The Role of Endoplasmic Reticulum Stress and Its Regulation in the Progression of Neurological and Infectious Diseases

Abstract

The unfolded protein response (UPR) is a cellular mechanism activated by endoplasmic reticulum (ER) stress, which ranges from inhibition of protein synthesis to apoptosis. ER stress is induced in general by aggregated autologous or foreign (e.g. viral) proteins, oxidative stress, mitochondrial dysfunction, disruption of intracellular calcium, or inflammation. In patients with Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS), the known stressors are aggregated amyloid-beta and superoxide dismutase (SOD-1), respectively, but autologous DNA released by trauma into the cytoplasm may also be involved in ALS. In HIV-1-associated neurocognitive disorders (HAND), ER stress is induced by HIV-1 and antiretroviral therapy. Additionally, in cases of epilepsy, ER stress has been implicated in neuronal dysfunction. In this chapter, we examine a clinical and immunologic approach to ER stress in the progression of neurological and infectious diseases. In addition, we will briefly discuss emerging treatments including omega fatty acids, progesterone, and DHA, which repair and favorably regulate UPR in some patients with neurological diseases.

1. Introduction

For decades, researchers in a diverse array of scientific fields have been working to understand the pathogenesis of neurodegenerative diseases including Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS). However, there is still much discourse within the scientific community on what exactly causes such diseases to occur. In recent years, the endoplasmic reticulum (ER), has been implicated in the progression of neurological diseases [1] including AD, ALS, epilepsy, and human immunodeficiency virus (HIV)-associated neurocognitive disorder (HAND). To understand how exactly this cellular organelle can lead to such detrimental neurological disorders, a basic understanding of the function of the ER is necessary.

The ER is a cellular organelle that functions to fold proteins correctly following their translation by ribosomes. The ER also plays an integral role in physiological homeostasis, primarily via calcium regulation [2] and protein synthesis [3]. Typically, correctly folded proteins are secreted by the ER and are transported to the Golgi body for further processing and sorting for transport to their eventual destinations. However, ER overload results in protein misfolding and accumulation of unfolded proteins in the ER lumen, which induces ER stress [4]. ER stress signals the cell to enter the survival pathway and initiates an unfolded protein response (UPR) [5] through three ER membrane-associated proteins: inositol requiring enzyme 1 (IRE1), pancreatic ER kinase (PERK), and activating transcription factor-6 (ATF6) [6].

IRE1 (encoded by the ERN1 gene) is an ER transmembrane receptor sensitive to misfolded protein aggregation in the ER lumen [7]. Activated IRE1 stimulates the translation of the X-box binding protein 1 (XBP1) into a transcription factor, which upregulates ER chaperones and ER-associated degradation (ERAD) [8]. Ideally in a functioning ER, these ERAD elements will assist with the degradation of unfolded proteins within the ER lumen to reduce ER stress.

PERK reduces protein synthesis by inactivation of eukaryotic initiation factor 2α, leading either to a pro-apoptotic pathway or a protective pathway involving chaperones and foldases [9]. Thus, PERK expression will mitigate the additional buildup of proteins within the ER. However, in extreme conditions, the cell suffers apoptosis in response to excessive misfolded protein aggregation [10].

Finally, ATF6 increases the transcription of several ER proteins including chaperones, foldases, and ERAD elements. These ER proteins increase the cell’s ability to fold proteins and reduce the load of unfolded proteins in the ER [11].

These ER stress proteins communicate between ER and mitochondria in the region called the mitochondria-associated ER membrane [9]. Activation of all three of these transmembrane proteins is prompted by unfolded protein buildup in the lumen of the ER, which in turn activates the nuclear transcription of ERAD elements (Figure 1).

Figure 1.

A scheme of the unfolded protein response in cells with ER stress. Illustration created with Biorender.com.

Thus, the role of ER stress in the progression of AD, ALS, epilepsy, and HAND are of rising interest in the scientific community and may serve as a possible explanation for their development. Due to the increasing evidence implicating ER stress in these diseases, researchers have shifted their focus to studying possible treatments that target the ER to mitigate disease progression. In this chapter, we will discuss the role of ER stress in AD, ALS, epilepsy, and HAND, and review the current therapeutic options for treating ER stress such as omega fatty acids including docosahexaenoic acid (DHA), and progesterone.

2. ER stress and the UPR in Alzheimer’s disease

AD is a progressive neurodegenerative disease characterized by the buildup of the cytotoxic protein aggregates amyloid-beta (Aβ) and fibrillary phospho-tau (P-τ). Within the brain, Aβ accumulates into plaques that can block synaptic function [12], induce ER stress [13], and ultimately cause neuronal apoptosis [14]. These cytotoxic proteins ultimately lead to widespread neurodegeneration leading to cognitive decline [12]. AD can arise via genetic factors in familial AD (fAD) or lifestyle factors in sporadic AD (sAD), but ER stress has been shown to play a role in the progression of both cases.

Many factors including oxidative stress, mitochondrial dysfunction, glutamate-induced neurotoxicity, and imbalance of calcium contribute to ER stress and UPR induction in AD patients [15].

Oxidative stress in the cell often results from the buildup of reactive oxygen species (ROS) from dysfunctional mitochondria [16] and can induce ER stress. In patients with AD, an imbalance in antioxidants can lead to an increase in ROS which can cause widespread cell damage [16]. In normal aging, this antioxidant imbalance is increasingly common and the body is often pushed into a “pro-oxidative state” meaning that the level of ROS is higher than normal [17]. However, in patients with AD, the aggregation of Aβ exacerbates this pro-oxidative state [18]. A 2001 study revealed that Aβ’s interaction with Iron within the brain causes increased ROS, and by treating aggregated Aβ with an Iron chelator there is a marked reduction of neural toxicity [18]. Typically, ROS levels are mediated by the ER proteins PERK and ATF6. However, when ROS levels exceed the normal range, PERK and ATF6 are unable to produce enough antioxidants, and the ER enters a state of ER stress [17], exacerbating cellular dysfunction and leading to neuronal apoptosis (Figure 2).

Figure 2.

A scheme depicting the effect of decreased antioxidants, glutamate neurotoxicity (GNT), and amyloid-beta (Aβ) aggregation on the buildup of ROS and ER stress in AD patients. Image created with Biorender.com.

Additionally, glutamate has been implicated in the buildup of ROS in the AD brain. When glutamate binds to NMDA receptors, this prompts glutamate-induced neurotoxicity (GNT). GNT causes a major influx of calcium into the cell and prompts the release of ROS from the mitochondria [19]. A buildup of GNT in the AD brain can impair glutamate receptors necessary for metabolism [20], lead to increased production of ROS, and cause an imbalance of cytosolic calcium leading to cell death (Figure 2) [19].

However, the exact etiology of AD concerning ER stress is still under investigation. Recent studies have pointed to the buildup of Aβ and P-τ as the cause of the development of ER stress and the UPR [13]. On the other hand, some believe that ER stress arises from increased ROS in the brain, leading to the buildup of Aβ and P-τ, which are released into the brain following cellular apoptosis [21]. Therefore, the sequence of biochemical events in AD is still very much under debate.

Additionally, the presenilin proteins have been implicated in ER-stress-mediated AD pathogenesis. Presenilin 1 (PS1) and presenilin 2 (PS2) are part of the γ-secretase complex which mediates the cleavage of the amyloid-beta precursor protein (APP) [22]. In fAD, mutations in these proteins have been found to alter the amyloid precursor protein (APP) cleavage process, resulting in higher levels of cytotoxic Aβ [21]. The mutant PS1 affects the ER stress response attributed to the inhibited activation of the ER stress pathways IRE1, PERK, and ATF6. Cells expressing PS1 mutants also display increased Aβ production and increased sensitivity to apoptosis caused by ER stress [21]. Therefore, the damage associated with the PS1 mutant proteins cannot be reversed by chaperones and folding proteins, and apoptosis is the most common outcome. In patients with sAD, mutations in the PS2 gene are also linked to disease progression by downregulation of the UPR pathways [23].

The target genes of the transcription factor XBP1 are also linked to AD. XBP1 affects the expression of at least one of the key proteins in the γ-secretase complex, primarily UBQLN1, the gene coding for ubiquitin, which is a negative regulator of presenilins. UBQLN1 plays a role in the control of APP trafficking. Therefore, in the production of Aβ, reduced expression of XBP1 in AD increases the production of Aβ and causes apoptosis [24].

Thus, the combined effects of a buildup of ROS, mitochondrial dysfunction, GNT, and presenilin mutation exacerbate the effects of normal aging in patients with AD and lead to an increase in ER stress. Thus, ER stress has been shown to play an integral role in the pathogenesis of AD through several pathways.

3. ER stress in epilepsy

Epilepsy is a neurological disease that involves chronic seizures as well as abnormal brain activity, which causes periods of unusual behavior or sensations [25]. There are different types of epileptic seizures, including generalized, focal, and unknown onset epilepsy, which differ in the area in which seizures occur [26]. The most common type of epilepsy among adults is focal epilepsy known as temporal lobe epilepsy (TLE), however, patients who continue to experience seizures are at risk of other areas of the brain becoming damaged, such as the hippocampus [27].

Current research incriminates ER stress as an important factor in the pathogenesis of epilepsy [25]. Neuronal death after a seizure is caused by apoptotic signaling pathways, of which there have been two gene families identified to be critical. These gene families are the Bcl-2 family proteins and the caspases [27], which have both been linked to ER stress generation [28]. Bcl-2 proteins are apoptosis regulators, and caspases are proteases that cleave certain proteins and enzymes that induce apoptosis. A study on the hippocampus of patients with epilepsy in 2006 revealed that caspases 6, 7, and 9 were higher in patients with epilepsy than in controls and were localized to the ER-containing region of the temporal lobe [27]. Additionally, patients’ hippocampal regions contained altered levels of Bcl-2 protein and an increase in both the ER stress-related motif KDEL and calnexin [27]. KDEL and calnexin are markers for ER stress and were used here to examine levels of ER stress in epilepsy patients [27, 29]. Finally, the researchers showed co-localization of the ER stress marker KDEL and caspases within the epileptic patients’ brain [27]. Thus, this study found that apoptotic pathways in epilepsy are directly linked to the ER and can induce ER stress.

The ER stress-related pathways discussed earlier were also abnormal in animal studies. In a rat model of status epilepticus (SE), phosphorylated PERK (p-PERK), phosphorylated eIF2α (p-eIF2α), and C/EBP homologous protein (CHOP) [30] were increased. Although the CHOP protein was considered as causing cell death, it has been reinterpreted as protective against neuronal death after a seizure [31]. In another study, the hippocampus of mice with status epilepticus (SE) was found to have elevated mRNA levels of spliced XBP1 compared to controls [31], suggesting an activation of the IRE1 branch. Higher XBP1 levels lead to an increase in the expression of chaperones for ER stress reduction. In the same study, it was found that these mice also had increased levels of ATF in the hippocampus [31]. Therefore, ER stress in epilepsy appears to induce both injurious pro-apoptotic and beneficial anti-apoptotic pathways.

4. ER stress in amyotrophic lateral sclerosis

The neurologic disorder ALS is characterized by its neuropathology demonstrating neuronal apoptosis in the spinal cord and brain, and inflammatory attack of neurons induced by aggregated superoxide dismutase (SOD-1) and other stimuli [32, 33]. The progression of ALS is associated with a gradual decrease in movement, paralysis, and overall weakness [34]. ALS has a sporadic form (sALS) with an unknown cause and a familial form (fALS) [35].

ER stress in individuals with fALS can be triggered by the Cu/Zn SOD-1 protein malfunction. This protein is responsible for degrading ROS, therefore, a mutation in SOD-1 promotes the disease [36]. As previously described, the increase of ROS results from the increase in GNT and a greater amount of calcium within the cell, damaging both the ER and mitochondria [1]. Disease progression of ALS is identified by mitochondrial dysfunction and a change in the mitochondrial membrane structure due to a release in molecules that initiate apoptosis [21]. Due to the aggregation of mutated SOD1 in the mitochondria of motor neurons, motor skills are adversely affected [35]. The protein previously implicated in neuronal apoptosis in epilepsy, Bcl-2, is also responsible for maintaining the structural integrity of the mitochondrial membrane [37]. When mutated SOD-1 interacts with the Bcl-2 protein in ALS patients, the membrane is weakened. Thus, there is an increase in ROS production due to the mutation of the SOD-1 protein creating an increase in ER stress in individuals with ALS (Figure 3) [21].

Figure 3.

A scheme depicting the cycle of ER stress buildup in ALS. Illustration created with Biorender.com.

In ALS individuals, caspase activation is also common [38]. We know from caspase’s role in epilepsy that caspases have been implicated in neuronal apoptosis and ER stress genesis [28]. Caspase activation is especially prevalent when the SOD1 mutation is present. In a mouse model of mutant Cu/Zn SOD-1 exhibiting symptoms of ALS, an increase in oxidative stress was related to activation of caspase 12, 9, and 3 in the spinal cord [38]. Caspase 12 is located in the endoplasmic reticulum [39] wherein the cleavage of this enzyme overall increases the oxidative stress further causing an exaggeration of ALS symptoms in the mice. This cleavage of caspase-12 may be a result of the activation of calpain, a calcium-dependent enzyme, in the mice’s spinal cord [21].

Thus, SOD-1 aggregates in individuals with ALS exacerbate ROS release and mitochondrial dysfunction, which induces ER stress and leads to eventual neuronal apoptosis.

5. ER stress in human immunodeficiency virus 1

Human immunodeficiency virus 1 (HIV-1) is the result of acquired immunodeficiency syndrome (AIDS), characterized by the destruction of CD4 T cells [40]. In addition to the dramatic opportunistic infection Pneumocystis carinii pneumonia, AIDS is associated with degenerative complications in the brain and the heart, HIV-1 encephalitis, and cardiomyopathy, which display inflammatory damage by virus-infected monocyte/macrophages [40]. HIV-1 particles are carried into the brain and the heart in monocytes through interendothelial gaps opened by virus envelope gp120, TNF-α, and the antiretroviral (ART) drug azidothymidine [41]. Because of this, besides encephalitis, the neuropathology of HIV-1 includes subtler HIV-associated neurocognitive disorders (HAND) [42].

ER stress and UPR activation play a large role in the neurodegeneration of AIDS and HIV-1 patients and, thus, are a potential therapeutic target. In HAND, the expression of the three UPR pathways in astrocytes leads to the opening of the mitochondrial permeability transition pore (mPTP) linked to apoptosis [42]. The inflammatory cytokine IL-1β along with ART drugs increases cytosolic calcium and triggers ER stress through upregulation of UPR pathways, mitochondrial depolarization, excitotoxicity, and increased ROS [43]. A recent study in 2020 examined the effects of ART on the ER stress pathway in HAND. The researchers found that HIV along with IL-1β increased the UPR transcripts IRE1, PERK, and ATF6. Another HAND signal, the nucleotide reverse transcriptase inhibitor (NRTI) abacavir, upregulated AEG-1 transcription and regulated calcium signaling and ER quality control by co-localizing with calnexin, an integral calcium-dependent chaperon protein in the ER [43]. Together, IL-1B and abacavir induce increased intracellular calcium (via ER calcium release) in astrocytes to levels comparable to that of the known ER stressor thapsigargin [43]. A prolonged intracellular increase in calcium triggers mitochondrial depolarization and ER stress response, as it increases mitochondrial permeability transition pore (mPTP). The mPTP opening leads to increased ROS as well as calcium-dependent exocytosis of glutamate, which causes GNT and neuronal damage experienced in HAND [43].

Additionally, HIV-1 upregulates the Tat protein, which induces GNT, ER stress, mitochondrial dysfunction, and UPR [44]. Therefore, astrocyte ER stress could act as a therapeutic target for HIV-1 neuronal infection: IL-1β and abacavir induce intracellular calcium dysregulation and mitochondrial dysfunction (mPTP opening), which can lead to apoptosis via triggering ER stress and increase in UPR signaling pathways [43].

6. Therapies for treating ER stress in neurodegenerative patients

Due to its implications in a variety of neurodegenerative diseases, ER stress and UPR proteins are growing targets for immunotherapy treatment for several neurological disorders. Various lipid-based molecules such as omega-3 fatty acids and progesterone induce neuroprotective effects against ER stress, regulating Aβ-induced neuroinflammation.

Omega-3 fatty acids have been found to modulate UPR counteracting ER stress. In macrophages of patients with AD and mild cognitive impairment (MCI), in vitro supplementation with fish-derived ω-3 fatty acids resulted in downregulation of ER stress signature genes CHOP, DDIT3, and CASP3 (caspase-3), which typically promote apoptosis [15]. Further, fatty acid treatment upregulated genes associated with UPR proteins including IRE1, ATF6, and ATF4 [15]. Thus, the researchers concluded that the phospho-PERK pathway was heterogeneously affected by omega-3 fatty acids via an increase in immunoenhancement but a decrease in pro-apoptosis, but omega-3 can enhance UPR genes necessary for combating ER stress in patients with AD [15].

Docosahexaenoic acid (DHA), a specific omega-3 fatty acid beneficially responds to the ER stress induced by traumatic brain injury (TBI), which triggers calcium homeostatic disruption. The mechanism by which DHA prevents ER stress is through its protectins, which block ER stress-inducing IP₃R-mediated ER Ca²⁺ depletion [45]. Protectins such as neuroprotection D1 (NPD1), a DHA derivative, upregulate anti-apoptotic factors and downregulate pro-apoptotic factors [45]. In addition, DHA’s resolving derivatives also directly decrease the inflammation responses to ER stress by blocking pro-inflammatory cytokines TNF-α and ILβ1 as well as reducing pro-inflammatory mediators prostaglandin E2, thromboxanes, and leukotrienes [45]. DHA administration reduced post-TBI increase of CHOP-gene expressing microglia and macrophages [46]. Thereby, DHA as an omega-3 fatty is seen to decrease the triggering and the effects of ER stress through various mechanisms and is, therefore, a potential therapy.

The neurosteroid progesterone has additionally been seen to improve ER stress in its applications in AD astrocytes with the release of IL-1 and TNF-α [47]. In these AD cells, Aβ-induced ER stress and inflammation were mediated by progesterone. Progesterone is associated with the downregulation of pro-inflammatory cytokines which in turn reduces ER stress activation. This effect is further shown through progesterone’s attenuation of GRP78 expression, which is implicated in amyloid-beta-induced ER stress response [47].

Though therapies targeting neurodegenerative diseases such as ALS or epilepsy have not implicated ER stress as a potential therapeutic target, substantial research on ER stress treatment in AD brains has uncovered omega-3 fatty acids such as DHA and neurosteroid progesterone as potential supplemental therapies for ER stress and neuroinflammation.

7. Conclusions

ER stress has been implicated in several neurological and infectious diseases, primarily AD, ALS, epilepsy, and HIV-1. In AD, mutant PS1 and PS2 induced amyloid-beta aggregation and an increase in ROS lead to ER stress and neuronal death. In ALS, aggregated SOD-1 increases the buildup of ROS in the cytoplasm, inducing ER stress. Patients with epilepsy have also shown elevated ER stress with increased levels of pro-apoptotic chaperones, yet the exact mechanism is still under investigation. Finally, HIV-1-associated neurocognitive decline leads to a buildup of the pro-inflammatory cytokine IL-1B which induces ER stress and exacerbates neurocognitive decline. The apparent role of ER stress in the progression of these neurological and infectious diseases has prompted scientists to explore treatments targeting ER stress and the UPR. Treatment with omega fatty acids, progesterone, and DHA have shown positive effects on ER stress levels in patients with AD, yet a true treatment has not been developed.

Conflict of interest

The authors declare no conflict of interest.