The relationship between Al exposure and neurodegenerative diseases, especially cognitive impairment, has been the focus of extensive scientific investigation for many years (Kandimalla et al., 2016). Our findings are in concordance with the existing literature, which consistently demonstrates the neurotoxic potential of Al and its adverse impact on cognitive function. In a large-scale cohort study involving over 1,200 participants, a strong link was identified between Al accumulation in the brain and cognitive decline, particularly in domains related to memory and executive function (Rondeau et al., 2009). Our study supports these findings. Al workers were found to have elevated Al levels, which could lead to lower cognitive function MoCA scores (Fig. 1). Meanwhile, consistent outcomes were also exhibited in Al-exposed rat model, with changes in various behavioral indicators of rats being caused by Al exposure (Fig. 2 A, B, C), which strengthens the argument for Al's neurotoxicity and its detrimental impact on cognitive function.
Zinc is a critical metal element in the central nervous system, playing a pivotal role in neuroprotection and cognitive function through its involvement in the maintenance of metal ion homeostasis (Dike et al., 2023). Our result analysis indicates that the content of Zn in the hippocampus of the high-dose group was significantly lower than the control group, while the content of Al was significantly higher (Fig. 2D). It is important to note that various factors, such as age, health condition, diet, and intake of other nutrients, may influence the levels of Zn (Dziedzic et al., 2022). Both a deficiency and an excess of Zn can have detrimental effects on cognitive function (Babic Leko et al., 2021).
Metal homeostasis imbalance has been proven to be a crucial mechanism leading to neuronal dysfunction and triggering neuronal cell death, subsequently becoming one of the key factors for the progression of neurodegenerative diseases (Singh et al., 2020). In this study, a clear opposite correlation between Al concentration and Zn concentration was observed: as Al levels increased, the concentration of Zn showed a declining trend (Fig. 2 D). Further investigations into the relationship between Al and Zn have revealed that Al exposure not only induces MCI in rats but also causes disturbances in Zn content. One possible mechanism for this perturbation is the competitive relationship between Al and Zn. For instance, Al might compete with Zn for cellular uptake pathways, leading to a decrease of Zn concentration in intracellular. Additionally, Al could potentially interfere with the transport and distribution of Zn, hindering its arrival to critical neurons or specific brain regions. Most notably, Al might disrupt the metabolism and utilization of Zn, resulting in its deficiency or inefficient level in the nervous system (McAllister et al., 2020). Apart from the prior-mentioned studies on the correlation between Al and Zn concentrations, this research further highlights the role of Zn as a pivotal mediator in the relationship between Al exposure and cognitive function. Its negative mediation effect accounts for 17.82% of the total effect (Fig. 1 A). This finding aligns with the earlier studies by Neha and colleagues, who observed that Al exposure could induce expression changes in neuro-related proteins such as reversed tau, APP, GFAP, ubiquitin, and α-synuclein, while Zn exhibited an opposing regulatory effect on these changes (Singla and Dhawan, 2017). Among the various MoCA scores sub-item related cognitive function, Zn particularly demonstrated a significant mediating effect in the domains of abstract (completely negative mediating effect, 95% CI: -0.0680~-0.0119, 36.98%) and executive/visuospatial abilities (partial negative mediating effect, 17.71%, 95% CI: -0.2088~-0.0151) (Fig. 1 B, C). It is noteworthy that prior research has confirmed that deficits in abstract and executive functions would precede memory decline and were considered early indicators of neurodegenerative diseases. Therefore, the mediation role of Zn in cognitive impairments induced by Al appears to be particularly crucial, further supplementing the research framework on the role of metal in neurodegenerative diseases.
ZNF and their involvement in cognitive impairment have garnered significant attention in recent research (Leduc-Galindo et al., 2019). Plenty of earlier studies have demonstrated the crucial functions of ZNF in the nervous system, such as nerve cell development, synaptic transmission, antioxidant defense, and neuronal survival(Al-Naama et al., 2020). Additionally, these proteins play a significant role in the regulation of gene expression, with a particular focus on genes associated with cognitive abilities. In our Al-exposed rat model, transcriptomic and proteomic analysis explored extensive changes in gene and protein expression profiles. Notably, we focused on the subset of proteins related to ZNF and identified key members like ROCK1, DMD, DHX57, SEC24A, TSHZ3, and UTRN (Fig. 3). Then the expression of ROCK1, DMD, and DHX57 in the high-dose group was downregulated according to the WB experiment (Fig. 7). This strengthens the evidence for their involvement in Al-induced cognitive impairment. ROCK1 was a phorbol-ester (PE) / Diacylglycerol (DAG)-type ZNF, DMD was a ZZ-type ZNF, and DHX57 was a ZnF_C3H1-type ZNF (Craft et al., 2013). Among the proteins identified, ROCK1 got special attention due to its central role in our PPI and integrated enrichment analysis findings (Fig. 4, 5, 6). ROCK is widely distributed in various brain regions, such as the prefrontal cortex, hippocampus, and dorsal striatum. As isoforms of the ROCK protein, both ROCK1 and ROCK2 play roles in excitatory postsynaptic transmission and synaptic plasticity, subsequently influencing hippocampus-dependent learning and memory functions in mice (Yan et al., 2019). Earlier literature on ROCK1 also suggests it downregulated in various neurodegenerative diseases (Lu et al., 2020). Its integral part in neurite outgrowth and synaptic plasticity makes it a prime candidate as a potential mediator in cognitive impairment (Kambe et al., 2021). The DAG/PE-binding domain of ROCK1 attaches to two zinc ions using the six conserved cysteines and two histidine as likely ligands (Ahmed et al., 1991), which were related to the mechanisms that lead to neurodegenerative diseases. Furthermore, considering ROCK1 as a pivotal node, its interactions with other proteins and pathways become crucial. Any disruption in ROCK1, be it structural or functional, can have ripple effects on the whole cognitive process (Tyszka-Czochara et al., 2014).
Besides, it is important to identify that metal homeostasis did not operate in isolation. Their interactions with proteins or pathways in specific tissues could dramatically alter the spatial structures of these macromolecules. Such structural alterations didn't just reshape the physical protein but can also redefine its functionality, influencing biomolecular recognition, self-assembly, and an array of other biophysical properties directly linked to its biological function. In many proteins, the presence of metal and their complexes can induce the surrounding peptide segments to fold into their proper structures, a phenomenon known as the template-mediated structural motif (TMSM) (Shalev, 2022). Now, considering the changes in Zn concentrations after Al exposure, it becomes pertinent to discuss its implications on ROCK1 or other ZNF proteins, with a known Zn binding site or any metal-associated site. The binding of metal to these specialized sites often provides structural integrity to the protein, and any change in the concentration or the nature of these ions can lead to a deviation from the protein's native structure (Rao and Horne, 2020). Such deviations can manifest in numerous ways, from misfolding to loss of function or even gain of toxic function (Jacques et al., 2013). This finding not only strengthens the known literature on the role of ZNF and ROCK1 in cognitive impairment but also beckons further, more detailed mechanistic investigations.
The Integrated co-enrichment analysis of KEGG and GO pathways underscored the significance of the decreased RHOA/ROCK1 pathway (Fig. 5). These results align with previous research emphasizing the role of the RHOA/ROCK1 pathway in cognitive function and neurodegenerative diseases (Koch et al., 2018). Rho GTPase belongs to the Ras superfamily and was involved in processes such as cell migration, phagocytosis, contraction, and adhesion (Humphries et al., 2020). Among them, ROCK (Rho-associated kinase) was the most detailed downstream effector of Rho. RHOA/ROCK1 pathway was closely related to multiple neurobiological processes such as neuronal survival, migration, synapse formation and function, and blood-brain barrier integrity (Wang et al., 2022). The results of this study show that the increased content of Al and the decreased content of Zn were related to the downregulation of the expression of RHOA/ROCK1 pathway, which leading to the MCI (Fig. 7). Possible mechanisms related to the change in Al and Zn content and its association with the RHOA/ROCK1 pathway, which induced MCI, include the possibility that Al exposure could disrupt the homeostasis of the cytoskeleton. The RHOA/ROCK1 pathway also played a central role in regulating the stability of the cytoskeleton, especially microtubules and microfilaments (Strassheim et al., 2019). Zn serves as a key regulatory element for multiple neurotransmitter receptors, and a reduction in its concentration might affect the strength and plasticity of synapses (Krall et al., 2021). Concurrently, the RHOA/ROCK1 pathway was associated with synaptic stability and efficiency (Martin-Camara et al., 2021). Moreover, changes in the concentrations of Al and Zn in the brain could exacerbate neuroinflammation. The RHOA/ROCK1 pathway also played a pivotal role in regulating neuroinflammation, particularly the activation of astrocytes and microglia (Glotfelty et al., 2023). Such concentration changes could also trigger neuronal apoptosis, and the RHOA/ROCK1 pathway was connected to various cell survival and death pathways.
In addition, this study found that the expression of Myl9, miR431, and miR182 related to ROCK1 was reduced in the high-dose Al exposure group, and these genes were closely associated with the RHOA/ROCK1 pathway (Fig. 7). Previous studies have confirmed that Myl9 functioned as a light chain myosin acidase involved in cytoskeletal remodeling and contraction. ROCK1 regulated these functions by phosphorylating Myl9. If the expression of Myl9 decreases, it might affect the cell's mechanical properties and migration. The reduction in miR431 and miR182 could weaken the cell's response to external stress and inflammatory factors, increasing cell vulnerability. ROCK1 might influence the gene expression of Myl9, miR431, and miR182 through regulated chromosome modification and structure (Farooqi et al., 2022).
This demonstrated the critical role of the RHOA/ROCK1 pathway in Zn-mediated Al-induced cognitive impairment, underscoring the importance of ROCK1 as a potential therapeutic target. It has been demonstrated that Fasudil was a small molecule ROCK non-specific inhibitor that could be used as a drug targeting RHOA/ROCK. However, it might have caused side effects such as intracranial hemorrhage, leukopenia, and damage to liver and kidney functions. The ROCK inhibitor L-F001 also exhibited effects of resisting oxidative stress, scavenging reactive oxygen species, and reducing intracellular glutathione levels, and it had the potential to emerge as a new drug for the treatment of Parkinson's disease and other neurodegenerative diseases (Peng et al., 2022). Nonetheless, its specific effect still needed verification through large-scale data. Overall, research specifically focusing on ROCK1 as a target was relatively limited, and further exploration of its impact and mechanism in the context of neurodegenerative diseases was still required.
In summary, our findings suggest that the role of Zn in Al-induced cognitive impairment might be attributed to the following mechanistic pathway: Al3+ could interfere with the transportation and absorption of Zn2+ across the blood-brain barrier and within neuronal cells in the hippocampus, resulting in a reduced Zn2+ content. This change of Zn2+ may subsequently inhibit the activity of the ROCK1 protein by inactivating its catalytic site and altering the conformation of its zinc finger domain, which consists of Zn2+ bound to two cysteine and two histidine residues within the active site of the C1 structure. Continue, such alterations could compromise the overall stability and functionality of ROCK1, leading a decreased expression levels of upstream and downstream genes in the RHOA/ROCK1 signaling pathway. Furthermore, alterations in Al3+ content in brain tissue may also directly impact other molecules and regulatory mechanisms associated with the RHOA/ROCK1 pathway, consequently disrupting its activity. This down-regulated RHOA/ROCK1 pathway could likely impair nerve cell skeleton remodeling, synapse formation, and maintenance, among other processes. Then interference would occur with nerve signal transmission, leading to a series of neurological cognitive function impairments, such as learning and memory. We will further verify this hypothesis in the next studies.