Copper loading may affect rat neurobehaviour by impairing mitochondria-associated endoplasmic reticulum membranes in hippocampal neurons

(2025) 21:32 Sun et al. Behavioral and Brain Functions https://doi.org/10.1186/s12993-025-00277-y Behavioral and Brain Functions Open Access RESEARCH Copper loading may affect rat neurobehaviour by impairing mitochondria‑associated endoplasmic reticulum membranes in hippocampal neurons Zhengzhe Sun1†, Shan Jin2†, Xiang Fang2,3*, Wenming Yang2 and Huaizhen Chen2 Abstract Background To observe the effects of copper sulfate (CuSO4)-induced copper loading on neurobehaviour, mitochondria-associated endoplasmic reticulum membranes (MAMs) and related regulatory proteins in the hippocampal CA1 region of Sprague–Dawley (SD) rats. Methods Forty SD male rats were randomly divided into control and copper loading groups of 20 rats each. The control group rats were fed with normal feed and water; rats in the copper loading group were fed high copper feed (containing 1g/kg of CuSO4) and CuSO4 deionized water (concentration of 0.185%). After 12 weeks of rearing, the morris water maze (MWM) task and novel object recognition (NOR) test were conducted to compare the neurobehavioral characteristics of the two groups of rats. Morphological changes of neuronal MAMs in the hippocampal CA1 region of copper-loaded rats were observed using a transmission electron microscope (TEM) and immunofluorescence double-labelling techniques. Western-blot analysis was used to detect the expression of MAMs proteins VDAC1, IP3R, GRP75 and Mfn2. Results The results revealed that rats in the copper-loading group had significantly prolonged escape latency and reduced number of platform crossings in the MWM task (p < 0.01). The percentage of novel objects explored (also known as the Discrimination Ratio, DR) and the discrimination index (DI) were significantly reduced in the NOR test (p < 0.01). In addition, electron microscopy shows increased disruption of neuronal endoplasmic reticulum (ER)mitochondrion coupling in the hippocampal CA1 region of rats in the copper-loading group (p < 0.05), and the percentage of MAMs in mitochondrial circumference decreased (p < 0.05), the colocalization coefficients between the ER and mitochondria was significantly reduced (p < 0.05). Moreover, the protein expression levels of VDAC1, IP3R, and GRP75 in rat hippocampal tissue were detected to be significantly increased (p < 0.01), while the protein expression level of Mfn2 was significantly decreased (p < 0.01). Conclusions In this study, it is speculated that the neurobehavioral changes in rats may be related to the increased expression levels of the MAMs proteins VDAC1, IP3R, and GRP75, the reduced expression level of Mfn2, and the disruption of the structural integrity of MAMs in the hippocampal CA1 region of rats caused by copper loading. Keywords Copper loading, Neurobehavior, MAMs, Wilson’s disease † Zhengzhe Sun and Shan Jin should be regard as co-first authors. *Correspondence: Xiang Fang Full list of author information is available at the end of the article © The Author(s) 2025. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/. Sun et al. Behavioral and Brain Functions (2025) 21:32 Introduction Copper metabolism disorders can cause various neurological diseases, among which Wilson’s disease (WD) is the most common. The copper loading present in the WD can cause neurological damage, exhibiting a variety of neurobehavioural impairments [1, 2]. Among them, cognitive impairment has a hidden onset but the risk is relatively high, which has a significant impact on the long-term quality of life of patients, and its severity is positively correlated with the duration of the disease, whereas cognitive deficits of the patients can be significantly improved after decoppering chelation treatment [3]. Copper is highly oxidatively toxic, and the neurological damage caused by copper loading is closely related to free radical production [4]. Copper loading can induce free radical overproduction, leading to mitochondrial and DNA damage, energy metabolism disorders in the body, and cytotoxic effects [5]. In particular, mitochondrial damage is the initial target site for copper-mediated neuronal injury and an important contributor to the development of several neurodegenerative diseases [6, 7]. The changes in mitochondrial function are closely related to cognitive impairment [8], but MAMs play an important role in cognitive related diseases by regulating processes such as mitochondrial fusion, fission, and transport, which affect mitochondrial function [9]. MAMs are enriched with various junctional proteins, among which GRP75, an important protein at the interface of MAMs, affects Ca2+ signaling and maintains mitochondrial calcium homeostasis by binding to IP3R and VDAC1 to form the IP3R-GRP75-VDAC1 protein complex [10, 11]. Mfn2 is localized in the mitochondrial outer membrane and ER, enriched in MAMs, and mainly functions as a physical linkage to maintain the structural stability of MAMs [12]. The combined action of IP3R, GRP75, VDAC1, and Mfn2 is of great significance for stabilizing the structure and function of MAMs [10]. Based on the important role of mitochondria-MAMs interactions in maintaining cognitive function, and the close relationship between MAMs junction proteins and the maintenance of MAMs structure and function, it is speculated that structural and functional changes of MAMs involving multiple connectivity proteins may be an important part of the development of high copperinduced cognitive impairment [13]. In this study, we intend to investigate the possible mechanism of MAMs and related proteins involved in high copper-induced abnormal neurobehaviour by observing the effects of copper loading on neurobehavior, structural homeostasis of MAMs, and expression of functionally related proteins in rats. Page 2 of 10 Materials and methods Ethical statement The rat experiments in this study were conducted at the Animal Experiment Centre of Anhui University of Traditional Chinese Medicine. All animal experiments were conducted in accordance with the National (...truncated)