monitoring the change in absorbance at 442 nm over time. Enzyme activity was determined by the Michaelis and Menten (Michaelis Menten 1913). In brief, the absorbance of bilirubin was monitored over time for each concentration of biliverdin. The slope of each curve in the linear range was recorded and plotted against the corresponding substrate concentration. This data was then fit to the Michaelis -Menten equation to determine the Km and Vmax of the enzyme-substrate reaction (1). Methylthiazoletetrazolium (MTT) assays WT and BVRA -/- MEFs were plated at a density of 75,000 cells/well in a 24 -well plate. After 24h, BVRA-/- cells were transfected with empty vector, WT -BVRA, and G17A -BVRA respectively. The following day, the culture media was supplemented with varying H2O2 concentrations (0 to 250uM) for 6 hours, after which the media was replaced with fresh media containing 0.5 mg/mL MTT for 1 h. The media was then removed before dissolving the reduced cellular MTT in DMSO. Cell viability was determined by measuring the absorbance at 570 nm and normalizing absorbances to the vehicle condition. Single-Cell RNA Sequencing Single-cell RNA-sequencing was performed as previously described (2). Briefly, whole brains from postnatal day 0 pups were dissected and dissociated for sequencing. Single cell RNA-seq libraries were constructed using the 10X Chromium Single Cell 3′ Reagent Kits v2 according to manufacturer’s descriptions and subsequently sequenced on a NovaSeq 6000. Reads were aligned to the mm10 genome using the 10X CellRanger pipeline with default parameters to generate the feature -barcode matrix. 610 Downstream quality control and analyses on fe ature- barcode matrices were performed with Seurat v3. Genes that were not detected in at least 4 cells were excluded. Cells with fewer than 1,000 genes or more than 5,000 genes were also excluded from analysis. We also excluded cells in which greater than 15% of reads mapped to mitochondrial genes. The filtered matrices were log -normalized and scaled to 10,000 transcripts per cell. Using the variance-stabilizing transformation in the FindVariableFeatures function, we identified the top 2,000 most variable genes per sample. We then harmonized gene expression across datasets prior to clustering by identifying anchors between samples in each dataset using the 620 FindIntegrationAnchors function and then computing an integrated expression matrix from these was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 8, 2025. ; https://doi.org/10.1101/2025.06.04.657936doi: bioRxiv preprint anchors as input to the IntegrateData function. We then linearly regressed the number of UMIs per cell and percentage of mitochondrial reads using the ScaleData function and performed dimensionality reduction using PCA. For each dataset, we selected the top 30 dimensions to compute a cellular distance matrix, which was used to generate a K -nearest neighbor graph. The KNN was used as input to the Louvain Clustering algorithm implemented in the FindClusters function. We chose a resolution parameter of 0.8 for clustering via Louvain. To annotate and merge clusters, we performed differential gene expression analysis on the integrated expression values between each cluster using the default parameters in the FindMarkers function RNA isolation, reverse transcriptase PCR (RT-PCR), and quantitative-PCR (q-PCR) RNA isolation and qPCR were performed as previously described (3). In brief, WT and BVRA -/- cells were treated with vehicle or 50 μM H 2O2 for 8 h. Total cellular or tissue RNA was extracted using the RNeasy Plus Universal Kit (Qiagen) per the manufactu rer’s instructions. RT -PCR was performed with the SuperScript III One -Step RT-PCR System (Invitrogen), whereas q -PCR was performed with the TaqMan RNA-to-Ct 1-Step Kit (Applied Biosystems). Immunoprecipitation HEK cells were co-transfected with the plasmids of interest as indicated above. Two days later, the culture media was supplemented with 10 μM MG-132 (Sigma M744) for 4 h. Cells were then lysed in immunoprecipitation buffer (1% Triton -X100, 50 mM Tris, 150 mM NaCl, 5% glycerol, 25 mM NaF, 1 mM Na 3VO4) supplemented with protease inhibitors (Sigma) and placed on a shaker at 4°C for 15 min. The lysates were then centrifuged at 16,000 g for 15 min at 4°C. IP samples were incubated with 1 mg of protein lysate and 40 μL of either Glutathione Sepharose 4B (GE Healthcare) or anti-FLAG M2 (Sigma) beads overnight at 4°C. 5% of each sample was set aside as input prior to addition of beads. The following day, beads were pelleted by centrifugation at 2,500g for 2 min at 4 °C. The supernatant was aspirated, and beads were washed 5 times in immunoprecipitation buffer by centrifugation, aspiration, and resuspension. Proteins were eluted from beads by boiling for 5 min at 100°C in LDS sample buffer (Thermo Fisher) and the eluates analyzed by Western blot as described above. Nrf2 ARE Luciferase Reporter Assay Nrf2 activity was monitored with a reporter cell line in which changes in Nrf2 activity are coupled to the expression of firefly luciferase (Nrf2/ARE Luciferase Reporter HEK293 Stable Cell Line). Nrf2 ARE HEK293 cells were infected with lentivirus encoding either empty vector, WT-BVRA, or G17A- BVRA were plated at 125,000 cells/well in a 24 -well plate. The following day, cells were treated with 200 μM H 2O2, 10 μM SFN, and 25 μM TBHQ for 6 h. Cells were then lysed in pas sive lysis buffer (Promega E1941) supplemented with protease inhibitors (Sigma) and let shake at 4°C for was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 8, 2025. ; https://doi.org/10.1101/2025.06.04.657936doi: bioRxiv preprint 15 minutes. The lysates were then centrifuged at 16,000 g for 15 minutes at 4°C and protein estimated using the BCA assay. 15 μL of lysate was mixed with 5 μL of luciferase assay substrate LARII (Promega) and the amount of light produced was measured using a luminometer for a 10 second integration time with a delay of 2 seconds. The luciferase measurement was normalized to protein and reported normalized to the vehicle condition. GST Firefly Dual Luciferase Assay WT and BVRA-/- MEFs were plated in a 24-well plate and transfected with NQO1 or GST ARE firefly luciferase and renilla luciferase. Two d post -transfection, cells were treated with H 2O2, SFN, and tBHQ for 8 hours. Cells were then lysed in passive lysis buffer (Promega E1941) supplemented with protease inhibitors (Sigma) and let shake at 4°C for 15 minutes. The lysates were then spun down at 16,000g for 15 min at 4°C and protein estimated using the BCA assay. 15 μL of lysate was mixed with 5 μL of luciferase assay substrate LARII (Promega) and the amount of light produced was measured using a luminometer for a 10 second integration time with a delay of 2 seconds. Afterwards, 5 μL of Stop&Glo Reag ent (Promega) was added and measured for 10 seconds with a delay of 2 s to estimate the amount of Renilla luciferase produced. The Renilla measurement and was used as the protein loading control to normalize the signal given by LARII. Data was normalized to the vehicle condition. Chromatin immunoprecipitation-sequencing (ChIP-seq) Chromatin immunoprecipitation (ChIP) assays were performed on cultured FLAG -BVRA mouse embryonic fibroblasts from 10 million cells per IP as described previously (4). The chromatin was immunoprecipitated with 10 μg anti -FLAG-M (Sigma Aldrich), 10 μg anti -Nrf2 (D1Z9C -XP Cell Signaling Rabbit mAB #1271) antibodies. 5 ng of the immunoprecipitat ed chromatin was used for ChIP-seq library preparation using NEBNext library kit (New England Biolabs) and sequenced in Illumina sequencer (50 million paired end reads per sample). Bowtie2 software (5) was used to map the sequence reads to the mm10 mouse genome. Peak calling and motif analysis were done with HOMER v3 software (6). Global annotation of peaks with respect to TSS and gene body was performed with the R Bioconductor package ChIPseeker (7). Computational analysis Homologous sequences of BVRA were retrieved using sequence -profile searches done with the PSI-BLAST program (8) against a curated database of completely sequenced genomes as well as a non-redundant protein database from the National Center for Biotechnology Information clustered down to 50%. Retrieved sequences were aligned with the Kalign2 (9) and MAFFT programs (10). For the MAFFT program alignment, the local -pair algorithm was combined with the following parameters: maxiterate, 3,000; op, 1.5; ep, 0.2. The aligned sequences were then used to perform was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 8, 2025. ; https://doi.org/10.1101/2025.06.04.657936doi: bioRxiv preprint phylogenetic maximum likelihood analyses using the FastTREE2 program (11). Cartoon structures were rendered with the Mol* (12) or PyMOL (The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC) software. Alphafold3 (13) was used to model predicted interactions of BVRA with its NADPH, Biliverdin and Nrf2 Statistical Analysis. Results are presented as means ± SEM for at least three independent experiments. The sample sizes used were based on the magnitude of changes and consistency expected. Statistical significance was reported as appropriate. P values were calculated with Student’s t test. was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 8, 2025. ; https://doi.org/10.1101/2025.06.04.657936doi: bioRxiv preprint Fig. S1. Domains in BVRA and alignment of BVRA from various species reveals conserved residues in the NAD(P)H and biliverdin binding sites. Multiple sequence alignment of BVRA homologs from eukaryotes to bacteria. Proteins are labeled by their gene names or accession numbers and species names. Secondary structure is provided above the alignment with helices represented by cylinders and strands by arrows. Residue columns are colored by their consensus with absolutely conserved residues shaded red, h (hydrophobic), l (aliphatic), and a (aromatic) residues shaded in yellow; p (polar) residues shown in blue; c (charged) and + (basic) residues are shown in pink font; s (small) is shown in green font; u (tiny) is shown on a green background; b (big) is shaded gray. The NADPH binding residues (N) and biliverdin binding residues (B) were derived from the structure of the Synechocystis BvrA (pdb: 5B3V). The few additional residues that also bind NADPH in the Rat BVRA (PDB: 1LC0) are also included. C292 of Human BvrA is also indicated. was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 8, 2025. ; https://doi.org/10.1101/2025.06.04.657936doi: bioRxiv preprint Fig. S2. BVRA does not interact with ubiquitin or modulate either Nrf2 stability or its nuclear translocation. (A) BVRA does not interact with Ubiquitin Provide details for each of these. (B) Ubiquitination of Nrf2 does not play a role in interaction with BVRA. (B) BVRA does not regulate the stability of Nrf2. (C) BVRA does not regulate the nuclear localization of Nrf2. A B C was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 8, 2025. ; https://doi.org/10.1101/2025.06.04.657936doi: bioRxiv preprint Fig. S3. BVRA Nrf2 coregulated genes are critical in neurophysiology. (A) K means clustering of expression of BVRA-Nrf2 associated genes in BVRA and WT (treated with either vehicle (PBS) or H2O2. (B) Pathway analysis of KEGG on cluster 1 and 3 that shows maximal enrichment with neuronal pathways. A B was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 8, 2025. ; https://doi.org/10.1101/2025.06.04.657936doi: bioRxiv preprint SI References 1. Michaelis L, Menten ML, Johnson KA, & Goody RS (2011) The original Michaelis constant: translation of the 1913 Michaelis-Menten paper. Biochemistry 50(39):8264-8269. 2. Dhindsa RS, Zoghbi AW, Krizay DK, Vasavda C, & Goldstein DB (2021) A Transcriptome- Based Drug Discovery Paradigm for Neurodevelopmental Disorders. Ann Neurol 89(2):199-211. 3. Paul BD, et al. (2014) Cystathionine gamma-lyase deficiency mediates neurodegeneration in Huntington's disease. Nature 509(7498):96-100. 4. Sharma SM, et al. (2007) MITF and PU.1 recruit p38 MAPK and NFATc1 to target genes during osteoclast differentiation. J Biol Chem 282(21):15921-15929. 5. Langmead B, Trapnell C, Pop M, & Salzberg SL (2009) Ultrafast and memory -efficient alignment of short DNA sequences to the human genome. Genome Biol 10(3):R25. 6. Heinz S , et al. (2010) Simple combinations of lineage -determining transcription factors prime cis -regulatory elem ents required for macrophage and B cell identities. Mol Cell 38(4):576-589. 7. Yu G, Wang LG, & He QY (2015) ChIPseeker: an R/Bioconductor package for ChIP peak annotation, comparison and visualization. Bioinformatics 31(14):2382-2383. 8. Altschul SF, et a l. (1997) Gapped BLAST and PSI -BLAST: a new generation of protein database search programs. Nucleic Acids Res 25(17):3389-3402. 9. Lassmann T, Frings O, & Sonnhammer EL (2009) Kalign2: high -performance multiple alignment of protein and nucleotide sequences allowing external features. Nucleic Acids Res 37(3):858-865. 10. Katoh K & Standley DM (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 30(4):772-780. 11. Price MN, Dehal PS, & Arkin AP (2010) FastTree 2 --approximately maximum -likelihood trees for large alignments. PLoS One 5(3):e9490. 12. Sehnal D, et al. (2021) Mol* Viewer: modern web app for 3D visualization and analysis of large biomolecular structures. Nucleic Acids Res 49(W1):W431-W437. 13. Abramson J, et al. (2024) Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature 630(8016):493-500. was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted June 8, 2025. ; https://doi.org/10.1101/2025.06.04.657936doi: bioRxiv preprint