ZIP14 deletion disrupts divalent metal homeostasis in mouse cerebrospinal fluid

Abstract

ZIP14 is a member of the SLC39A (ZIP) family of metal transporters, primarily facilitating the cellular influx of divalent metals including manganese (Mn), zinc (Zn), and iron (Fe). Previous studies have demonstrated that Zip14 knockout (Zip14^−/−) mice exhibit significant increases in whole blood and brain Mn levels. However, the impact of ZIP14 deletion on metal homeostasis within the cerebrospinal fluid (CSF) remained unexplored. In this study, we comprehensively assessed Mn, Zn, Fe, sodium (Na), potassium (K), and calcium (Ca) levels in whole blood, serum, and CSF of male and female Zip14^−/− mice to elucidate both systemic and central nervous system (CNS)-specific alterations in metal homeostasis. Our findings reveal that Zip14^−/− mice exhibit pronounced Mn accumulation, with CSF Mn levels increasing by approximately 15-fold in males and 46-fold in females compared to wild-type controls. Correspondingly, blood Mn levels rose 23-fold in males and 17-fold in females, while serum Mn levels increased 10-fold and 15-fold, respectively. In contrast, Zn and Fe levels in whole blood and serum remained comparable between Zip14^−/− and wild-type mice. However, significant elevations in CSF Zn were observed, with a 7-fold increase in males and a 16-fold increase in females, alongside a 3-fold rise in CSF Fe levels in females. The CSF to serum ratios of Zn and Fe remained below 1 but were increased in the knockout mice, suggesting the activation of alternative metal transporters in the absence of ZIP14, which may contribute to the increased Mn accumulation in the CSF as well. Importantly, Na⁺ and K⁺ levels in whole blood, serum, and CSF were unaltered in Zip14^−/− mice, indicating that ZIP14 deletion does not broadly disrupt systemic electrolyte balance or compromise blood–CSF barrier integrity. Conversely, CSF Ca²⁺ levels were significantly reduced by 33% in male and 23% in female Zip14^−/− mice, highlighting a specific effect of ZIP14 on calcium homeostasis within the CNS.

1. Introduction

The blood-cerebrospinal fluid (CSF) barrier forms at the choroid plexus. It separates blood in small capillaries from the CSF inside the brain’s ventricles. Specialized cells called choroid plexus epithelial cells line this barrier. These cells secrete fluid with balanced ions and nutrients, while reabsorption mechanisms return fluid to the bloodstream. They use tight junctions to restrict the passage of large or harmful molecules. This barrier helps maintain a stable composition of CSF, ensuring that essential substances are delivered, metabolic waste is removed, and the brain remains cushioned against physical damage [1–3].

Metal homeostasis within the central nervous system (CNS) is vital for numerous physiological processes, including enzymatic function, neurotransmission, and antioxidant defense. Essential metals such as manganese (Mn), zinc (Zn), and iron (Fe) are tightly regulated, as both deficiencies and excesses of these metals can lead to significant neurological dysfunction [4, 5]. Mn is a cofactor for several enzymes involved in neurotransmitter synthesis and antioxidant defense but can induce neurotoxicity at elevated levels [6, 7]. Zn plays a pivotal role in synaptic transmission and neuroplasticity but is also implicated in excitotoxicity and oxidative stress when dysregulated [8, 9]. Fe is essential for myelination and mitochondrial respiration but can catalyze the formation of reactive oxygen species, contributing to oxidative damage when accumulated excessively [10, 11].

The SLC39A (ZIP) family of metal transporters, particularly ZIP14, has emerged as a key player in regulating divalent metal ions within the CNS. ZIP14 facilitates the cellular uptake of Mn, Zn, and Fe, thereby influencing the intracellular concentrations and distributions of these essential metals [12–14]. In the brain, ZIP14 is localized at the basolateral membrane of choroid plexus epithelial cells and choroid plexus tissue, indicating its strategic position for regulating metal transport between the blood and the CSF [15, 16]. This localization suggests that ZIP14 plays a critical role in mediating the entry of essential metals into the CSF, thereby impacting metal homeostasis within the CNS.

Previous studies using Zip14 knockout (Zip14^−/−) mice have demonstrated significant alterations in systemic metal levels. Specifically, Zip14^−/− mice exhibit an over ten-fold increase in whole blood Mn levels and an eight- to ten-fold rise in brain Mn content compared to wild-type controls [17, 18]. These findings emphasize the critical role of ZIP14 in maintaining Mn homeostasis; however, the impact of ZIP14 deletion on other essential metals and their distribution within the CNS remains incompletely understood. While previous research has focused on systemic and brain changes in metal concentrations, the status of these metals within the CSF of Zip14^−/− mice has not been explored. Understanding CSF metal levels is crucial, as CSF directly interacts with the extracellular milieu of the brain, influencing neuronal and glial cell function. Alterations in CSF metal concentrations could have profound implications for neuronal signaling, neurotoxicity, and the pathophysiology of neurodegenerative diseases [19, 20]. Moreover, since ZIP14 also transports Zn and Fe, it is essential to investigate whether the deletion of ZIP14 affects the homeostasis of these metals within the CSF, despite previous reports indicating no significant differences in their serum [18] and total brain levels in Zip14^−/− mice [17].

In addition to divalent metals, the balance of other cations such as sodium (Na), potassium (K), and calcium (Ca) is fundamental for CSF secretion and overall fluid balance within the CNS [21]. Na+/K+-ATPase pump plays a pivotal role in the transcellular movement of Na+ and K+. Located at the apical membrane facing the CSF, the Na+/K+-ATPase maintains the necessary electrochemical gradients by actively transporting Na+ out of the CSF into the blood and K+ into the CSF, creating an osmotic gradient that facilitates water movement and CSF production [22, 23]. Any disruption in Na⁺ and K⁺ homeostasis can impair CSF dynamics, neuronal excitability, and signal transmission, potentially leading to neurological deficits [23, 24]. Therefore, it is essential to assess whether ZIP14 deletion impacts these primary electrolytes, which could indirectly influence metal homeostasis and CNS function. Calcium (Ca) is a pivotal secondary messenger in various cellular processes, including neurotransmitter release, synaptic plasticity, and neuronal excitability [25, 26]. Proper regulation of Ca²⁺ levels is essential for maintaining neuronal function and preventing excitotoxicity [27], which can lead to cell death and is implicated in numerous neurodegenerative conditions [28].

This study aims to comprehensively evaluate the impact of ZIP14 deletion on metal homeostasis within the CNS by measuring Mn, Zn, Fe, Na, K, and Ca levels in the whole blood, serum, and CSF of Zip14^−/− mice. By focusing on CSF metal concentrations for the first time, we seek to elucidate the localized effects of ZIP14 on metal regulation within the CNS, independent of systemic changes. Additionally, by assessing Na, K, and Ca levels, we aim to determine whether ZIP14 influences the fundamental ionic environment necessary for CSF secretion and neuronal function. These investigations will facilitate the understanding of ZIP14’s role in maintaining metal and electrolyte balance in the brain, with potential implications for neurodegenerative disease mechanisms and therapeutic interventions targeting the metal transport pathways.

2. Materials and Methods

2.1. Animals

All mice were housed at the University of Arizona’s central animal facility, and all procedures were approved by the Institutional Animal Care and Use Committee (Approval number 16–172). Mice were maintained on a 12 hour light/dark schedule and received water and standard rodent diet (Teklad 7913, Envigo, Indianapolis, IN, USA). To perform genotyping, tail snips were collected from 3-week-old mice. The collected tissue samples were processed for digestion and polymerase chain reaction (PCR) using the Mouse Direct PCR Kit (Selleck Chemicals, Houston, TX, USA). The following primers were used for the genotyping PCR analysis: 5’-GTC GGC AGT CCC CAG ACA-3’ (Zip14 WT For), 5’-CAT GCC TCA TGC ACA TGA CCC-3’ (Zip14 WT Rev), 5’-GGC GGC GAA TGG GCT G-3’ (Zip14 Mut For), and 5’-CAG GAT CAC ACC ATT GAG GCC-3’ (Zip14 Mut Rev). The CSF was collected from 6- to 7-week-old mice following an established protocol [29]. Briefly, the skin and muscle were carefully dissected to expose the base of the skull and the cisterna magna. Using a fine-point glass capillary syringe, the dura was punctured to extract approximately 10 μL of CSF from each animal. The collection was performed under a microscope to ensure no blood vessel damage, confirming that the samples were blood-free. The collected CSF appeared clear and colorless. Samples were immediately snap-frozen in liquid nitrogen and stored at −80°C for future analysis.

2.2. Immunoblotting analysis

Liver samples were collected alongside blood and CSF to confirm that ZIP14 protein expression in each sample aligned with the genotyping results. Liver tissues were processed using a stainless steel tissue grinder in the presence of NETT lysis buffer consisting of 150 mM NaCl, 5 mM EDTA, 10 mM Tris, 1% Triton X-100, and 1× protease inhibitors (Bimake, Houston, TX, USA). The homogenized tissue lysates were subjected to centrifugation at 10,000× g for 10 minutes to separate nuclei and cellular debris. The clarified supernatant was collected for subsequent analysis. Protein concentrations in the lysates were quantified using the RC DC protein assay kit (Bio-Rad, Hercules, CA, USA), following the manufacturer’s instructions. Equal amounts of protein from each sample were mixed with a 1× Laemmli buffer, incubated at 37 °C for 30 minutes, and loaded onto 10% polyacrylamide SDS gels. The proteins were separated electrophoretically by running the gels at a constant voltage of 120 volts (V) for 90 minutes. After electrophoresis, the proteins were transferred to nitrocellulose membranes at 100 V for 2 hours. To reduce nonspecific binding, the membranes were blocked in a blocking solution containing 5% nonfat milk in tris-buffered saline (TBS) with 0.1% Tween-20 (TBST) for 1 hour at room temperature. Following blocking, the membranes were incubated overnight at 4 °C with primary antibodies targeting mZIP14 (1:1000), which was developed in our laboratory [30, 31]. After washing 4 times with TBST, the membrane was incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Millipore Sigma, Burlington, MA, USA) at a dilution of 1:4000 for 1 hour at room temperature. For normalization of protein expression, the membranes were probed with an HRP-conjugated anti-GAPDH antibody (Proteintech, Rosemont, IL, USA) at a dilution of 1:20,000. The signals were visualized using the ChemiDoc MP imaging system, and images were analyzed with Image Lab software (Bio-Rad, Hercules, CA, USA).

2.3. Metal level measurement by inductively coupled plasma mass spectrometry (ICP-MS)

All samples were digested in concentrated nitric acid at room temperature overnight and then at 80 °C for 4 hours. After digestion, the samples were diluted with Milli-Q water to achieve a final concentration of 3% nitric acid. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) analysis was conducted at the Arizona Laboratory for Emerging Contaminants (ALEC). The sample volumes were as follows: 50 μL of blood or serum per individual mouse and 10 μL of cerebrospinal fluid (CSF) per individual Zip14^−/− mouse. Mn levels in the CSF of individual wild-type mice were below the mass spectrometer’s detection limit. To address this, we pooled 80–120 μL CSF samples from multiple wild-type mice of the same sex to obtain one pooled sample. Each sex had three such pooled samples, which were then analyzed alongside the individual Zip14^−/− mouse samples.

2.4. Statistical analysis

All data were analyzed in GraphPad Prism software (GraphPad Software, Boston, MA, USA). For two-group comparison statistic tests, we first performed an F-test to assess whether the two groups had equal variances. If they did, we used an unpaired Student’s t-test. If they did not, we applied Welch’s t-test. A p-value below 0.05 is considered statistically significant.

3. Results

3.1. Validation of Zip14 knockout

To ensure the accuracy and reliability of our analyses on CSF metal levels, we first confirmed the successful knockout of the Zip14 gene in the Zip14^−/− mice used in our study. Since ZIP14 is prominently expressed in the liver [32, 33], we used liver tissues to detect ZIP14 protein. Our immunoblotting results demonstrated the absence of ZIP14 protein in the liver tissues of Zip14^−/− mice, indicating successful gene deletion (Figure 1). This confirmation ensured that any changes we noted in CSF metal levels could be attributed to the absence of ZIP14 rather than an incomplete knockout.

Figure 1. Validation of Zip14 knockout.

Representative immunoblotting images of liver samples from wild-type (WT) and Zip14 knockout (Zip14^−/−) mice. Each lane was loaded with the same amount of total protein. The ZIP14 band appears in WT samples but is absent in knockout samples. GAPDH was used as a loading control. All mice used in this study were validated by this method.

3.2. Increased manganese levels in the CSF, blood, and serum of Zip14^−/− mice

Previous studies show that, at around 9 to 12 weeks of age, Zip14^−/− mice had about a ten-fold increase in whole blood Mn levels and an eight- to ten-fold rise in brain Mn levels compared to wild-type animals [17, 34]. However, the CSF Mn level has not been reported. While total brain measurements provide an overall snapshot of Mn content in the CNS, assessing CSF Mn levels can capture more immediate or dynamic changes in Mn availability within the CNS. To address this gap, we conducted the first assessment of CSF Mn levels in Zip14^−/− mice. We found that Zip14^−/− mice display striking increases in Mn levels (Figure 2). Male knockout mice showed approximately a 15-fold increase in CSF Mn, while female knockout mice displayed an even more substantial 46-fold rise compared to wild-type animals. Blood Mn levels were elevated by 23-fold in males and 17-fold in females. Serum Mn levels also increased significantly, with a 10-fold rise in males and a 15-fold increase in females. These findings highlight a pronounced Mn accumulation in Zip14^−/− mice.

Figure 2. Manganese levels in male and female Zip14 knockout mice.

(A–C) show total blood, serum, and CSF manganese levels, respectively, in male mice. (D–F) present these same measurements in female mice. All panels compare Zip14^−/− (Zip14 KO) mice to wild-type (WT) controls. Sample sizes: whole blood and serum measurements—n = 5–7 individual WT and Zip14^−/− mice; CSF measurements—n = 3 grouped WT samples and n = 6–7 individual Zip14^−/− mice. Error bars represent mean ± standard deviation (SD). Asterisks indicate significant differences from WT (** p < 0.01, *** p < 0.001, **** p < 0.0001).

3.3. Zinc and iron levels in the CSF of Zip14^−/− mice increase

ZIP14 can mediate the cellular influx of other divalent metals besides Mn, including Zn and Fe [12–14]. Although a previous study reported no significant differences in the serum levels of Zn and Fe in Zip14^−/− mice by atomic absorption spectrophotometry [18], and another study found no differences in the total brain levels of these two metals by ICP-MS analysis [14], the status of these metals in the CSF remained unknown. To address this critical gap, we measured Zn and Fe levels in whole blood, serum, and CSF. By looking at CSF levels, we aim to detect any localized changes in the CNS that might not be evident when only measuring total brain or serum levels. This comparison will help to clarify how ZIP14 affects metal homeostasis and whether transport into the CSF follows the same pattern as blood or brain tissue.

We found that Zn and Fe levels in whole blood and serum of Zip14^−/− mice remained comparable to that of wild-type controls for both male (Figure 3A–B and 4A–B) and female mice (Figure 3D–E, and 4D–E). Despite these similarities in the systemic circulation, significant alterations were observed in the CSF. Specifically, CSF Zn levels were significantly elevated in both male and female Zip14^−/− mice compared to wild-type controls. Male knockout mice exhibited a 7-fold increase (0.140 ± 0.108 ng/ μL vs. 0.020 ± 0.009 ng/ μL) (Figure 3C), while female knockout mice showed a 16-fold increase in CSF Zn (0.276 ± 0.248 ng/ μL vs. 0.017 ± 0.004 ng/ μL) (Figure 3F). In contrast, Fe levels in the CSF displayed a sex-dependent response. Male Zip14^−/− mice demonstrated a trend toward elevated CSF Fe levels (Figure 4C); however, this increase did not reach statistical significance; while female knockout mice had a significant increase in CSF Fe levels, showing a 3-fold rise compared to wild-type animals (0.171 ± 0.049 ng/ μL vs. 0.054 ± 0.008 ng/ μL) (Figure 4F).

Figure 3. Zinc levels in male and female Zip14 knockout mice.

(A–C) show total blood, serum, and CSF zinc levels, respectively, in male mice. (D–F) present these same measurements in female mice. All panels compare Zip14^−/− (Zip14 KO) mice to wild-type (WT) controls. Error bars represent mean ± SD. Asterisks indicate significant differences from WT (ns p > 0.05, * p < 0.05).

Figure 4. Iron levels in male and female Zip14 knockout mice.

Panels A–C display total blood, serum, and CSF iron levels in male mice, respectively. Panels D–F present the corresponding measurements for female mice. All comparisons are made between Zip14^−/− mice (Zip14 KO) and wild-type (WT) controls. Data are shown as mean ± SD. Asterisks denote statistically significant differences from WT (** p < 0.01), while “ns” indicates no significant difference.

These results indicate that ZIP14 deletion leads to substantial alterations in Zn homeostasis within the CNS of both sexes and affects Fe regulation more prominently in females. The significant elevation of CSF Zn and Fe in female Zip14^−/− mice emphasizes the role of ZIP14 in maintaining metal balance in the CNS and suggests potential sex-specific mechanisms in metal transport and regulation.

3.4. Sodium and potassium levels do not change in Zip14 knockout mice

Building upon our investigation of Mn, Zn and Fe homeostasis, we next examined Na and K levels in whole blood, serum, and CSF of Zip14^−/− mice. CSF secretion is an active process that does not rely on osmotic gradients or ultrafiltration across the choroid plexus epithelium. Instead, it depends on ion transport mechanisms involving Na and K [35–37]. Understanding their levels is essential to determine whether ZIP14 deletion affects not only divalent metal transport but also the fundamental ionic environment of the CNS. Our ICP-MS analysis revealed that Na and K levels in whole blood (Figure 5A,5D and 6A, 6D), serum (Figure 5B and 6B, 6E), and CSF (Figure 5C,5F and 6C, 6F) were largely similar between Zip14^−/− mice and wild-type controls, except that serum Na levels were about 4% lower in female Zip14^−/− mice(Figure 5E). These findings suggest that the deletion of ZIP14 does not substantially disrupt the homeostasis of these critical electrolytes, indicating that ZIP14’s role is specific to the transport of divalent metals rather than the regulation of primary monovalent ions essential for CSF secretion and neuronal function.

Figure 5. Sodium levels in whole blood, serum, and CSF of male and female Zip14 knockout mice compared to wild-type controls.

Panels A and D illustrate sodium levels in the whole blood of male and female mice, respectively, while panels B and E display serum sodium concentrations. Panels C and F depict sodium levels within the CSF for male and female mice. Data are shown as mean ± SD, with “ns” indicating no significant difference. Across all measured compartments and both sexes, sodium levels were largely similar between Zip14^−/− mice and WT mice except for serum Na in females (* p < 0.05), indicating that ZIP14 deletion does not disrupt systemic or central sodium homeostasis.

Figure 6. Potassium levels in whole blood, serum, and CSF of male and female Zip14 knockout mice compared to wild-type controls.

Panels A and D illustrate potassium levels in the whole blood of male and female mice, respectively, while panels B and E display serum sodium concentrations. Panels C and F depict sodium levels within the CSF for male and female mice. Data are shown as mean ± SD, with “ns” indicating no significant difference. Across all measured compartments and both sexes, potassium levels remained consistent between WT and Zip14^−/− mice, indicating that ZIP14 deletion does not disrupt systemic potassium homeostasis.

3.5. CSF Calcium levels decrease in Zip14 knockout mice

Ca is a secondary messenger in various cellular processes, including neurotransmitter release, synaptic plasticity, and neuronal excitability. We found that whole blood and serum Ca levels remained unchanged between Zip14^−/− and wild-type mice (Figure 7A–B, 7D–E), while CSF Ca levels were significantly decreased in both male and female Zip14^−/− mice compared to wild-type controls (Figures 7C, 7F). Specifically, CSF Ca levels were reduced by 33% in male (43.09 ± 4.71 ng/ μL vs. 29.01 ± 2.98 ng/ μL) and 23% in female Zip14^−/− mice (44.11 ± 0.81 ng/ μL vs. 33.83 ± 7.71 ng/ μL). Together, these findings indicate that ZIP14 deletion disrupts Ca homeostasis within the CSF while maintaining stable Na and K levels systemically.

Figure 7. Calcium levels in whole blood, serum, and CSF of male and female Zip14 knockout and wild-type mice, measured by ICP-MS.

Panels A–F display calcium levels in whole blood (A, D), serum (B, E), and CSF (C, F) for males and females. Data are shown as mean ± SD. Calcium levels in whole blood and serum were unchanged between Zip14 knockout (Zip14^−/−) and wild-type (WT) mice. However, CSF calcium levels were significantly lower in Zip14^−/− mice. Data are presented as mean ± SD. Asterisks indicate significant differences from WT (* p < 0.05, *** p < 0.001), while “ns” indicates no significant difference.

4. Discussion

The CSF plays a crucial role in maintaining CNS homeostasis by regulating the extracellular environment, facilitating the distribution of essential substances, and removing metabolic byproducts. The production and regulation of CSF are primarily mediated by the choroid plexus, where ion transporters maintain the electrochemical gradients necessary for fluid secretion and ionic balance. In the present study, we assessed whole blood, serum, and CSF metal levels in Zip14^−/− mice to capture both systemic and CNS-specific changes in metal homeostasis. Assessing whole blood provides an overall view of systemic metal levels, serum measurements reflect the transportable fraction of these metals, and CSF analysis allows us to detect localized changes within the CNS. This comprehensive approach enables a detailed understanding of how ZIP14 influences metal homeostasis both systemically and within the CNS.

Our results demonstrate that at 6–7 weeks of age, Zip14^−/− mice exhibited significant Mn accumulation in the CSF, blood, and serum, with CSF Mn levels increasing by approximately 15-fold in males and 46-fold in females compared to wild-type controls. Blood Mn levels rose 23-fold in males and 17-fold in females, while serum Mn levels increased 10-fold and 15-fold, respectively. In contrast, Zn and Fe levels in whole blood and serum remained comparable between Zip14^−/− and wild-type mice. However, CSF Zn levels were significantly elevated by 7-fold in males and 16-fold in females, alongside a 3-fold rise in CSF Fe levels in females. Importantly, the levels of Zn and Fe in whole blood and serum remained comparable between Zip14^−/− and wild-type mice for both sexes. This indicates that the alterations in Zn and Fe are specific to the CSF and are not reflected in the systemic circulation.

In Zip14^−/− mice, the loss of ZIP14-mediated transport at the blood–CSF barrier likely leads to compensatory activation of alternative metal transport systems. These systems may facilitate the influx of Zn and Fe into the CSF to maintain essential metal availability within the CNS despite the absence of ZIP14. To further understand the mechanisms driving the elevated metal levels in the CSF of Zip14^−/− mice, we calculated the CSF to serum ratios for Mn, Zn, and Fe (Figure 8). In wild-type mice, the CSF to serum ratio for Mn was lower than, but close to one, indicating a balanced distribution across the blood-CSF barrier. However, in Zip14^−/− mice, this ratio exceeded one, suggesting that active transport mechanisms are responsible for the increased Mn levels in the CSF. We noted that although the CSF to serum ratios for Zn and Fe remain below one in both wild-type and Zip14^−/− mice—indicating that their concentrations in the CSF are lower than in the blood—the absolute CSF levels of Zn and Fe tend to be higher in the Zip14^−/− mice. This apparent increase in knockout animals suggests an underlying compensatory mechanism. Specifically, in the absence of ZIP14, alternative divalent metal transporters may be activated to facilitate the influx of Zn and Fe into the CSF. Indeed, one study showed that dietary zinc supplementation decreases brain manganese loading in Zip14^−/− mice [18], which supports the theory that an alternative divalent metal transport pathway is activated when ZIP14-mediated transport is disrupted. This compensatory mechanism ensures that essential metals continue to enter the CNS despite the loss of ZIP14 function. The sex-specific increase in CSF Fe levels in female Zip14^−/− mice, contrasted with the non-significant trend in males, points to possible hormonal influences or differential regulation of metal transport pathways between sexes. This highlights the need for further investigation into sex-dependent mechanisms that modulate metal homeostasis in the CNS.

Figure 8. CSF to serum ratios of manganese, zinc, and iron in male and female mice.

Illustration of the ratios of manganese (Mn), zinc (Zn), and iron (Fe) concentrations in cerebrospinal fluid (CSF) relative to serum for both male and female mice. These ratios were calculated using the average metal concentrations from each respective group. The blue dashed line represents a ratio of 1, indicating equal concentrations of the metal in CSF and serum. The numerical ratio values are displayed above each group symbol for clarity.

The choroid plexus maintains the necessary electrochemical gradients by actively transporting Na⁺ from choroid plexus epithelial cells into the CSF and reabsorbing K⁺ from the CSF into these cells, thereby facilitating water movement and CSF production [38, 39]. The unaltered Na and K levels in whole blood and serum suggest that ZIP14 deletion does not broadly affect systemic electrolyte balance. Furthermore, the stability of these primary electrolytes in the CSF indicates that the fundamental mechanisms responsible for CSF secretion and ionic balance, such as the Na⁺/K⁺-ATPase pump, remain functional despite the absence of ZIP14. Moreover, the stability of these primary monovalent ions implies that the blood–CSF barrier remains intact and uncompromised in Zip14^−/− mice. The maintenance of normal electrolyte levels underscores the specificity of ZIP14’s role in regulating divalent metal ions without impacting the essential balance of monovalent electrolytes. In addition to changes in Mn, Zn, and Fe, we observed a modest but significant reduction in CSF Ca levels in Zip14^−/− mice. Ca ions are essential for numerous neuronal functions, including synaptic transmission and plasticity [40]. A decrease in CSF Ca levels could have implications for neuronal signaling and may contribute to neurological deficits observed in conditions of metal dysregulation. Future studies are needed to elucidate the underlying mechanism and the functional consequences of lowered CSF Ca.

5. Conclusion

In summary, ZIP14 is essential for maintaining metal homeostasis with the CNS. ZIP14 deletion disrupts Mn balance and leads to increased levels of Zn and Fe in the CSF, likely through the activation of alternative metal transport systems. Importantly, Na and K levels in whole blood, serum, and CSF remained unaltered in Zip14^−/− mice, indicating that ZIP14 deletion does not broadly disrupt systemic electrolyte balance or compromise blood–CSF barrier integrity. A reduction in CSF Ca levels was observed in Zip14^−/− mice, suggesting that ZIP14 deletion may influence Ca regulation within the CNS. These findings underscore the multifaceted role of ZIP14 in neuronal health. Future research should aim to identify the specific compensatory transport pathways involved and further elucidate the mechanisms and functional consequences of altered Ca levels in Zip14^−/− mice.

Significance statement:

This is the first study to investigate metal levels in the CSF of Zip14^−/− mice. We discover that ZIP14 deletion leads to significant accumulation of Mn, Zn, and Fe in the CSF, as well as reduced calcium levels, highlighting its critical role in regulating metal homeostasis across the blood–CSF barrier. These findings provide new insights into the mechanisms of metal regulation in the CNS and have potential implications for understanding and addressing neurological disorders associated with ZIP14 dysfunction and metal dysregulation.