Effect of vitamin D and omega-3 on the expression of endoplasmic reticulum-associated protein degradation and autophagic proteins in rat brain

Introduction

The rat hippocampus is a C-shaped structure that is settled in the caudal part of the brain. Three different subregions can be noticeable: the dentate gyrus (DG), the hippocampus proper (composed of cornu ammonis CA3, CA2, and CA1), and the subiculum [1].

Vitamin D is special because it can be produced in the skin by exposure to sunlight [2]. It has been determined that this vitamin serves many purposes than its well-known functions in bone metabolism, calcium absorption, and bone integrity [3]. Little focus has been placed on the effects of vitamin D on the growth and operation of the nervous system, although numerous papers have described the cytoprotective impacts of vitamin D and its function in the development of diabetes, cardiovascular disease, and other malignancies [4]. Vitamin D has steroid-like actions and carries a cholesterol backbone. This fat-soluble hormone is responsible for playing a crucial part in maintaining brain health [5]. In recent years, vitamin D has been understood as an essential neuro-steroid with a variety of functions in the brain [6]. Regarding proper brain growth and function, adequate vitamin D levels may be highly significant [7], 8]. Because it may lower the incidence of central nervous system illnesses, an adequate supply of vitamin D at various life stages, especially the prenatal period, seems to be especially significant [9]. According to a study by Di Somma et al. [10], the adult brain has to have adequate levels of vitamin D in the bloodstream to maintain neurological growth. Vitamin D is essential for brain growth in fetal life. According to the study by Yates et al. [11], vitamin D insufficiency in mothers and offspring leads to developmental abnormalities, such as memory issues and learning, as well as grooming behaviors.

The omega-6 and omega-3 fatty acids are essential fatty acids that are important nutrients found in the diet [12], 13]. These critical fatty acids are concentrated at high levels in the brain, where they affect cognitive, social, and linguistic capabilities, as well as fundamental growth and development processes. A lack of alpha-linolenic acid and its omega-3 derivatives has been linked to behavioral changes, eyesight impairment, tingling in the limbs and extremities, loss of motor coordination, and growth retardation, according to previous research [14], [15], [16], [17].

Despite the imperfection of the folding process, it is estimated that about 30 % of freshly produced proteins are misfolded [18]. Misfolded proteins are subsequently kept in the endoplasmic reticulum, where they are destroyed via the endoplasmic reticulum-associated degradation pathway (ERAD). Retrotranslocation, proteasomal degradation, and ubiquitination are some of the procedures used in ERAD to choose the target protein [19]. Misfolded proteins are targeted for proteasomal breakdown via ERAD. Misfolded substrates are removed from the ER membrane by the valosin-containing protein (p97/VCP) complex and transported to the 26S proteasome for destruction [20]. VCP, also known as p97, performs a variety of tasks by joining with more than 40 distinct adaptor proteins. Both p97/VCP and cofactors often rely on particular protein domains for these interactions. Small p97/VCP-interacting protein (SVIP) and ubiquitin ligase (gp78) are two cofactors that contain the VCP-interacting motif, which is one such domain [21], 22]. Recent research has shown that SVIP is primarily localized to the central and peripheral nervous systems [23]. p97/VCP has been related to a variety of physiological processes, including cell cycle control, Golgi biogenesis, the formation of nuclear membranes, the ubiquitin-proteasome system (UPS), apoptosis, and autophagosome maturation [24], 25].

It has been reported that groups treated with vitamin D3 in human sperm had higher levels of Hsp70 protein than control groups [26]. Given that the Hsp70 chaperone is also one of the ERAD proteins for this reason, we investigated the relationship of SVIP and p97/VCP, two ERAD proteins with vitamin D supplementation in the brain.

Cargo molecules are captured by autophagy and transported to lysosomes in the form of the double-membrane vesicles known as autophagosomes. When an autophagosome is finished, it fuses with a lysosome to generate an autolysosome. P62 and LC3B have received the most attention among autophagy markers [27], [28], [29].

Many studies conducted have shown the relationship between ERAD and autophagic proteins. The specific mechanisms of omega-3 and vitamin D’s effect on the brain are not yet elucidated. Given the significance of p97/VCP, SVIP, and LC3B, p62, the current study aimed to examine the effects of omega-3 and vitamin D on the expression of ERAD and autophagic proteins in the rat brain. We hypothesize that ERAD markers (p97/VCP and SVIP) and autophagy markers (LC3B and p62) may have a relationship when given omega-3 and vitamin D supplements.

Materials and methods

Results

Cellular expressions of vitamin D receptor

We first conducted a thorough analysis of VDR localization. Although the immunostaining of VDR was seen in all groups, the staining intensity was increased in the neuronal cells of the hippocampus and cortex of brain sections in the vitamin D treated group compared to the control group. Additionally, moderate immunoexpression of VDR was detected in vitamin D+omega-3, and this expression was decreased in the hippocampus and cortex of the brain in the control and omega-3 groups (Figure 1). The H-score analysis revealed that the immunoexpression level of VDR was significantly increased in the rat hippocampus and the cortex of the brain from the vitamin D-treated group compared to the control (p<0.05). There was no significant difference considering the expression of VDR in omega-3 and vitamin D+omega-3 groups compared to the control group (p>0.05) (Figure 2).

Figure 1:

Immunolocalization of VDR protein in the hippocampus and cortex of rat brain in control and vitamin D, omega-3, and vitamin D+omega-3 treatment groups. Black arrows indicate the VDR-positive cells. Three independent experiments were performed. The micrographs were taken with 10× and 40× objectives. Scale bars: 100 μm, 200 μm. VDR, vitamin D receptor.

Figure 2:

The H score analysis of the immunoexpression levels of VDR, p97/VCP, SVIP, LC3B and p62 in the hippocampus and cortex of rat brain in control and vitamin D, omega-3, and vitamin D+omega-3 treatment groups. The immunoexpression levels of VDR and p97/VCP were considerably raised in the rat hippocampus and cortex of the brain from the vitamin D-treated group compared to the control. The immunoexpression level of LC3B was significantly decreased in the rat hippocampus of the brain from the vitamin D-treated group compared to the control group. Three independent experiments were performed. The data are demonstrated as mean ± SEM. *p<0.05. VDR, vitamin D receptor.

Altered expression of ERAD (p97/VCP and SVIP) markers in brain tissue

In addition to VDR, we have also investigated the expression of ERAD markers in brain tissues. The immunolocalization of p97/VCP (Figure 3) and SVIP (Figure 4) was detected in the neuronal cells in the hippocampus and the cortex of the rat brain in all groups. The H-score analysis revealed that the immunoexpression level of p97/VCP was significantly boosted in the rat hippocampus and cortex of the brain from the vitamin D-treated group compared to the control group (p<0.05). The immunoexpression levels of p97/VCP did not show any significant differences in omega-3 and vitamin D+omega-3 groups compared to the control group (p>0.05). Furthermore, the SVIP immunoexpression was higher in the vitamin D group compared to others. However, there was no significant difference considering the expression of SVIP in vitamin D, omega-3, and vitamin D+omega-3 groups compared to the control group (p>0.05) (Figure 2).

Figure 3:

Immunolocalization of p97/VCP protein in the hippocampus and cortex of rat brain in control and vitamin D, omega-3, and vitamin D+omega-3 treatment groups. Black arrows indicate the p97/VCP-positive cells. Three independent experiments were performed. The micrographs were taken with 10× and 40× objectives. Scale bars: 100 μm, 200 μm.

Figure 4:

Immunolocalization of SVIP protein in the hippocampus and cortex of rat brain in control and vitamin D, omega-3, and vitamin D+omega-3 treatment groups. Black arrows indicate the SVIP-positive cells. Three independent experiments were performed. The micrographs were taken with 10× and 40× objectives. Scale bars: 100 μm, 200 μm.

Altered expression of autophagy (LC3B and p62) markers in brain tissue

We examined the expression of autophagic markers in rat brain cortex and hippocampus in addition to ERAD markers. Autophagic protein expressions LC3B (Figure 5) and p62 (Figure 6) tended to decrease in vitamin D and omega-3 treated groups compared to control. The H-score analysis revealed that the immunoexpression level of LC3B was significantly decreased in the hippocampus of the brain from the vitamin D-treated group compared to the control group (p<0.05). There was no significant difference considering the expression of LC3B in omega-3 and vitamin D+omega-3 groups compared to the control group (p>0.05). Furthermore, p62 was decreased in the hippocampus and the cortex of the rat brain from the vitamin D-treated group compared to the control group. However, the immunoexpression levels of p62 showed no significant differences in vitamin D, omega-3, and vitamin D+omega-3 groups compared to the control group (p>0.05) (Figure 2).

Figure 5:

Immunolocalization of LC3B protein in the hippocampus and cortex of rat brain in control and vitamin D, omega-3, and vitamin D+omega-3 treatment groups. Black arrows indicate the LC3B-positive cells. Three independent experiments were performed. The micrographs were taken with 10× and 40× objectives. Scale bars: 100 μm, 200 μm.

Figure 6:

Immunolocalization of p62 protein in the hippocampus and cortex of rat brain in control and vitamin D, omega-3, and vitamin D+omega-3 treatment groups. Black arrows indicate the p62-positive cells. Three independent experiments were performed. The micrographs were taken with 10× and 40× objectives. Scale bars: 100 μm, 200 μm.

Discussion

In the present study, we examined the effects of vitamin D and omega-3 supplements on the ERAD and autophagic protein expressions in the hippocampus and cortex of the rat brain. We demonstrated for the first time the expression of p97/VCP and SVIP proteins in rat brains with vitamin D and omega-3 supplements. In addition, we present evidence that vitamin D supplements importantly changed both the expression level of ERAD and autophagy proteins in rat brains. The histopathological results revealed that the cellular architectures of the hippocampus and cortex were normal throughout the groups.

Circulating 25(OH) vitamin D penetrates glial cells and neuronal cells after crossing the blood-brain barrier, where it is transformed into 1,25(OH)2 D, the active form of vitamin D [32]. The vitamin D receptor, through which vitamin D works in the brain, is a nuclear steroid receptor. It has been discovered that the brain produces and destroys vitamin D. Additionally, VDR is present in several parts of the brain and necessary for vitamin D to have its effects [33].

For the brain to develop into its full functional potential, omega-3 fatty acids are crucial. Neurodevelopmental disorders in humans have been related to low maternal n-3 polyunsaturated fatty acids (PUFA) intake [34]. Concerns about the potential negative consequences of low n-3 PUFA consumption on human child neurodevelopment [35] and the prevalence of neurodevelopmental illnesses, including Autism spectrum disorder and schizophrenia [36], have been raised globally.

Moreover, the central nervous system’s microglia, or nonneuronal cells, also contain nuclear VDRs [37]. Neuroprotection has been associated with the activation of the vitamin D pathway [38]. In the post-mortem human brain, neurons and glial cells express VDR, showing that vitamin D can be locally activated [39], 40]. The central nervous system has been shown to express the vitamin D receptor and the enzymes that bioactivate vitamin D, notably in regions where neurodegenerative diseases are prevalent, such as the hippocampus. Gezen-Ak et al. [41] have investigated the presence of 25-OH vitamin D-24-hydroxylase enzyme required for vitamin D catabolism in cortical and hippocampal neurons. They also contrast the expression of 24-hydroxylase and VDR in cortical and hippocampal neurons. They concluded that increased 24-hydroxylase and VDR gene expression may imply a greater requirement for vitamin D in the hippocampus and the possible impacts of vitamin D shortage on cognitive decline, neurodegeneration, and Alzheimer’s disease. To determine the VDR expression in the brain, we performed immunohistochemistry. The cellular localization of VDR was shown in the neuronal cells of the hippocampus and cortex of the rat brain. Additionally, the immunoexpression of VDR was significantly increased in the vitamin D-treated group.

The valosin-containing protein, which is ubiquitously expressed and supports numerous cellular processes in a cofactor-dependent manner, is encoded by the VCP gene. p97/VCP performs apoptosis, mitochondrial quality control, and protein homeostasis maintenance [24]. The ubiquitin-proteasome system and autophagy lysosomal systems work in conjunction with p97/VCP to regulate the cell cycle, produce organelles, sort vesicles, and maintain protein homeostasis, among other cellular activities [42]. Endoplasmic reticulum-associated protein production is decreased by VCP knockdown in primary cultured hippocampal neurons, which reduces the development of dendritic spines [43], 44]. The multisystem disease known as inclusion body myopathy associated with Paget’s disease of bone and frontotemporal dementia, which is defined by inclusion bodies in the brain or muscle, is related to mutations in the human VCP [45], 46]. According to Hirabayashi et al. [47], p97/VCP serves as both a pathogenic effector for several neurodegenerative symptoms and a recognition factor for proteins that are folded improperly. For the therapy of neurodegenerative diseases, p97/VCP may be the perfect molecular target.

SVIP has been associated with several physiological functions, including the regulation of autophagy, vacuole formation, and ERAD suppression. It serves as the adaptor protein for the multifunctional p97/VCP [23], 48], 49]. The study conducted by Y. Wang et al. [23] has shown that SVIP is primarily localized in the central and peripheral nervous systems. According to Jiawen Wu et al. [50], SVIP and p97/VCP were seen to co-localize in the cell bodies of neurons. It is important to note that they also discovered that in compact myelin, where p97/VCP was not present, SVIP co-localizes with myelin basic protein.

The molecular effects of 1,25(OH)2 D3 on the unfolded protein response (UPR), ERAD, and androgenic signaling were studied by Erzurumlu et al. [51]. In LNCaP prostate cancer cells, they discovered that 1,25(OH)2 D3 distinctly controlled the inositol-requiring enzyme 1 (IRE1) and protein kinase RNA-like ER kinase (PERK) branches of the UPR and adversely affected the expression level of ERAD components.

A recent study has indicated that SVIP plays a regulatory function in p97 subcellular localization and is a new regulator of autophagy [23]. Moreover, p97/VCP eases the degradation of misfolded proteins through UPS and ERAD, and it is essential for aggresome formation and protein aggregate trafficking. It has been previously reported that p97/VCP is in the regulation of autophagy [52]. Previous studies have indicated that vitamin D3 decreases cell apoptosis, protects cell survival, and reduces disease severity by autophagy regulation. Also, the study showed that vitamin D3 treatment develops autophagy by decreasing the expression of p62, LC3-II, and Beclin-1 proteins in the myocardium of rats treated with vitamin D3 [53]. In the present study, LC3B and p62 revealed reduced expressions in vitamin D-treated group compared to control in the rat brain and hippocampus.

In conclusion, we have identified the cellular localization and immunoexpression of ERAD pathway proteins (p97/VCP and SVIP) and autophagic proteins (LC3B and p62) in the rat hippocampus and cortex of the brain tissue. The immunolocalization of p97/VCP and SVIP was detected in the neuronal cells of the hippocampus and cortex of the brain. Additionally, it was established that vitamin D increased the immunoexpression of the ERAD pathway’s p97/VCP and SVIP proteins. Also, in this study, SVIP expressions were shown in the rat hippocampus and cortex of the brain tissue. Autophagic protein expressions LC3B and p62 tended to decrease in vitamin D and omega-3 groups compared to control. Accordingly, a negative correlation between ERAD markers (SVIP, p97/VCP) and autophagic markers (LC3B, p62) was observed, recommending a relationship between ERAD and autophagy.

In summary, the current study represented that vitamin D supplements activated the VDR pathway. To define autophagic flux and protein activities, cell culture studies with varied inhibitors and activators of ERAD and autophagy will absolutely explain the interrelationship between them with supplementations. To conclude, our results suggest that ERAD and autophagy protein expressions differ between the experimental groups. Therefore, we can recommend that more research is required to ascertain and determine the relationship between vitamin D and omega-3 supplementation and the pathways.