NON-REDUNDANT ROLES OF COPPER TRANSPORTERS ATP7A AND ATP7B IN NORADRENERGIC SIGNALING

Menkes disease and Wilson disease are debilitating neurometabolic disorders caused by mutations in the copper (Cu) transporters ATP7A and ATP7B, respectively. In either disease, normalization of systemic Cu levels often does not eliminate neurological deficits, suggesting dysregulated Cu homeostasis within vulnerable neuronal populations. However, the specific roles of ATP7A and ATP7B and the extent of their functional redundancy in neurons remain poorly defined. Here, we selectively deleted Atp7a or Atp7b in noradrenergic neurons, which express both transporters and require Cu for catecholamine biosynthesis. ATP7A deletion reduced Cu levels in the locus coeruleus, disrupted dopamine -β-hydroxylase localization, impai red norepinephrine synthesis, and induced proteomic signatures of defective vesicular trafficking and proteostasis, culminating in neurodegeneration and impair ed regulation of energy balance and adaptive thermogenesis. In contrast, ATP7B deletion preserved Cu levels but altered intracellular Cu utilization, resulting in catecholamine imbalance, α-synuclein upregulation, aberrant dopamine-β-hydroxylase distribution, and dysregulated thermogenesis. These findings establish ATP7A and ATP7B as non -redundant reg ulators of noradrenergic function within neural circuits governing metabolic and energy homeostasis and provide a mechanistic framework for persistent neurological pathology independent of systemic Cu levels . (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint

Copper (Cu) is essential for human development and health. By serving as an enzymatic cofactor, Cu supports mitochondrial respiration, neurotransmitter synthesis, antioxidant defense, and other cellular processes, making adequate Cu availability especially critical for the nervous system. Copper-transporting ATPases ATP7A and ATP7B are major regulators of Cu homeostasis in cells and tissues: they deliver Cu to the cuproenzymes within the secretory pathway and export excess Cu to maintain intracellular Cu ba lance(Lutsenko et al., 2024) . Loss -of-function mutations in ATP7A cause Menkes disease (MD) , a fatal neurodegenerative disorder characterized by systemic Cu deficiency, widespread inactivation of essential cuproenzymes, catecholamine misbalance, and profound developmental delay s(Fujisawa et al., 2022; Ramani and Parayil Sankaran, 2024; Tumer and Moller, 2010) . Mutations in ATP7B cause Wilson disease (WD) , in which Cu accumulates in the liver, brain, and other tissues, resulting in hepatic failure, movement disorders, psychiatric symptoms, and metabolic disturbances , including changes in catecholamines (Czlonkowska et al., 2018) . MD and WD patients have neurological deficits that are traditionally attributed to altered Cu levels in the brain, i.e. low Cu in MD and elevated Cu in WD (Dusek et al., 2019; Fujisawa et al., 2022) . Indeed, treatments aimed at restoring Cu levels can improve neuronal function in either disease (Mohr and Weiss, 2019; Sarkar et al., 1993) . However, despite early Cu supplementation in M D, neurological improvement remains incomplete . Similarly, Cu chelation in WD corrects but does not fully reverse neurologic symptoms and can even trigger worsening (Litwin et al., 2024). These

indicate that mechanisms beyond systemic Cu imbalance contribute to neuropathology . How specific neurons regulate their Cu homeostasis and what happens when these homeostatic mechanisms are disrupted remain poorly understood. To uncouple the contribution of ATP7A and ATP7B to neuronal physiology at the cellular and circuit levels , we focused on noradrenergic neurons . These neurons express both ATP7A and ATP7B ((Lutsenko et al., 2019; Schmidt et al., 2018; Washington -Hughes et al., 2023) and Fig. 1a), rely on Cu -dependent dopamine-β-hydroxylase (DBH ) for biosynthesis of norepinephrine (NE) (their primary effector molecule) , and control organism -level processes such as adaptive thermogenesis. Adaptive thermogenesis is mediated by brown adipose tissue (BAT) and inducible beige adipocytes within white adipose tissue (WAT). BAT is the major thermogenic organ, whereas beige adipocytes expand thermogenic capacity in response to environmental stimuli (2 –5). NE release from noradrenergic neurons activates the β-adrenergic receptors on adipocytes, triggering lipolysis and inducing uncoupling protein -1 (UCP1 ), which dissipates the mitochondrial proton gradient to generate heat. Cu is required for this pathway as a cofactor for cytochrome c oxidase , which establishes the mitochondrial proton gradient, and for DBH, which converts dopamine to NE. Thus, systemic Cu imbalance (either deficiency or excess ) simultaneously perturbs neuronal signaling, mitochondrial bioenergetics, and peripheral metabolism, making it difficult to disce rn the specific neuronal functions of ATP7A and ATP7B. In this study, we clarified these functions by individually deleting Atp7a or Atp7b in noradrenergic neurons in mice and characterizing biochemical, cellular, and physiological consequences of their deletion. We found that Atp7a activity is essential for neuronal survival , noradrenergic signaling, (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint and adaptive thermogenesis , independent ly of systemic Cu status. These findings reveal a neuronal contribution to thermogenic failure in MD and establish a role for neuronal Cu homeostasis in maintaining energy balance. We also uncovered a previously unrecognized role for Atp7b in supporting thermogenic responses and demonstrated dependence of DBH abundance on cellular Cu levels, especially at the cell periphery . Beyond their relevance to M D and WD, our results have implications for mechanistic studies of other neurodegenerative disorders for which Cu imbalance has been reported, such as Fatal Congenital Copper Deficit, Parkinson’s disease, Alzheimer’s disease, and aging.

Targeted inactivation of Atp7a but not Atp7b alters Cu levels in the locus coeruleus (LC) To investigate the physiological importance of Cu homeostasis in noradrenergic (NE) neurons, we individually deleted Atp7a or Atp7b in Dbh -expressing cells by crossing Atp7aLoxP/LoxP or Atp7bLoxP/LoxP females, respectively, with transgenic males expressing Cre recombinase under the Dbh promoter (Yamaguchi et al., 2018) (Fig.1b). Offspring were genotyped by PCR to verify the presence of the Cre allele and confirm successful recombination ( Fig. S1 ). Both knockouts (Atp7aDbh and Atp7bDbh) were born at expected Mendelian frequencies; neither showed postnatal lethality or the lifespan reduction when compared to their respective LoxP/LoxP controls, indicating that the loss of either Cu transporter in NE neurons does not compromise animal survival under basal conditions. Locus coeruleus (LC) is the brain’s main source of norepinephrine (Schwarz and Luo, 2015) and an anatomically distinct and easily identifiable cluster of NE neurons . Immunostaining of coronal brain sections containing LC confirmed deletion of Atp7a or Atp7b proteins specifically in the NE neurons (Fig. 1c-d). The deletion of either Atp7a or Atp7b did not significantly alter the total Cu content of the brain, as determined by atomic absorption spectroscopy of whole brain homogenates (Fig. 2a). Similarly, the peripheral tissues, such as liver and adipose tissue, had Cu levels similar to those of respective LoxP/LoxP controls in both knockout strains ( Fig. 2b). Thus, systemic Cu balance is unaffected by the deletion of either Atp7a or Atp7b in Dbh -expressing cells, allowing us to examine specific consequences of Cu dis -homeostasis in neurons. The NE neurons constitute only a small fraction of the total brain cell population, and a whole - brain analysis may not detect changes in their Cu content. Consequently, we examined Cu levels specifically within the LC region using laser ablation –inductively coupled plasma -time of flight - mass spectrometry (LA -ICP-TOF-MS). Adjacent sections were used for immunostaining of tyrosine hydroxylase (Th) to identify LC; the Th immunofluorescence images were then co - registered with the metal maps (Fig. 2c and Fig.S2). This analysis revealed a significant reduction of Cu levels in the LC of Atp7aΔDbh mice relative to the age -matched 20 -week-old wild -type controls (Fig. 2d and Table S1). The Cu levels outside of LC (such as medulla oblongata) also showed a mild decrease ( Fig. 2e ), indicating that the loss of Cu in Atp7aΔDbh LC impacts the surrounding medullary region. Broader perturbations in metal homeostasis in the Atp7aΔDbh LC were further highlighted by unexpected decreases in iron (Fe) and zinc (Karczewski et al.) (Fig. 2d and 2e ). In contrast, neither Atp7bDbh LC nor the surrounding areas showed significant changes in copper or other metals ( Fig. 2d and 2e). Dbh protein abundance is reduced in Atp7a ΔDbh and Atp7bΔDbh mouse brains Since the biochemical functions of Atp7a and Atp7b are very similar, we investigated whether the abundance of either transporter changed to compensate for the loss of the other. Immunostaining of Atp7b in Atp7aΔDbh LC reveals no increase in the Atp7b signal; in fact, the intensity of Atp7b staining appeared lower in Atp7aΔDbh cells (Fig. S3 ). Similarly, Atp7a levels were not noticeably (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint increased in Atp7bΔDbh mice ( Fig. 2f -g). By contrast, the abundance of Dbh protein changed significantly. The 20-week-old Atp7aΔDbh whole-brain homogenates and LC both showed marked loss of the Dbh signal compared to control ( Fig. 3 a-d). In young er Atp7aΔDbh animals (4 weeks after birth), Dbh expression was also diminished ( Fig. 3e-f), suggesting that low Dbh abundance is likely a direct result of Cu misbalance during brain development, rather than a secondary long - term change. Unlike the Atp7aΔDbh LC, the Atp7bΔDbh brain sections that include LC showed no difference in Dbh staining compared to the control. Since the Dbh protein is produced in cell bodies and then transported to the cell periphery to synthesize NE in secretory granules, we also measured Dbh levels in the entire brain. In whole -brain homogenates from Atp7bΔDbh animals, Dbh levels were significantly reduced - by about 50% - compared to the age -matched controls ( Fig. 3a -b and Fig.S4). In both Atp7aΔDbh and Atp7bΔDbh mice, the reduction in Dbh levels was greater in whole brain homogenates than in the LC cell bodies, suggesting that Cu imbalance negatively impacts DBH delivery to cell periphery, likely diminishing NE synthesis/neurotransmission. Direct measurements of dopamine (DA) and norepinephrine (NE) confirmed a marked increase in the DA/NE ratio in Atp7aΔDbh brains (Fig. 3g-h), indicative of impaired DA -to-NE conversion. A less dramatic, but statistically significant increase in the DA/NE ratio was observed in Atp7bΔDbh brains (Fig. 3g). Serotonin levels remained unchanged in both Atp7aΔDbh and Atp7bΔDbh animals ( Fig. S5), indicating that Cu imbalance in the NE neurons specifically dysregulates NE/DA homeostasis without broadly affecting monoamine metabolism. Inactivation of Atp7a dysregulates cell adhesion and vesicular trafficking, and increases autophagy markers To better understand the molecular effects of Atp7a and Atp7b deletions and compare these effects, we analyzed the LC proteomes of knockout animals and respective LoxP/LoxP controls (3 animals per group for each of four genotypes). The LC regions, identified by immunostaining for tyrosine hydroxylase (Th), were isolated using laser -capture microdissection and subjected to tandem mass tag (TMT)-labelling and liquid chromatography-mass spectrometry ( Fig.S6). A total of 3,963 proteins were detected in all 12 s amples, and their relative abundances were quantified. The LC with inactivated Atp7a showed a greater number of significantly changed proteins, 131 (1.2-fold change, q < 0.05), compared to the respective control, whereas Atp7bΔDbh LC had 43 proteins changed (1.2 -fold change, q < 0.05) ( Fig. 4a-d and Table S2). In Atp7aΔDbh LC, the most down -regulated proteins included tyrosine hydroxylase, Dbh, and catecholamine transporter Slc18a2 – all required for norepinephrine biosynthesis. We also observed a decrease in proteins involved in RNA synthesis (Rps24, Polr2m, Sf3b6) and lip id and energy balance (Slc25a3, Lactb), suggesting metabolic deficits in the neurons. The largest category of dysregulated proteins included proteins involved in cell adhesion, vesicular trafficking, neuronal guidance, and synapse formation ( Table 1 and T able S2). Significant changes were also observed in proteins involved in inflammatory response (Serpinb1a, Nrlx1, Map3k7) and apoptosis/protein degradation (Slk, Tmem9, Tmem192, Ube2a, Ddrgk1). The list of all significantly changed proteins is included in Table S2. Together, these changes suggested a potential axonal atrophy and /or loss of neurons. To test this, we co -immuno-stained the LC - containing brain sections with antibodies against p62 ( SQSTM1), which promotes the recruitment of ubiquitinated proteins to autophagosomes and tyrosine hydroxylase (Th) Although in the entire LC proteome, SQSTM1 was not significantly changed, in Th -positive neurons of Atp7aΔDbh mice, SQSTM1/p62 signal was strongly increased , consistent with cellular stress and autophagy ( Fig. 4e). Strong increase in the immunostaining of Iba, a marker of activated microglia/macrophages (Fig. 4f-h) further pointed to neuronal degeneration and an increased inflammatory response. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint Inactivation of Atp7b affects the LC proteome differently from Atp7a deletion Changes in the proteome of Atp7bΔDbh LC were markedly different. Not only were fewer proteins changed, but an overlap between the differentially expressed proteins in Atp7aΔDbh and Atp7bΔDbh LC was minimal. Commonly changed proteins include TMEM192 (lysosomal function) (Park et al., 2025), which was down -regulated in both strains, and Scyl1 (Kaeser-Pebernard et al., 2022) (involved in Golgi maintenance and vesicular trafficking), which was commonly upregulated. The abundances of Dbh and Th were not changed in Atp7b KO in agreement with the LC immunostaining data. Also, upregulation of Iba1 levels was not observed ( Fig. 4g-h). Instead, we found a highly significant increase in the abundance of α-synuclein (3-fold, p-value = 0.0017), which was confirmed by immunostaining ( Fig. S7 ), pointing to potential changes in neurotransmission in NE neurons lacking Atp7b. Changes in t he abundances of Scyl1, Dennd4b, Rmdn3, and Mylk further pointed to altered vesicle trafficking and recycling as well as dysregulation of axonal outgrowth (Burman et al., 2008) . Other categories of proteins with changed abundance included metabolic enzymes and proteins involved in immune response. Sqstm1 (Gallagher and Holzbaur, 2023; Tominaga et al., 2017) was downregulated, which coincides with the upregulation of Pzp, a protein that facilitates protein folding (Cater et al., 2019). Taken together, these data suggested that inactivation of Atp7b in NE neurons leads to metabolic adaptation and potentially altered secretory vesicular trafficking, but, unlike Atp7a, this deletion does not cause significant neuronal loss. Atp7aΔDbh and Atp7bΔDbh mice show distinct metabolic changes To evaluate the functional consequences of noradrenergic Atp7a or Atp7b deletion , we first examined the known physiological effects of NE (Smythe et al., 1984; Walters et al., 1997) . Previously, Dbh-/- mice that lack NE were shown to have hypoglycemia and hyperinsulinemia, implicating catecholaminergic circuits in metabolic control (Arnold et al., 2017). Consequently, we first examined glucose homeostasis in our knockouts. At steady -state, Atp7aΔDbh mice showed hypoglycemia, whereas Atp7bΔDbh did not ( Fig. 5a ). Following intraperitoneal glucose administration, Atp7aΔDbh mice had lower blood glucose levels at all time points. Lower levels of blood glucose in Atp7aΔDbh mice suggested either increased glucose clearance or impaired glucose production by the liver. To distinguish between these possibilities, we performed an insulin tolerance test and a pyruvate tolerance test, respectively. Although glucose levels at 30 -, 60-, and 90 minutes post -insulin administration were lower in Atp7aΔDbh animals, the kinetics of glucose clearance was unchanged ( Fig. 5b). In contrast, fo llowing pyruvate injection, Atp7aΔDbh mice showed less glucose at 15, 30, 60, and 90 minutes, compared to control, indicative of impaired hepatic glucose production. Atp7bΔDbh mice's responses to glucose or insulin administration were similar to controls (Fig. 5a ). In the pyruvate tolerance test, Atp7bΔDbh animals initially showed a higher surge of blood glucose than controls, but their glucose levels returned to normal levels over time. Despite metabolic changes, body weight, fat mass, and lean mass in Atp7aΔDbh and Atp7bΔDbh mice were similar to controls (Fig. 5d). Notably, Atp7aΔDbh mice ate more food (hyperphagia) (Fig. 5e), along with a significantly increased oxygen consumption (VO₂) and carbon dioxide production (VCO₂), indicating a higher metabolic rate ( Fig. S8 ). This rise in energy expenditure ( Fig. 5f ) occurred independently of changes in ambulatory or overall physical activity during both light and dark phases (Fig. 5g, h). Therefore, the Atp7a function in NE neurons is essential for NE signaling that regulates glucose balance and energy use. In contrast, Atp7bΔDbh mice exhibited a modest reduction in locomotor activity without corresponding changes in metabolic rate ( Fig. 5h); these differences were not statistically significant. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint Cu misbalance in NE neurons is associated with BAT metabolic quiescence To identify physiological processes in Atp7aΔDbh mice that require increased expenditure of metabolic energy, we examined their adaptive thermogenesis, which is an NE-dependent process (Garside et al., 2022) . Under standard temperature conditions ( ∼22-24°C), both Atp7aΔDbh and Atp7bΔDbh mice displayed a warmth -seeking behavior, characterized by frequent and prolonged burrowing beneath nestlets within their cages (Fig.6a). This behavior was absent in the respective LoxP/LoxP controls, which remained active and exposed ( Fig. 6a ). The behavior to seek insulation suggest ed impaired thermoregulatory capacity or heightened cold sensitivity in Atp7aΔDbh and Atp7bΔDbh mice, even at ambient temperatures. To evaluate thermoregulatory capacity, we examined the major thermogenic tissue, BAT. We also analyzed WAT (including anatomically distinct inguinal (iWAT) and gonadal (gWAT) depots), which can adopt thermogenic characteristics under certain physiological stimuli (Finlin et al., 2018; Huang et al., 2017) . Histological analysis of BAT from Atp7aΔDbh mice revealed enlarged lipid droplets, suggesting impaired lipid utilization ( Fig. 6b). A milder increase in lipid droplet size was also observed in Atp7bΔDbh mice (Fig. 6c). The mRNA levels of thermogenic markers PGC1α and UCP1 were significantly lower in BAT of Atp7aΔDbh animals, consistent with the suppressed NE signaling to β-adrenergic receptors and decreased thermogenic capacity ( Fig. 6d& e ). In Atp7bΔDbh BAT, the PGC1α mRNA levels were significantly lower than in control tissue, whereas the UCP1 transcript was unchanged, suggesting that some thermogenic capacity was preserved in these mice. In agreement with the histological findings, BAT from Atp7aΔDbh mice weighed significantly more than BAT of wild -type controls, whereas Atp7bΔDbh mice showed only a mild, non-significant increase in the BAT weight ( Fig. S9). Despite impaired lipid mobilization in BAT, the total body weight, fat mass, and lean mass of knockout mice were comparable to controls ( Fig. 5d), prompting us to investigate whether WAT depots underwent compensatory remodeling (beiging). Inguinal and gonadal WAT from Atp7aΔDbh mice showed multilocular, smaller lipid droplet size when compared to controls, resulting in darker eosinophilic cytoplasm in H&E staining. These histological findings suggested beiging of WAT, a morphological feature consistent with a compensatory thermogenic activation (Fig. 6f-i). A similar, although less robust phenotype was observed in Atp7bΔDbh mice (Fig. 6f-i). Atp7aΔDbh and Atp7bΔDbh mice fail to maintain body temperature during acute cold exposure To directly test how well Atp7aΔDbh and Atp7bΔDbh mice maintain their core body temperature, we first acclimated them to thermoneutral conditions for 10 days and then exposed mice to cold (6 °C). In response to cold, the core body temperature of both Atp7aΔDbh animals and respective controls declines ( Fig. 7a ). After the initial drop, the control mice maintained their body temperature. In contrast, the body temperature of Atp7aΔDbh mice dropped below the critical threshold (28°C) by 5 hours, which terminated the experiment. The body weights of control and Atp7aΔDbh mice were similar either pre -cold or post -cold ( Fig. 7b). However, the Atp7aΔDbh BAT was significantly heavier after cold exposure than the BAT of Atp7aLoxP/LoxP controls, suggesting that Atp7aΔDbh BAT could not utilize fat stores (Fig. 7c and Fig. S10). In support of impaired lipid mobilization, Atp7aΔDbh BAT exhibited enlarged lipid droplets and defective lipolysis ( Fig. 7h–i). BAT dysfunction in Atp7aΔDbh mice was apparent even under prolonged thermoneutral conditions (30 °C, 10 days) (Fig. 7f-g and Fig. S11), pointing to a severe deficit in NE signaling. In response to cold exposure, Atp7bΔDbh animals also exhibited a significant decrease in core body temperature at 2h and 5h. However, they survived and at later time points were able to maintain (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint a core body temperature comparable to that of the Atp7bLoxP/LoxP controls ( Fig. 8a). Their weight at thermoneutrality trended to be lower than in controls, but the pre - and post -exposure body weights showed no significant differences ( Fig. 8b). After cold exposure, Atp7bΔDbh BAT weighed more than control BAT, whereas other adipose depots remained unchanged ( Fig. 8c- e and Fig. S10). Taken together, these findings identify Atp7a as a key player in the noradrenergic regulation of adaptive thermogenesis, with Atp7b playing a more limited, likely fine -tuning role.

This study uncovered the specific roles of Atp7a and Atp7b in the structural and functional integrity of NE neurons. Atp7a is essential for the survival and function of these neurons, enabling NE biosynthesis, maintaining local metal homeostasis, and coordinating thermogenic and metabolic responses. In contrast, Atp7b plays a more subtle modulatory role, reflected in its milder impact on the NE:DA ratio and thermogenesis. However, the clear thermogenic phenotype in Atp7bΔDbh mice, even in the presence of Atp7a, underscore s a non-redundant, specialized role for Atp7b within catecholaminergic circuits . In both knockout strains, loss of one Cu transporter fails to induce compensatory upregulation of the other, and functional substitution does not occur. Notably, both Atp7aΔDbh and Atp7bΔDbh mice exhibit decreased Dbh protein abundance, indicating that DBH is vulnerable to Cu dyshomeostasis. Our results provide a mechanistic insight into MD patients’ often -reported hypothermia (Fujisawa et al., 2022) . We demonstrate that the loss of ATP7A function in NE neurons alone impairs thermoregulation. By linking Cu -dependent NE biosynthesis to systemic energy balance, our findings reframe MD hypothermia as a circuit -level pathology that occurs even when Cu status of peripheral tissues is unaffected (or restored). Additionally, our results highlight potential contributions of noradrenergic Cu misbalance to metabolic and neurologic phenotypes in neurodegenerative disorders involving Cu and/or catecholaminergic d ysfunction. Our findings may help elucidate the role of LC neurons in driving metabolic phenotypes in Wilson disease, providing a framework to dissect central versus peripheral contributions to copper -related metabolic dysfunction. Thermogenic neural circuitry comprises three components: thermosensory afferents, central processing regions, and sympathetic efferent outputs. Because both Atp7aΔDbh and Atp7bΔDbh mice exhibit warmth -seeking behavior associated with hypothermia, their thermosensory inputs appear intact. In contrast, both central and sympathetic efferent outputs are malfunctioning, especially in Atp7aΔDbh mice. This is evidenced by hyperphagia, indicative of abnormal NE signaling to hypothalamic nuclei (Sayar-Atasoy et al., 2023; Sciolino et al., 2022) , neuronal loss, and proteomic changes. High fat content in the BAT of both knockout strains before and after cold exposure is consistent with a deficit in NE signaling by sympathetic neurons. Taken together, our data identify a Cu -modulated noradrenergic pathway linking brainstem activity to peripheral metabolic responses. Within this framework, Cu emerges as an essential determinant of brain - body communication. This integrative paradigm illustrates that trace metal transport can directly shape neural net work function and systemic adaptation, with broad implications for metabolic regulation and neurodegenerative disease pathogenesis.

Animal studies: All animal experiments were conducted based on ARRIVE guidelines and followed protocols approved by the Johns Hopkins University Animal Care and Use Committee (ACUC; Protocol No. M023M337). Mice were housed in the Johns Hopkins Animal Care Facilities under specific pathogen-free (SPF) conditions with a controlled temperature of 22 -24 °C, relative humidity of (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint 40–60%, and a 14-hour light/10-hour dark cycle. Animals were group-housed (up to five per cage) in individually ventilated cages containing corn cob bedding or paper nesting material. Standard rodent chow (Teklad 2018 or Picolab5V75) and water were available ad libitum. Static cages were changed once per week, while ventilated cages were changed every two weeks. All animals were monitored daily by trained animal care staff for their general health, grooming, and activity levels . All animals used in this stu dy were on a C57BL/6J (B6) genetic background. Atp7aLoxP/LoxP mice and Atp7bLoxP/LoxP mice were described previously in (Xiao et al., 2018) and (Muchenditsi et al., 2017), respectively. To generate mice with a targeted deletion of Atp7a or Atp7b in noradrenergic cells, female Atp7aLoxP/LoxP or Atp7bLoxP/LoxP animals were crossed with males expressing Cre recombinase under the control of the dopamine β -hydroxylase ( Dbh) promoter (Dbh -Cre; Jackson Laboratory strain B6.Cg-Dbhtm3.2(cre)Pjen/J). Genotypes were confirmed by PCR analysis of genomic DNA isolated from tail biopsies for the presence of Cre, Atp7aLoxP/LoxP, or Atp7bLoxP/LoxP alleles (Fig. S1). Primer sequences are listed in Table S3. All experiments were performed using age - and sex- matched 4- to 20-week-old adult male mice. Age -matched littermates lacking Cre served as the control group. Wild -type, Atp7aLoxP/LoxP or Atp7bLoxP/LoxP age-matched male mice showed no significant differences in Atp7a, Atp7b, or Dbh expression in the locus coeruleus, nor in tissue Cu levels. Therefore, wild -type and LoxP/LoxP (Atp7aLoxP/LoxP, or Atp7bLoxP/LoxP) animals were used interchangeably as controls in subsequent experiments unless otherwise specified. Only animals confirmed by PCR to have the correct genotype and within the designated age range were included in the study. No animals were excluded after th e inclusion criteria were met, unless humane endpoints were reached. Allocation was based on genotype, and blinding was applied during data collection and analysis where possible. Copper measurements Cu levels were measured from the freshly dissected mouse brain using Atomic Absorption Spectroscopy (AAS) (Perkin Elmer Pinnacle 900T). Approximately 30mg of tissue washed and perfused in 1X Phosphate Buffered Solution (PBS) was digested in 200 μl of trace element-grade nitric acid (HNO3) (Fisher Scientific, USA) for 2 hours at 95°C in metal -free 15 ml tubes (Labcon). The digested sample was further diluted using MilliQ water to adjust the nitric acid concentration to less than 0.2%. Metal content was quant ified using calibration standards 20 ppb and 10, 20, 50, and 100 ppb and divided by tissue weight. Enzyme-linked immunosorbent assay (ELISA) Serum norepinephrine levels were measured using NA/NE(Noradrenaline/Norepinephrine) ELISA Kit (Elabscience; E -EL-0047) following the manufacturer’s protocol. The color intensity was measured at 450 nm using a BMG Labtech Microplate reader. The concentratio n of norepinephrine in the samples was determined by comparing the OD of the samples to the standard curve. A reference standard curve was prepared using concentrations of 0, 0.31, 0.63, 1.25, 2.5, 5.0, 10.0, and 20.0 ng/mL Catecholamine analysis using mass spectrometry Serum and brain concentrations of dopamine, norepinephrine, epinephrine, and serotonin were quantified by liquid chromatography -mass-spectrometry (LC –MS) following protein precipitation with methanol. For serum, 50 µL was extracted with 150 µL methanol; b rain tissue was homogenized in 1 mL methanol with ceramic beads, and both were spiked with 5 µL of 1 µg mL ⁻¹ dopamine-d₄ as internal standard. Supernatants were centrifuged (4 °C, 45 min), dried under vacuum, and reconstituted in 50 µL water. Calibration standards (0.2–200 ng mL⁻¹) were prepared from authentic stocks. LC separation was performed on an Agilent 1290 Infinity II UHPLC with a (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint Waters Acquity BEH C18 column (2.1 × 100 mm, 1.7 µm) at 45 °C, using water with 0.1% formic acid (A) and methanol (B) at 0.40 mL min ⁻¹, with a gradient of 2 –98% B over 4 min. Comprehensive Laboratory Animal Monitoring System (CLAMS) analysis Whole-body energy expenditure and metabolic parameters were assessed by indirect calorimetry using the Comprehensive Lab Animal Monitoring System (CLAMS; Columbus Instruments) at the Center for Metabolism and Obesity Research, Johns Hopkins University (Bal timore, MD, USA). The measurements included oxygen consumption (VO ₂), carbon dioxide production (VCO ₂), respiratory exchange ratio (RER), and energy expenditure (EE). Food and water intake, as well as physical activity, were also recorded. Age-matched adult male mice (16 weeks old) (Wild -type; n=8, Atp7aΔDbh; n=12, Atp7bΔDbh; n=4) were individually housed in chambers under standard environmental conditions (22 –24°C; 12 - hour light/dark cycle) with ad libitum access to food and water. Mice were acclimated to the chambers for 24 hrs before data collection. Measurements were obta ined over 48 hrs and analyzed across light and dark phases. Ambulatory activity was quantified based on consecutive X -axis infrared beam breaks. Energy expenditure was calculated using the Weir equation: EE (kcal/day) = VO2× [3.815 + (1.232×RER)]. To account for variation in body composition, energy expenditure, and metabolic parameters were normalized to both total and lean body mass (Sarver et al., 2020) . Glucose Tolerance Test (GTT) To assess systemic glucose handling, male mice ( Atp7aΔDbh, n=12; wild-type littermates, n=8; Atp7bΔDbh, n=4) were fasted for 6 h before testing. D -glucose (1 mg/g body weight) was administered intraperitoneally, and blood glucose concentrations were determined at baseline (0 min) and at 15-, 30-, 45-, 60-, 90-, and 120-min post-injection using a handheld g lucometer (NovaMax glucometer). For each time point, blood was obtained from awake mice by tail snipping on Novamax glucose t est strips. Insulin Tolerance Test (ITT) To evaluate insulin sensitivity, the same cohorts of male mice ( Atp7aΔDbh, n=12; wild-type, n=8; Atp7bΔDbh, n=4) were fasted for 2 h before testing. Human recombinant insulin (1.0 U/g body weight; intraperitoneal) was administered, and blood glucose was measured at 0, 15, 30, 45, 60, 90, and 120 min using Novamax glucometer using Novamax glucose test strips. T he blood was collected onto the strips by tail snipping on awake mice. Pyruvate Tolerance Test (PTT): To assess hepatic gluconeogenic capacity, male mice ( Atp7aΔDbh, n = 12; wild-type, n = 8; Atp7bΔDbh, n = 4) were fasted for 6 h, followed by intraperitoneal injection of sodium pyruvate (1 – 2 mg/g body weight). Blood glucose concentrations were monitored at baseline and 15, 30, 45, 60, 90, and 120-min post-injection using an Accu -Chek glucometer. Retro-orbital sampling was used for blood collection. Thermoneutral Housing To assess metabolic responses under thermoneutral conditions, 10-week-old male mice, Atp7aFlox(n=5) and Atp7aΔDbh (n=5) were housed at 30°C in a static cage with a filtered top in a temperature-controlled environmental chamber with a 12 -hour light/dark cycle and ad libitum access to food and water. Mice were acclimated for 10 days before physiological or metabolic measurements to ensure full adaptation to thermoneutrality, the ambient temperature at which thermogenic demand is minimize d. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint Cold Tolerance Test Cold-induced thermogenesis was evaluated by exposing mice to acute cold stress following acclimatization in thermoneutral conditions. Age-matched (10 weeks) adult male mice Atp7aFlox (5), Atp7bFlox (n=6), Atp7aΔDbh (n=5), and Atp7bΔDbh (n=4) were individually housed at 6°C in cages without nesting material and with minimal bedding. Access to food was restricted during acute cold exposure. Core body temperature was measured using a rectal probe (Physitemp RET-3 Rectal Probe; Cat. No. NC9713069) at baseline (room temperature) and at 1 -hour time intervals for up to 6 hours following cold exposure. Mice had free access to water throughout the experiment. The cold exposure was terminated earlier if a mouse’s core temperature dropped below 30°C, at which point the animal was euthanized by cervical dislocation to prevent hypothermic distress. Following cold exposure, animals were sacrificed, and tissues were harvested for downstream analyses. Haematoxylin and Eosin (H&E) staining and histology analysis The brown adipose tissue (BAT), inguinal white adipose tissue (iWAT), and gonadal adipose tissue (gWAT) isolated from mice were fixed overnight in 10% formalin and embedded in paraffin wax as previously described(Lee et al., 2012). Sections (8 μm) were stained with hematoxylin and eosin at the Johns Hopkins University Reference Histology Core, Baltimore, MD, USA. Images were acquired using an Olympus light color microscope (Johns Hopkins University, Microscope Facility, Baltimore, MD, USA). ImageJ was used for the quantification of adipocyte diameters from H&E-stained sections of iWAT, gWAT, and BAT collected from 4 mice in each group (Wild type, Atp7aFlox, Atp7bFlox, Atp7aΔDbh, and Atp7bΔDbh). Membrane Protein Extraction and Immunoblotting Whole mouse brains (~450 mg) were homogenized in ice -cold homogenization buffer (3 ml) containing 0.25 M sucrose, 10 mM Tris -HCl (pH 7.5), and 1 mM EDTA, supplemented with a protease inhibitor cocktail (Roche, Basel, Switzerland) and 30 μl of each phosphat ase inhibitor cocktails (Phosphatase inhibitor cocktail 2; Cat. No. P5726 -1ML and Phosphatase inhibitor cocktail 3, Cat. No. P0044 -1ML; Sigma -Aldrich). Homogenization was performed using a glass Dounce homogenizer with 50 strokes on ice. The homogenate was centrifuged at 100,000 × g for 20 minutes at 4°C using a Beckman Coulter benchtop ultracentrifuge. The pellet was resuspended in 100 μl homogenization buffer containing 0.1% Triton X -100. Protein concentrations were determined using the BCA assay (Pierce BCA Kit; Cat. No. 23227; Thermo Fisher Scientific) following the manufacturer’s instructions. Equal amounts of protein (15 µg) were resolved by 10% SDS-PAGE and transferred to PVDF membranes using semi-dry transfer in Tris- Buffered Saline transfer buffer f or 10 min (Bio -Rad, California, USA). Immunoblotting was performed using the appropriate primary and secondary antibodies ( Table S4 ). The PVDF membrane was blocked using blocking buffer (5% Milk in 1X TBST). The primary antibodies were diluted in 3% Milk in 1X TBST and incubated overnight at 4°C. The secondary antibodies were diluted in 3% Milk in 1X TBST and incubated for 1 h at room temperature. Relative expression levels of target proteins were quantified by densitometry using ImageJ software and normalized to the corresponding loading control. RNA Extraction and Quantitative PCR Total RNA was extracted from fresh -frozen BAT, iWAT, and gWAT using TRIzol reagent (Life Technologies, Cat. No. 15596026), following the manufacturer’s protocol. RNA concentration and purity were assessed by 260/280 nm ratio using a BMG Labtech LVi plate r eader. Reverse transcription was performed using the HiScript® IV RT supermix for qPCR with gDNA wiper (Vazyme, Catalog No. R423) to synthesize cDNA. Quantitative PCR (qPCR) was performed using the SupRealQ Ultra -HSYBR qPCR Master Mix (Vazyme) on a QuantSt udio 6 Flex (Applied (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint Biosystems) real -time PCR system. Target gene’s transcript levels were normalized to Gapdh mRNA, and relative transcript abundance was calculated using the comparative Ct ( ΔΔCt) method(Livak and Schmittgen, 2001) . Laser Ablation -Inductively Coupled Plasma -Time of Flight -Mass Spectrometry (LA-ICP- TOF-MS). Spatial distribution of metals in 4% PFA fixed coronal brain sections was determined using LA-ICP-TOF-MS. Fresh -frozen brains from 20 -week-old male mice were cryosectioned at 10 µm and mounted on Superfrost PLUS slides. Frozen sections (prepared as describ ed below) were air-dried and ablated using an ImageBIO 266 laser ablation system (266 nm laser, 10 µm spot size, 200 Hz, 1.8–2.3 J/cm²) coupled to a TOFWERK ICPTOF S2 mass spectrometer. Helium was used as the carrier gas, and daily tuning was performed with NIST SRM612 glass. Elemental images for 63Cu, 56Fe, and 66Zn were acquired using TofPilot, calibrated against gelatin standards in Iolite (v4.8.6), and co -registered with confocal images to define regions of interest in the locus coeruleus. Immunofluorescence and confocal microscopy Mice were anesthetized with 5% isoflurane and transcardially perfused with 1× sterile phosphate - buffered saline (PBS; 0.1 M, pH 7.4). Following perfusion, animals were sacrificed by cervical dislocation. Brains and adrenal glands were promptly dissected an d post -fixed in 4% (w/v) paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB) for 12 hours at 4 °C. Tissues were placed in 30% (w/v) sucrose prepared in 0.1 M PB for a minimum of 48 hours until they sank. Subsequently, tissues were embedded in Optimal Cut ting Temperature (OCT) compound and stored at –80 °C until sectioning. Frozen tissues were sectioned at a thickness of 10 µm using a Leica cryostat (Leica CM3050 S) and mounted on Superfrost Plus slides (Fisherbrand; Catalog No. 1255015) for immunofluoresc ence. Sections were blocked and permeabilized in a buffer containing 2% bovine serum albumin (BSA), 0.2% saponin, and 15% fetal bovine serum (FBS; HiMedia) for 1 hour at room temperature. Primary antibodies were applied overnight at 4 °C in blocking buffer, using the following dilutions: Anti -Dopamine β-Hydroxylase (1:500) Anti -Tyrosine Hydroxylase (1:300) Anti -ATP7B (1:200) Anti-ATP7A (1:50) Anti-Iba1 (1:300) Anti -GFAP (1:200). After washing, sections were incubated with the appropriate secondary antibodie s conjugated to Alexa Fluor 488, Alexa Fluor 568, or Alexa Fluor 647 (1:500; Goat anti -Rabbit or Donkey anti - Sheep) for 1 hour at room temperature in the dark. Nuclei were counterstained using ProLong ™ Gold Antifade Mountant with DAPI (Thermo Fisher Scient ific), and coverslips were applied. Images were acquired using a Zeiss LSM 800 confocal microscope and processed using ImageJ (NIH) software for analysis and visualization. Laser capture microdissection Mouse brain tissue sections were prepared from TH -immunostained specimens sectioned at 30 μm thickness using a microtome and mounted on polyethylene naphthalate membrane glass slides (Carl Zeiss Microscopy GmbH, Jena, Germany). Sections were placed into th e dish holder of the microscope, and laser capture microdissection (LCM) was performed using the PALM MicroBeam Ultraviolet Laser Microdissection system on the AxioObserver Z.1 system. TH-positive locus coeruleus regions were identified and micro -dissected from both left and right hemispheres of each tissue section at 10× magnification while keeping laser power to a minimum. A total of four locus coeruleus samples were collected per anim al (bilateral dissection from two sections per animal). Isolated tissue samples were directly collected in the cap of collection tubes (AdhesiveCap 200 opaque). Samples in collection tubes were stored at -80°C until further processing. Sample Processing and Protein Digestion (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint To release tissue samples from the adhesive caps, 30 μL of lysis buffer containing 1% n-dodecyl- β-D-maltoside (DDM), 600 mM guanidine HCl, 150 mM NaCl, 10 mM HEPES (pH 7.4), and 15 mM TCEP was added to each collection tube. Samples were resuspended and transferred to new tubes. Sonication was performed using a Branson Sonifier 250 (Branson Ultrasonics), followed by centrifugation at 14,000 × g for 1 min. For decrosslinking, samples were incubated at 95°C for 30 min, then centrifuged again at 14,000 × g for 1 min. Proteins were reduced and alkylated with 10 mM Tris(2 -carboxyethyl) phosphine hydrochloride and 40 mM chloroacetamide at room temperature (22 –25°C) for 1 h. The proteins were then digested with Lys-C (Lysyl endopeptidase MS grade; Fujifilm Wako Pure Chemic al Industries Co, Ltd) at a final concentration of 10 ng/ μL and incubated at 37°C for 3 h. Subsequently, trypsin digestion was conducted by diluting the guanidine HCl concentration from 600 mM to 200 mM with 50 mM TEABC buffer, adding trypsin (sequencing grade modified trypsin; Promega) at a final concentration of 10 ng/ μL, and incubating at 37°C overnight (15 –18 h). TMT Labeling and Fractionation The resulting peptides were desalted using C18 StageTips (3M Empore; 3M) and labeled with 18-plex TMT reagents according to the manufacturer's instructions (Thermo Fisher Scientific). The labeling reaction was performed at room temperature for 1 h, followe d by quenching with 1/10 volume of 1 M Tris–HCl (pH 8.0). The labeled peptides were pooled and pre fractionated into 6 fractions using strong cation exchange (SCX) Stage-Tips (3M Empore) with the following elution conditions: fraction 1 (50 mM ammonium ace tate, 20% ACN, 0.5% FA), fraction 2 (75 mM ammonium acetate, 20% ACN, 0.5% FA), fraction 3 (125 mM ammonium acetate, 20% ACN, 0.5% FA), fraction 4 (200 mM ammonium acetate, 20% ACN, 0.5% FA), fraction 5 (300 mM ammonium acetate, 20% ACN, 0.5% FA), and frac tion 6 (80% ACN, 5% ammonium hydroxide). Subsequently, the fractionated samples were vacuum dried using a SpeedVac (Thermo Fisher Scientific) and stored at −80°C until use. Mass Spectrometry analysis of proteomes Mass spectrometry analysis was performed as previously described with minor modifications (22). Peptide samples were analyzed on an Orbitrap Fusion Lumos Tribrid Mass Spectrometer (Thermo Fisher Scientific) coupled to an Ultimate 3000 RSLCnano nanoflow liq uid chromatography system (Thermo Fisher Scientific). Each fraction was reconstituted in 16 μL of 0.1% formic acid (FA), and 15 μL was injected onto a trap column (Acclaim PepMap 100, C18, 5 μm, 100 μm × 2 cm, nanoViper; Thermo Fisher Scientific) at a flow rate of 8 μL/min. Peptides were separated on an analytical column (Easy -Spray PepMap RSLC C18, 2 μm, 75 μm × 50 cm; Thermo Fisher Scientific) using a linear gradient of solvent B (0.1% FA in 95% acetonitrile) at 0.3 μL/min. The column was interfaced with an EASY -Spray ion source operated at ~2.0 kV. Chromatographic separation was carried out over 120 min. Protein identification and quantification were performed using Proteome Discoverer (version 3.2.0.450; Thermo Fisher Scientific). Statistical analysis Statistical analysis was conducted in Perseus (version 1.6.0.7) (Tyanova et al., 2016) . Data normalization was achieved by dividing reporter ion intensities by median protein values, followed by log2 transformation and median centering across samples. Differential protein expressions were assessed by Student’s two -sample t -test, with protei ns showing q < 0.05 considered significantly different. For volcano plot generation, q -values were calculated using Significance Analysis of Microarrays (SAM) with permutation -based false discovery rate estimation and an S0 parameter of 0.1 (Tusher et al., 2001). Principal component analysis (PCA) was performed using (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint MetaboAnalyst (Pang et al., 2022). Functional enrichment of differentially expressed proteins was analyzed with Enrichr (Kuleshov et al., 2016) and Ingenuity Pathway Analysis (ThermoFisher).

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Copper regulates rest -activity cycles through the locus coeruleus -norepinephrine system. Nat Chem Biol 14:655-663. Yamaguchi, H., F.W. Hopf, S.B. Li, and L. de Lecea. 2018. In vivo cell type -specific CRISPR knockdown of dopamine beta hydroxylase reduces locus coeruleus evoked wakefulness. Nat Commun 9:5211. Acknowledgments We thank Dr. Martina Ralle (OHSU) for metal analysis, Dr. Betty Eipper for providing high -quality antibody, Mr. Benjamin Devenney for assistance with the initial animal work, and Dr. Dwight Bergles for helpful advice. CLAMS studies were made possible by t he Johns Hopkins IBBS Core Coins award to SL. Funding: This work was supported by the National Institute of Health grant R01NS134958 to SL. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint Author contributions: Conceptualization: SR and SL Methodology: KM, AW, AC, AT, MP, CT, KG Investigation: SR, YW, NS, KK, AM, SA, LTH Visualization: SR, YW, NS, AC, SA, KK Supervision: SL, CHN, AK, TO’H Writing—original draft: SR, YW, NS, KM, SL Writing—review & editing: SR, SA, AK, MP, TO’H, KG, SL Competing interests: None Data and materials availability: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD069564 and 10.6019/PXD069564 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint Figure 1. Targeted inactivation of Atp7a and Atp7b in noradrenergic neurons (a)The schematic of predicted functions of the ATP -driven Cu transporters in noradrenergic neurons. ATP7A is located in the trans -Golgi network and delivers Cu cofactor to DBH; ATP7B is in cytoplasmic vesicles, where it sequesters Cu from the cytosol. Cu -bound DBH is located in secretory granules where it converts dopamine (DA) to norepinephrine (NE), which is then released upon neuronal activation. ( b) Strategy for generating Atp7aΔDbh and Atp7bΔDbh mice: Atp7aLoxP/LoxP or Atp7bLoxP/LoxP female mice were crossed with male mice expressing Cre -recombinase under the DBH promoter ( DBH-Cre mice) for targeted deletion in noradrenergic neurons. ( c,d) Coronal brain sections from wild -type control and Atp7aΔDbh (c) or Atp7bΔDbh mice (d) were co-immunostained for tyrosine hydroxylase (Th) (Red), a marker of noradrenergic neurons, and either Atp7a (Green) or Atp7b (Green). In Atp7aΔDbh and Atp7bΔDbh mice, the respective transporter signal was undetectable in Th -positive neurons (outlined by dotted line), indicating successful deletion within the noradrenergic cell population. 3 coronal brain sections for n=3; animals were analyzed per genotype. b d a c Atp7a Dbh Atp7b Dbh Atp7b Dbh Atp7a Dbh (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint Figure 2: The effects of Atp7a and Atp7b deletion on Cu levels in locus coeruleus (a) Analysis of Cu content in whole brain homogenates by atomic absorption spectroscopy found no difference for the age -matched 20 -week-old wild-type, Atp7aΔDbh, and Atp7bΔDbh mice (b) Cu levels in peripheral tissues (liver, adipose) are unchanged across the genotypes. ( c) Representative coronal brain sections analyzed by laser ablation inductively coupled plasma mass spectrometry (LA -ICP- MS) are shown, with metal distribution maps overlaid on tyrosine hydroxylase immunofluorescence confocal images (TH is gree n, nuclei are blue). Less intense Cu signal is apparent in the Th -positive region. (d) Quantitation of LA -ICP-MS signals in coronal brain sections reveals reduced Cu, Fe, and Zn levels in the locus coeruleus of Atp7a ΔDbh mice compared to wild -type controls. ( e) Metal levels in the medulla oblongata (ROI) (indicated by white dotted lines) are compared across wild -type, Atp7aΔDbh, and Atp7bΔDbh mice. (f) Immunostaining for Atp7b (red) in noradrenergic LC neurons (TH, green; LC outlined by the white dotted line) shows reduced Atp7b signal in Atp7aΔDbh mice compared to wild type (Supplementary figure S2) ( g) Immunostaining for Atp7a (red) in noradrenergic LC neurons (TH, green; LC outlined by white dotted line) shows no change in Atp7bΔDbh mice compared to wild type. In all experiments, 3 -4 mice were used for each genotype, ns = not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. a b d c e f g Wild type (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint Figure 3. Inactivation of Atp7a or Atp7b in NE neurons reduces Dbh expression and alters catecholamine metabolism (a) Representative immunoblots of the whole -brain homogenates show markedly reduced Dbh expression in 20-week-old Atp7aΔDbh compared to Atp7aLoxP/LoxP (Atp7aFlox) and wild-type controls. Additional replicates are shown in the supplementary figure S11. ( b) Densitometry of DBH signal normalized to Na+/K+ ATPase confirms significantly reduced Dbh in 20-week-old Atp7aΔDbh brains. (c) Representative coronal brain sections (10 μm) from 20-week-old mice immunostained for Dbh (green) and tyrosine hydroxylase (Th, red) highlight noradrenergic neurons in the locus coeruleus (LC); Atp7aΔDbh brains display marked loss of Dbh signal compared to controls. ( d) Dbh-positive neurons were normalized to the total number of TH -stained neurons in the LC. Three coronal brain sections from n=3 animals per genotype were analyzed. ( e) Immunoblots at 4 weeks similarly show reduced Dbh expression in both Atp7aΔDbh and Atp7bΔDbh. (f) Densitometry of the Dbh signal normalized to Na +/K+ ATPase confirms significantly reduced Dbh in 4-week-old Atp7aΔDbh and Atp7bΔDbh brains. LC-MS metabolite analysis of whole-brain homogenates reveals a ( g) decreased norepinephrine (NE) and ( h) increased dopamine (DA)/NE ratio in Atp7aΔDbh mice relative to Atp7aFlox controls (20 weeks, n=5). ( i) increased DA/NE ratio in Atp7bΔDbh brain homogenates relative to Atp7bFlox controls (20 weeks, n = 3). Data are mean ± SEM. Statistical analysis: unpaired two -tailed Student’s t -test with Welch’s correction; *p < 0.05, **p < 0.01. Data points for Atp7aFlox, Atp7bFlox, Atp7aΔDbh, and Atp7bΔDbh are shown in black, blue, orange, and green, respectively. 20 weeksa b Atp7a DbhAtp7b Dbh Wild type c d e f g h i 4 week (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint Figure 4. Loss of Atp7a but not Atp7b induces autophagic and neuroinflammatory responses in locus coeruleus. (a, b) Volcano plots showing differentially expressed proteins in Atp7aΔDbh (a) and Atp7bΔDbh (b) laser- dissected locus coeruleus compared to the respective LoxP/LoxP controls. (c, d) Principal component analysis of proteomic datasets demonstrates clear segregation between mutant and control samples for Atp7aΔDbh (c) and Atp7bΔDbh (d). (e) Immunofluorescence staining of coronal brain sections demonstrates accumulation of the autophagy adaptor p62 (red) in Atp7aΔDbh neurons, colocalizing with tyrosine hydroxylase (TH, green). (f) Enhanced microglial activation in Atp7aΔDbh LC, indicated by increased Iba1 immunoreactivity (red). ( g) Iba1 staining in Atp7bΔDbh LC is similar to controls. ( h) Quantification of Iba1+ cells in the LC (n = 3 mice per genotype). Data are mean ± SEM. Statistical analysis: unpaired two - tailed Student’s t -test with Wel ch’s correction; *p < 0.05, **p < 0.01. Data points for Atp7aFlox, Atp7bFlox, Atp7aΔDbh, and Atp7bΔDbh are shown in black, blue, orange, and green, respectively. Wild typeDbh Atp7a a b e f g h c d Atp7bFlox Atp7a Dbh Atp7a Flox Atp7b Dbh Atp7a Dbh Atp7a Dbh Atp7b Dbh (Atp7a Dbh/Atp7aflox) (Atp7b Dbh/Atp7bflox) Atp7b DbhAtp7a Dbh (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint Figure 5: Altered glucose metabolism, enhanced energy expenditure, and food intake in Atp7aΔDbh mice. (a) Glucose tolerance test (GTT) shows reduced blood glucose levels in Atp7aΔDbh mice compared to the wild-type controls. ( b) Insulin tolerance test (ITT) shows that Atp7aΔDbh mice respond to insulin and remain hypoglycemic. ( c) The pyruvate tolerance test (PTT) demonstrates impaired gluconeogenesis in Atp7aΔDbh mice. (d) Body weight and composition (including fat and lean mass percentages) showed no significant differences between genotypes. (e) Daily food intake increased in Atp7aΔDbh mice. (f) Indirect calorimetric analysis reveals that energy expenditure (normalized to lean mass or body weight) increases in Atp7aΔDbh mice during light, dark, and whole -day (total) periods. ( g) Respiratory exchange ratio (RER) shows no significant difference between the genotypes. ( h) Ambulatory and total activity counts over light, dark, and whole -day periods show no significant differences between groups. Data are presented as mean ± SEM; p < 0.05; p < 0.01; p < 0.001; p < 0.0001; ns, not significant (n=5 -12 mice per genotype) a b c d e f hg GTT ITT PTT (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint Figure 6: Altered non-shivering thermogenesis and adipose tissue morphology and thermogenic gene expression in Atp7aΔDbh and Atp7bΔDbh (a) Images illustrating the warmth -seeking behaviour of Atp7aΔDbh and Atp7bΔDbh mice compared to the Atp7aFlox and Atp7bFlox animals. (b) Hematoxylin and eosin (H&E) -stained sections of brown adipose tissue (BAT) show enlarged adipocytes in Atp7aΔDbh and Atp7bΔDbh mice relative to the wild type. (c) Quantification of BAT adipocyte size confirms a significant increase in adipocyte size in both mutants. (d –e) Relative mRNA expression of thermogenesis -related genes in BAT: ( d) PGC1α and (e) UCP1 are reduced in Atp7aΔDbh compared to wild type. ( f) H&E-stained sections of inguinal white adipose tissue (iWAT) reveal enlarged adipocytes in Atp7aΔDbh. (g) Quantification of iWAT adipocyte size demonstrates significantly increased adipocyte size in Atp7aΔDbh mice compared to wild type. (h) H&E-stained sections of gonadal white adipose tissue (gWAT). ( i) Quantification of gWAT adipocyte size shows significantly increased adipocyte size in Atp7aΔDbh mice compared to wild type. Data represent mean ± SEM. Statistical analysis: unpaired Student’s t -test; ns = not significant; *p < 0.05, **p < 0.01, ****p < 0.0001. PGC1 , BAT UCP1, BAT a c d e g h i * (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint Figure 7: Atp7aΔDbh animals exhibit severe hypothermia under acute cold stress (a) Twelve-week-old Atp7aΔDbh male mice and Atp7aFlox controls (n = 5 -6) were exposed to acute cold stress (6°C) after prolonged thermoneutral housing (30°C for 10 days). Unlike the age -matched Atp7aFlox, Atp7aΔDbh mice were unable to maintain core body temperature (>30°C). ( b) The body weight of Atp7aΔDbh and Atp7aFlox controls showed no significant difference before and after cold exposure. ( c) The weight of brown adipose tissue (BAT) in Atp7aΔDbh animals is significantly higher compared to controls. ( d, e) Gonadal white adipose tissue (gWAT) weight did not differ significantly between Atp7aΔDbh and Atp7aFlox controls. ( f) Representative hematoxylin and eosin (H&E) - stained sections of BAT from Atp7aFlox and Atp7aΔDbh mice, maintained in thermoneutrality (30 °C for 10 days), reveal enlarged adipocytes indicative of defective lipolysis in mutant animals. (g) Quantification confirms significant increases in adipocyte size in BAT. ( h) H&E-stained sections of BAT isolated from Atp7aΔDbh and Atp7aFlox controls following acute cold stress at 6 °C for 5 hours show enlarged lipid droplets in BAT compared to the Atp7bFlox controls. ( i) Quantification supports these observations. Data are presented as mean ± SEM. Statistical significance was determined by unpaired Student’s t -test: p < 0.05, p < 0.01, p < 0.0001; ns = not significant. a b c d e g h i Thermoneutrality Cold exposure (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint Figure 8. Impaired lipolysis in adipose tissues of Atp7bΔDbh mice following acute cold stress. (a) Twelve-week-old male Atp7bΔDbh and Atp7bFlox animals (n = 4 per genotype) were exposed to cold exposure (6°C) for 6 hours and showed a mild thermoregulatory defect. Although partial recovery was observed at 6 hours in both groups, Atp7bΔDbh mice continued to trend toward hypothermia compared to Atp7bFlox controls. (b) The body weight of Atp7bΔDbh and Atp7bFlox controls remained unchanged before and after cold exposure. ( c) Atp7bΔDbh animals displayed significantly increased BAT weight relative to controls. ( d, e) gWAT and iWAT weights showed no significant difference between Atp7bΔDbh and Atp7bFlox controls. (f) Representative H&E -stained BAT sections from Atp7aFlox and Atp7aΔDbh mice following cold stress at 6°C for 6 hours revealed enlarged adipocytes in Atp7aΔDbh animals, consistent with impaired lipolysis. ( g) Quantification confirms significant increases in adipocyte size in BAT in Atp7aΔDbh animals compared to the Atp7bFlox controls. Data are shown as mean ± SEM ( n = 4 mice per group). Statistical significance was determined using Graphpad Prism Version 10, using an unpaired two-tailed Student’s t-test with Welch’s correction. *p < 0.05, **p < 0.01, ****p < 0.0001; ns = not significant. Cold exposurea b c d e gCold exposure (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint Table 1. The most significantly changed proteins (fold change >1.5, p-value<0.05) in Atp7aΔDBH locus coeruleus (proteins directly involved in NE metabolism are in blue) Symbol Protein name Fold Change Function Upregulated in Atp7aDBH VAC14 VAC14 component of PIKFYVE complex 2.49 synthesis and turnover of PI(3,5)P2, endosomal trafficking TSNAXIP1 translin associated factor X interacting protein 1 2.47 sperm motility and cilia function NDEL1 nudE neurodevelopment protein 1 like 1 1.76 neuronal migration, mitosis, and the maintenance of cell structures FRMD4A FERM domain containing 4A 1.74 scaffolding, cell polarity MEMO1 mediator of cell motility 1 1.66 cell migration SCYL1 SCY1 like pseudokinase 1 1.54 regulating Golgi morphology and COPI trafficking SCN2B sodium voltage-gated channel beta subunit 2 1.5 ion transport Downregulated in Atp7aDBH CLSTN3 calsyntenin 3 0.63 postsynaptic adhesion molecule ICA1 islet cell autoantigen 1 0.63 role in neurotransmitter secretion POLR2M RNA polymerase II subunit M 0.62 translation C12orf43 chromosome 12 open reading frame 43 0.62 regulation of Wnt signaling pathway CKAP4 cytoskeleton associated protein 4 0.61 cell adhesion and migration DBH dopamine beta-hydroxylase 0.61 NE synthesis CARMIL1 capping protein regulator and myosin 1 linker 1 0.6 role in cells protrusions AASDHPPT aminoadipate -semialdehyde dehydrogenase-phosphopantetheinyl transferase 0.58 biosynthesis of lysine and fatty acids. TMEM192 transmembrane protein 192 0.56 lysophagy SLC25A31 solute carrier family 25 member 31 0.56 mitochondria function, energetics ARFIP1 ARF interacting protein 1 0.55 biogenesis of secretory granules at the trans-Golgi network TH tyrosine hydroxylase 0.53 DOPA synthesis SLC18A2 solute carrier family 18 member A2 0.52 monoamine transporter LACTB lactamase beta 0.37 mitochondria lipid metabolism (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint Table 2. The most significantly changed proteins (fold change >1.5, p -value<0.05) in Atp7bΔDBH locus coeruleus Symbol Protein name Fold Change Function Upregulated in Atp7bDBH SNCA synuclein alpha 2.9896985 regulation of synaptic functions IgHg Immunoglobulin heavy chain (gamma polypeptide) 2.91399823 long term immunity IGKC immunoglobulin kappa constant 2.05765342 antibody stabilization ALB albumin 1.83400809 inhibition of apoptosis ERBIN erbb2 interacting protein 1.80875876 cell polarity, apoptosis SERPIN1A serine peptidase inhibitor, clade A, member 1A 1.59107297 protective, anti-inflammatory tole MVD mevalonate diphosphate decarboxylase 1.5822746 lipid synthesis, synaptic plasticity APOA1 apolipoprotein A1 1.54114222 cholesterol transport MPI mannose phosphate isomerase 1.53900722 glycosylation Pzp PZP, alpha-2-macroglobulin like 1.53368266 inhibitor of NGF-dependent neuronal outgrowth SCYL1 SCY1 like pseudokinase 1 1.50316148 regulating Golgi morphology and COPI trafficking Downregulated in Atp7bDBH TALDO1 transaldolase 1 0.63683874 pentose phosphate pathway CARMIL1 capping protein regulator and myosin 1 linker 1 0.6172813 actin polymerization SQSTM1 sequestosome 1 0.59832448 autophagy receptor SZRD1 SUZ RNA binding domain 1 0.59295725 nonsense -mediated decay DENND4B DENN domain containing 4B 0.55980644 Rab10 activator, vesicle trafficking TMEM192 transmembrane protein 192 0.37241937 lysophagy (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint Supplementary Materials for NON-REDUNDANT ROLES OF COPPER TRANSPORTERS ATP7A AND ATP7B IN NORADRENERGIC SIGNALING Shubhrajit Roy et al *Corresponding authors. Shubhrajit Roy ([email protected]) and Svetlana Lutsenko ([email protected]) This PDF file includes: Supplementary Text Materials and Methods: Figs. S1 to S12 Tables S1, S3 and S4 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint Supplemental Materials and Methods LA -ICP-TOF-MS: a) Preparation and quantification of gelatin standards Matrix-matched LA -ICP-ToF-MS gelatin standards were made by spiking a molten gelatin solution with multi-elemental stock solutions to convert counts from elements (m/z) from the LA -ICP-TOF-MS into concentrations (ppm). Briefly, 10% (w/v) porcine gelatin (Bloom 300, Sigma Aldrich, St. Louis, MO, USA) solution in ultrapure H2O kept above the melting temperature at 55 oC. Following gelatin dissolution (care was taken to mix thoroughly while introducing few bubbles via sonication), the first set of gelatin standards was spiked with IV - Stock-74434 (Inorganic Ventures, 1000 μg/mL: As, B, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Mo, Na, Pb, S, Se, V, Zn) at concentrations of 0, 20, 35 and 50 ppm to calibrate concentrations of 63Cu, 56Fe and 66Zn. The second set of standards was prepared to evalu ate Na/P at concentrations of 0, 2000, 4000, and 8000 ppm using monosodium phosphate (Reagent Plus grade, ≥99.0%, Millipore Sigma, St. Louis, MO, USA). Both sets of gelatin standards were maintained at 55 oC before pipetting 250 μl from each gelatin standar d directly on a pre -cooled cryotome chuck ( -20 oC). Gelatin was allowed to freeze completely in the cryostat for at least 5 minutes before sectioning. Sectioning was accomplished using a Leica CM3050S cryostat (Leica Biosystems, DerPark, IL, USA) with a chamber temperature of −22 °C and objective temperature of −20 °C. Once solidified, standards and murine brain tissues were sectioned at 10 μm thickness and adhered to positively charged glass slides (Superfrost Plus, Thermo Fisher Scientific, Waltham, MA, U SA). The elemental concentrations of the gelatin standards were confirmed with ICP -QQQ-MS and ICP -OES as previously described (21). b) Data acquisition and analysis Tissue samples from 20 -week-old male mice (n=4) and standards mounted on glass slides were ablated using the ImageBIO 266 laser ablation system (Elemental Scientific Lasers, Bozeman, MT, USA), which is equipped with a 266 nm laser, an ultra -fast, low-dispersion TwoVol3 ablation chamber, and a dual concentric injector. The aerosolized sample was transferred to the TOFWERK icpTOF S2 mass spectrometer (TOFWERK AG, Thune, Switzerland), where the elemental content was analysed in real time according to mass/char ge (m/z) ratio. Daily tuning was performed using the NIST SRM612 glass certified reference material (National Institute for Standards and Technology). High intensities for 140Ce and 55Mn were used to optimize torch alignment, lens voltages, and nebulizer g as flow while maintaining low oxide formation based on the 232Th16O+/232Th+ ratio (less than 0.5). Laser ablation sampling was performed at 50% laser power (no dosage) at a repetition rate of 200 Hz using a circular 10 μm spot size. Helium was used as the carrier gas (chamber and cup helium at 350 ml/min), and laser fluence was 1.80 - 2.26 J/cm2. Ten lines of gelatin standards were ablated using the same parameters but with the raster spacing set at 30 μm to ensure clean ablation of each raster. Scanning data was recorded using TofPilot (version 1.3.4.0, TOFWERK AG) and saved in the ope n-source Hierarchical Data Format 5 file format. Image generation and concentration calibration were conducted using the Iolite software package (version 4.8.6, Elemental Scientific Lasers). Finally, mass -calibrated laser images were overlayed with confoca l images (using house-built software, unpublished) to locate the LC, where ROIs were made around LC to quantify 63Cu, 56Fe and 66Zn. 31P was used to normalize each element’s concentration. CATECHOLAMINE MEASUREMENTS: a) Sample Preparation for Serum Serum concentrations of dopamine, norepinephrine, epinephrine, and serotonin were quantified by liquid chromatography–mass spectrometry (LC –MS) following protein precipitation. Briefly, 50 µL of serum was combined with 150 µL of methanol, vortexed to ensur e complete protein precipitation, and centrifuged at 4 °C for 45 min at high speed. LC vials were prepared by spiking 5 µL of 1 µg mL⁻¹ dopamine-d₄ as an internal standard. A 50 µL aliquot of the supernatant was transferred to the spiked vials, and 10 µL was diluted with 490 µL of methanol. After vortexing, 50 µL of the diluted supernatant was transferred to a new vial. Additional dilution was performed when necessary to bring serotonin (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint concentrations within the calibration range. All samples were dried under vacuum and reconstituted in 50 µL of water prior to analysis. b) Sample Preparation for Brain Tissue Brain tissue was homogenized using five 1.4 mm ceramic beads and 1 mL of methanol in a Bead Mill 4 Mini Homogenizer (speed 5, 30 s). Homogenates were centrifuged at 4 °C for 45 min. Supernatants were processed following the same procedure as serum samples: spiking with 5 µL of 1 µg mL⁻¹ dopamine -d₄, dilution with methanol, and additional dilution as needed to bring serotonin and dopamine within the calibration range. Samples were dried under vacuum and reconstituted in 50 µL of water prior to LC –MS analysis. c) Calibration Standards Calibration standards were prepared by serial dilution from 1 mg mL⁻¹ stock solutions of dopamine, norepinephrine, epinephrine, and serotonin. The calibration range for all analytes was 0.2 –200 ng mL⁻¹. d) Chromatographic Separation Liquid chromatography was performed on an Agilent 1290 Infinity II UHPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a Waters Acquity UPLC BEH C18 column (1.7 µm, 2.1 × 100 mm). The mobile phase consisted of water with 0.1% formic aci d (A) and methanol (B). The gradient program was as follows: 2% B (0 –1.0 min), linear increase to 98% B (1 –4 min), hold at 98% B (4–6 min), return to 2% B (6 –6.1 min), and re -equilibrate at 2% B (6.1 –10 min). The flow rate was 0.40 mL min⁻¹, the column temperature was maintained at 45 °C, and the injection volume was 10 µL. LC-MS PROTEOMICS ANALYSIS OF LASER -MICRO-DISSECTED LC a) Mass Spectrometry Detection Mass spectrometric analysis was performed using a Sciex 6500+ triple quadrupole mass spectrometer (Sciex, Framingham, MA, USA) operating in positive electrospray ionization (ESI) mode. Multiple reaction monitoring (MRM) was used for detection and quantific ation of target compounds (Table 1). The declustering potential was 60 V, entrance potential 10 V, and collision cell exit potential 8 V. Source conditions were as follows: curtain gas, 35 psi; ion spray voltage, 5500 eV; source temperature, 500 °C; ion source gas 1, 65 psi; ion source gas 2, 55 psi. Quantification was performed using Sciex MultiQuant 3.1 software, based on the peak area ratio of the analyte to the internal standard. b) Data acquisition Calibration curves were constructed by plotting the peak area ratio of each analyte to the internal standard against the nominal concentration of calibration standards. Linear regression with 1/x weighting was applied to all calibration curves. Analyte con centrations in samples were calculated from the corresponding calibration curve. Method performance was evaluated by assessing linearity (R² ≥ 0.99). Limits of detection (LOD) and quantification (LOQ) were determined based on a signal -to-noise ratio of 3:1 and 10:1, respectively. Mass spectrometric data acquisition was performed in data -dependent mode with full scan acquisition covering a mass-to-charge ratio (m/z) range of 300 –1800 using "Top Speed" mode with a 3 s cycle time. Precursor ion scans (MS1) were acquired at the resolut ion of 120,000 at m/z 200, while fragment ion scans (MS2) were obtained using higher -energy collisional dissociation (HCD) at 35% collision energy and detected at the resolution of 50,000 at m/z 200. Automatic gain control was set to 1 × 10⁶ ions for MS1 and 5 × 10⁴ ions for MS2, with maximum injection times of 50 ms and 100 ms, respectively. Precursor isolation was performed using a 1.6 m/z window with 0.4 m/z offset. Dynamic exclusion was applied for 30 s, and singly charged precursors were excluded. Internal mass calibration was achieved using the lock mass function (m/z 445.12002) from ambi ent air. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint c) Data Processing The mass spectrometry data analysis was conducted as described previously with minor modifications. Protein identification and quantification were performed using Proteome Discoverer software (version 3.2.0.450; Thermo Fisher Scientific). For MS2 preproces sing, the top ten peaks within each 100 Da window were selected for database searching. Tandem mass spectra were searched against the UniProt mouse protein database (UP000000589, which included both Swiss -Prot and TrEMBL and was released in January 2019 wi th 55,435 entries), which contained protein entries with common contaminants (115 entries) using SEQUEST HT search algorithms. Search parameters included: trypsin digestion with up to two missed cleavages allowed, precursor mass tolerance of 10 ppm, fragment mass tolerance of 0.02 Da, carbamidomethylation of cysteine (+57.02146 Da) and TMT labeling (+304.20715 Da) on lysine residu es and peptide N -termini as fixed modifications, and methionine oxidation (+15.99492 Da) as a variable modification. The minimum peptide length was set to six amino acids, with at least one peptide required per protein identification. Peptide-spectrum matches (PSMs) and proteins were filtered to achieve a 1% false discovery rate using Percolator and Protein FDR Validator nodes, respectively. For TMT quantification, reporter ion integration utilized the most confident centroid method wit h 20 ppm mass tolerance for MS2 -level HCD fragmentation. Both unique and razor peptides contributed to protein quantification, with a co -isolation threshold of 50%. Signal -to-noise ratios determined reporter ion abundances, with missing values replaced by minimum intensity values and an average signal -to-noise threshold of 50. Protein grouping followed strict parsimony principles, and final protein abundances were calculated by summing all corresponding PSM reporter ion intensities. d) Statistical Analysis Statistical analysis of mass spectrometry data was conducted using Perseus software (version 1.6.0.7). Data normalization was performed by dividing the reporter ion intensities by the median protein values, followed by a log2 transformation and median cent ering of the relative abundance values for each sample. Differential protein expressions between comparison groups were assessed using Student's two -sample t- test. Proteins exhibiting q -values < 0.05 were designated as significantly differentially express ed. For volcano plot generation, q -values were determined using Significance Analysis of Microarrays (SAM) with permutation-based false discovery rate estimation and an S0 parameter of 0.1. Principal Component Analysis (PCA) analysis was conducted using th e MetaboAnalyst tool. Functional enrichment analysis was performed using Enrichr to identify significantly enriched biological pathways and Gene Ontology (GO) terms associated with differentially expressed proteins. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint Fig. S1. PCR validation of mouse genotypes. (a) Agarose gel electrophoresis (2% agarose in 1× Tris -acetate-EDTA buffer) showing PCR amplification of the Cre transgene. (b) PCR validation of Atp7aFlox alleles. (c) PCR validation of Atp7bFlox alleles. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint Fig S2: Metal maps for Cu, Zn, Fe and P on coronal brain sections from Wild type, Atp7a ΔDbh and Atp7bΔDbh using LA-ICPMS (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint Fig. S3. (a) Quantification of Atp7b fluorescent intensity in noradrenergic neurons of the Locus Coeruleus (LC), identified by TH staining, in Atp7aΔDbh and Atp7aFlox coronal brain sections. n=3 animals were analyzed. Data were analyzed using GraphPad Prism 10 and are presented as mean ± SEM. Statistical significance was determined using an unpaired two -tailed Student’s t- test: p < 0.05, *p < 0.01, **p < 0.001. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint Fig. S4. Immunoblot showing (a) Dbh from the membrane fraction isolated from whole brain homogenate from Atp7aFlox (n = 4) and Atp7b ΔDbh (n=4) and Atp7bFlox (20 weeks) (b) Quantification of DBH normalized to Na+/K+ ATPase shows reduced expression in Atp7b ΔDbh compared to the Atp7bFlox controls. Statistical significance was determined using GraphPad Prism version 10 software using an unpaired Student’s t-test with Welch’s correction. *p < 0.05, **p < 0.01, ns = not significant . (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint Fig S5: Inactivating copper transporters in Dbh neurons affects brain and serum Norepinephrine levels. (a, b) LC–MS analysis of whole -brain homogenates shows no significant change in serotonin levels in Atp7aΔDbh (a) or Atp7bΔDbh (b) relative to LoxP/LoxP controls (20 weeks, n = 5). (c) Brain dopamine (DA) levels are significantly elevated in Atp7aΔDbh mice compared to Atp7aFlox. Data are mean ± S.E.M. Statistical significance was determined using unpaired two - tailed Student’s t-test with Welch’s correction; *p < 0.05, **p < 0.01, *** p < 0.001. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint Fig S6: Immunohistochemistry was performed on 30 µm -thick coronal brain sections using a sheep anti - tyrosine hydroxylase antibody. Tyrosine hydroxylase -positive regions were identified and isolated using Zeiss Laser Capture Microdissection (LCM). The dissected t issues were subsequently subjected to LC - MS-based proteomic analysis. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint Fig S7: Immunostaining for SNCA (red) in Atp7b ΔDbh shows upregulation of SNCA in TH+ve (green) noradrenergic neurons in the LC region compared to the Atp7bFlox controls (20 weeks) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint Fig S8: Conditional inactivation of Atp7a in Dbh neurons disrupts systemic energy metabolism. Whole - body metabolic parameters were measured in wild -type (black), Atp7a ∆Dbh , and Atp7b∆Dbh male mice using the Comprehensive Lab Animal Monitoring System (CLAMS; Columbus Instruments) over a continuous 48 -h recording period following 24 h acclimatization. (a) Oxygen consumption (VO₂) and (b) carbon dioxide production (VCO₂) normalized to either lean mass or body weight. (c) Respiratory exchange ratio (RER). Light and dark p hases are indicated, and whole -day averages are shown. Data represent mean ± SEM, with individual animals shown as dots. Statistical analyses were performed using two -way ANOVA followed by Tukey’s multiple comparison test; *p < 0.05, **p < 0.01, ***p < 0.0 01, ns = not significant (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint Fig S9: Weight of adipose tissues isolated from mice kept at room temperature (22 -24⁰ C) (a) BAT from Atp7aΔDbh animals shows significantly increased weight compared to the Wild type (20 weeks). Atp7bΔDbh did not show any significant change (b) iWAT (c) gWAT did not reveal any significant difference in adipose tissue weight in mutants compared to the wild type controls. Data were represented as Mean ± SEM. Statistical significance was determined using Grap hPad Prism version 10 software using an unpaired Student’s t-test with Welch’s correction. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint Fig S10: Downregulated thermogenic gene expression in adipose tissues in Atp7a ΔDbh and Atp7bΔDbh mice following cold exposure. (a) Immunohistochemistry for UCP1 of adipose tissues, such as BAT, gWAT and iWAT shows intense DAB staining in Atp7aFlox animals while Atp7a ΔDbh shows minimal staining suggesting reduced expression of UCP1 following acute cold stress; n=3. (b, c) Atp7a ΔDbh mice (n=5) show reduced PGC1α and UCP1 mRNA expression in BAT compared to Atp7aFlox mice (n=4) normalized to GAPDH mRNA expression. (e) Immunohistochemistry for UCP1 of adipose tissues, such as BAT, gWAT and iWAT shows intense DAB staining in Atp7bFlox an imals while Atp7bΔDbh shows minimal staining suggesting reduced expression of UCP1 following cold exposure; n=3). Statistical significance was determined using Graphpad Prism Version 9, using an unpaired two -tailed Student’s t-test with Welch correction; P < 0.05, P < 0.01. Atp7b ΔDbh (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint Fig S11: (a) Body weight of Atp7a∆Dbh kept at prolonged thermoneutrality (30 °C) shows a similar trend to that of Atp7aFlox (b) Adipose tissue morphology isolated from Atp7a∆Dbh and Atp7aFlox at thermoneutrality. Brown Adipose tissue (BAT) seems to be pale and enlarged. iWAT and gWAT seem to be comparable between Atp7a∆Dbh and Atp7aFlox. (c-e) Adipose tissue weight isolated from Atp7a∆Dbh and Atp7aFlox mice kept in prolonged thermoneutrality (30⁰C). Data were represented as Mean ± SEM. Statistical significance was determined using GraphPad Prism version 10 software using an unpaired Student’s t-test with Welch’s correction. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint Fig S12: Uncropped immunoblot showing (a) Na +K+ATPase and (b) DBH from the membrane fraction from whole brain homogenate. (c) Immunoblot for TH from soluble and membrane fractions from whole brain homogenate. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint Table S1: Elemental quantification at LC region in brain sections (copper is highlighted in blue) Genotype Element LC-Left LC-Rright LC AVG Wild type 1 Fe56_ppm 26.06644311 30.94934954 28.5079 Cu63_ppm 13.23363265 12.76365315 12.99864 Zn66_ppm 29.26250966 35.37463234 32.31857 Wild type 2 Fe56_ppm 24.13445349 22.82077292 23.47761 Cu63_ppm 8.453597428 7.352046195 7.902822 Zn66_ppm 26.54174485 25.39997018 25.97086 Wild type 3 Fe56_ppm 30.4119277 20.06864234 25.24029 Cu63_ppm 25.06456501 17.93140846 21.49799 Zn66_ppm 49.07467499 24.67689718 36.87579 Wild type 4 Fe56_ppm 30.55253144 28.13670101 29.34462 Cu63_ppm 10.92706818 11.44964021 11.18835 Zn66_ppm 27.39776815 27.6259467 27.51186 Atp7aΔDbh _1 Fe56_ppm 17.03560611 16.06287638 16.54924 Cu63_ppm 4.314214862 3.69060261 4.002409 Zn66_ppm 11.35299273 10.63358884 10.99329 Atp7aΔDbh _2 Fe56_ppm 7.887174834 9.943505808 8.91534 Cu63_ppm 2.48685707 3.316787145 2.901822 Zn66_ppm 7.346034496 8.684080172 8.015057 Atp7aΔDbh _3 Fe56_ppm 15.37261191 15.99855668 15.68558 Cu63_ppm 5.032425899 4.320818919 4.676622 Zn66_ppm 12.05653024 15.84153393 13.94903 Atp7bΔDbh _1 Fe56_ppm 41.54731144 34.726308 38.13681 Cu63_ppm 14.85223744 8.853790876 11.85301 Zn66_ppm 38.09642812 32.34389281 35.22016 Atp7bΔDbh_2 Fe56_ppm 33.00461541 34.67047944 33.83755 Cu63_ppm 13.94175298 17.03608233 15.48892 Zn66_ppm 38.49497998 39.02257119 38.75878 Atp7bΔDbh_3 Fe56_ppm 42.87421338 38.52041995 40.69732 Cu63_ppm 12.99967127 8.379113077 10.68939 Zn66_ppm 34.38534161 34.52487674 34.45511 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint Table S3: Primers used for mouse genotyping and qPCR Gene Forward Primer Reverse Primer Atp7b Flox AT1: 5’-GTTCCACAGAAACTATATGCCTGGG-3’ SDL2: 5’-GACAATACTACACTGACCATATTCA-3’ Atp7b Flox NDEL1: 5’-CGTCATAGCAAAGCTTGGTAACC-3’ NDEL2: 5-’CTTAAGTGCTGGATATGGGCATG-3’ Dbh-Cre Cre5: 5’-AATGCTTCTGTTCCGTTTGCCCGGT-3’ Cre3: 5’-CCAGGCTAAGTGCCTTCTCTACA-3’ Pdk4: 5′-GTTCCTTCACACCTTCACCAC-3 5′-CCTCCTCGGTCAGAAATCTTG-3′ Ucp1 5′-TGGAGGTGTGGCAGTGTTCAT-3′ 5′-TGACAGTAAATGGCAGGGGAC-3′ Pgc1α 5′-GGAGCCGTGACCACTGACA-3′ 5′-TGGTTTGCTGCATGGTTCTG-3′ Gadd45g 5′-TTCGTGGATCGCACAATGACT-3′ 5′-GGACTTTGGCGGACTCGTAGA-3′ Dio2 5′-TCCCTCACCCCCCTCCCAACC-3′ 5′-GCCCCATCAGCGGTCTTCTCC-3′ (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint 45 Table S4 Antibodies used in immunofluorescent and immunoblot experiments Antibody Host animal Dilution Vendor Anti-tyrosine hydroxylase Sheep Western blot: 1:5000 IF/IHC: 1:400 NB300-110 Novus Biologicals Anti-Dopamine-β- hydroxylase Rabbit Western bot: 1:5000 IF: 1:400 Gifted by Dr. Betty Eipper Sodium Potassium Atpase α1 Mouse Western blot: 1: 10,000 sc-21712, Santa -Cruz Anti-UCP1 Rabbit Western blot: 1: 5,000 IHC: 1:300 23673-I-AP, Proteintech Anti-P62/SQSTM1 Rabbit Western blot: 1:10,000 IF: 1:300 A19700, Abclonal Anti-Iba1 Rabbit IF: 1:300 GTX635363, Genetex Anti-Atp7a Rabbit IF: 1:50 HP8040, Hycult Anti-Atp7b Rabbit IF: 1:200 ab124973, Abcam (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted January 16, 2026. ; https://doi.org/10.64898/2026.01.15.699802doi: bioRxiv preprint