Dementia is a chronic disease leading to disability and dependency among the elderly worldwide. For clinical classification, dementia can be divided into the following major categories: Alzheimer’s disease (AD), vascular dementia (VaD), dementia with Lewy bodies (DLB), frontotemporal dementia (FTD), and mixed dementia (Raz et al., 2016). Due to the complexities of neurodegenerative dementia, precise diagnosis remains a challenge in daily clinical practice. Eventually, patients with dementia will present a catastrophic situation for the entire community and society, leading to highly expensive long-term medical care. Moreover, evidence indicates that sustained brain inflammation due to excessive microglial activation contributes to cerebral aging (Hickman et al., 2018; Sanada et al., 2018), neurodegenerative disease (Maragakis and Rothstein, 2006; Hickman et al., 2018), and neurodegenerative dementia (Meraz-Ríos et al., 2013; Bhaskar et al., 2014).

Microglia stabilize and balance the central nervous system (CNS) by mediating immune responses and providing neurotrophic support for neurons (Wake et al., 2011). Embryologically, microglia are derived from the same origin as peripheral macrophages (myeloid progenitors), whereas other resident CNS cells (e.g., astrocytes, oligodendrocytes, and neurons) are derived from neuroepithelial precursors (Zhang et al., 2018). Microglia can be regarded as a specialized macrophage in the CNS owing to the aforementioned close association. Microglia exert biphasic effects that either benefit the neuron survival (M2 phase) or undermine it (M1 phase) under microenvironmental changes. As such, it is representative of the spectrum of M1 (proinflammation)/M2 (anti-inflammation) phenotypes (Song and Suk, 2017; Zhang et al., 2017). In the microenvironment of the CNS, M2 microglia function to maintain CNS homeostasis and neural protection by moving dynamically throughout the entire CNS parenchyma and performing phagocytosis when they encounter pathological changes initiated by pathogens, plaques, protein aggregates, and infectious agents (Thomas et al., 2015). However, under the circumstances of neurodegeneration (non-stressed status) or CNS trauma (injury-challenged status), temporally inappropriate, prolonged, or excessive inflammation indicates the pathological activation of detrimental M1 microglial cells involving the release of cytokines and chemokines (Tator and Fehlings, 1991; Luo et al., 2002). Aberrantly stimulated microglia produce nitric oxide (NO) in conjunction with reactive oxygen species (ROS), which can amplify the inflammatory cascade. In a deleterious pathological cycle, cytokines promote inflammation by inducing the production of additional cytokines, chemokines, NO, and ROS (Li et al., 2006). Sustained microglia-driven inflammation can eventually lead to apoptosis and the killing of oligodendrocytes and neurons, which can impair neuronal functioning and lead to permanent neurological damage (Wood, 1995; Mattson, 2000; Byrnes et al., 2007). Any novel strategies involving the attenuation of microglial inflammatory response could be beneficial in the management of neurodegenerative disease and dementia.

CISD2 (CDGSH iron-sulfur domain 2) has garnered interest for its protective role against calcium excitotoxicity (Shen et al., 2017), apoptosis (Chen et al., 2009), and inflammation (Lin et al., 2015). CISD2 has been referred to as a longevity gene, aiding in the preservation of mitochondrial integrity, thereby preventing mitochondrial malfunction and, ultimately, cell death. In response to stress, CISD2 exerts protective effects against autophagy/apoptosis by enhancing BCL2-BECN1 interactions and by the formation of a complex with BCL2 (Chang et al., 2010). Moreover, CISD2 attenuates the excitotoxic Ca²⁺ surge at the endoplasmic reticulum by binding to BCL2 and the inositol 1,4,5-triphosphate receptor (Chang et al., 2012). Furthermore, overexpression of CISD2 has been demonstrated to attenuate neuronal loss and β-amyloid-induced mitochondrial dysfunction in the AD mouse model (Chen et al., 2020). Thus, CISD2 holds promise as a potential therapeutic target for neurodegenerative dementia.

In a previous study, we identified a potential role of CISD2 in exhibiting the anti-inflammatory effects. In a CISD2 knockdown model developed with siCISD2, we observed that CISD2 deficiency led to a significant increase in iNOS production and a reduction in BCL2 levels in SH-SY5Y cells stimulated with lipopolysaccharide (LPS; Lin et al., 2015). No previous study has been reported on the anti-inflammatory effects of CISD2 in microglia. In the current study, we sought to delineate the protective effect of CISD2 in microglial inflammatory reactions associated with neurodegenerative dementia. It is a preliminary investigation into the anti-inflammatory effects of CISD2 in the EOC microglial cell culture model with a specific aim of determining whether it plays a role in the alternation of microglial M1/M2 polarization.

As many protective molecules in nature exhibiting biphasic effects (Lin et al., 2011a,b), microglia-mediated neuroinflammation has been described as a double-edged sword (Hu et al., 2015). It demonstrates protective and restorative effects in the nonpathological and healthy CNS. However, it may also exacerbate and destroy under excessive inflammation under microenvironments of non-stressed (as a proxy for chronic inflammation driven by aging and neurodegenerative disease) and injury-challenged CNS (as a proxy for high-grade impact from neurotrauma). During the early stages of neural pathological conditions of the CNS (e.g., aging and neurodegenerative disease), protective M2 microglia tend to be dominant in the spectrum of proinflammatory/anti-inflammatory phenotypes. During low-grade inflammation, the activated M2 microglia scavenge fatal pathogens to confer neuronal protection. However, when the damage exceeds the range of homeostasis, a decline is observed in the proportion of activated M2 microglia. Accordingly, a delayed reactivation of detrimental M1 microglia can lead to a deleterious inflammatory cycle (Karve et al., 2016), which can be detrimental to neuronal survival and potentially cause CNS disease, such as dementia. Figure 4 presents a schematic illustration showing the proposed sequence of events associated with the anti-inflammatory effects of CISD2 in microglia, as determined in this study.

During the process of aging, the cerebral immune system remains relatively active resulting in the upregulation of inflammatory genes associated with the aging brain. Under such an immunocompetent response, cerebral microglia are primed to be activated and remain resistant to immune adaptive regulation (Norden and Godbout, 2013). This type of sustained microglial dysfunction can cause low-grade chronic neuroinflammation in the brain and thereby exacerbate the cerebral aging process (Simen et al., 2011). Unlike normal aging, neurodegenerative diseases (due to genetic or environmental influences) can lead to misfolding and accumulation of native cerebral proteins, such as prion, tau, β-amyloid, α-synuclein, and huntingtin. Conformational changes in these proteins are resistant to proteolysis resulting in the formation of so-called inclusion bodies in the brain (Jucker and Walker, 2011). When stimulated by toll-like receptors, the aggregation of such atypical proteins promotes the release of microglial-derived inflammatory mediators at high concentrations resulting in long-lasting inflammatory stimuli. Such effects are observed in AD, multiple sclerosis (MS), and Parkinson’s disease (PD; Ross and Poirier, 2004). Thus, microglia-driven inflammatory cascades have definitively been linked to aging, neurodegenerative diseases, and neurodegenerative dementia.

The precise function of CISD2 has not been elucidated. Interestingly, CISD2 expression is attenuated during non-stressed or injury-challenged conditions in vivo and in vitro (Lin et al., 2019). We observed a significant reduction in CISD2 expression with age in the mouse brain and spinal cord (Lin et al., 2019). In mouse models, spinal cord injury has been demonstrated to attenuate the expression of CISD2 (Lin et al., 2015). Moreover, we previously observed that CISD2 deficiency exacerbated inflammatory responses and mitochondrial dysfunction in cultured cells (with or without injury). Specifically, CISD2 expression was lower in non-stressed 35 DIV astrocytes than in 7 DIV cells, and the expression of iNOS along with RANTES (regulated on activation, normal T cell expressed and secreted) was higher (Lin et al., 2019). In LPS-stimulated CISD2 knockdown SH-SY5Y cells, the deficiency of CISD2 led to elevated iNOS levels and significant mitochondrial dysfunction (Lin et al., 2015, 2019). Resident immune cells (i.e., microglia) would be the apparent focus while investigating inflammatory cascades in the CNS.

Priming and activation in microglia can promote the proinflammatory signaling associated with aging, neurodegenerative disease, and traumatic CNS injury (Norden et al., 2015). Data obtained in this study demonstrated that CISD2 deficiency (induced by the knockdown of CISD2 expression) augmented the detrimental effects associated with the M1 phase and led to neuroinflammation and apoptosis in non-stimulated EOC microglial cells. Hence, we postulate that CISD2 exerts anti-inflammatory effects in microglia under non-stressed microenvironments (as a proxy for microglial inflammation driven by aging and neurodegenerative dementia). The attenuation of microglia-mediated inflammatory cascades can be beneficial under such pathological conditions. The beneficial effect of the dementia-CISD2-microglial anti-inflammation axis provides a strong rationale for further development of CISD2-based anti-inflammatory therapies for patients with the aforementioned disorders.

Our data provide novel evidence that the anti-inflammatory effect of microglial CISD2 is associated with the regulation of NFκB signaling. At present, the molecular mechanism underlying the anti-inflammatory effects of CISD2 has yet to be elucidated. When located in the cytoplasm and mitochondrial intermembrane space, the activation of NFκB, in addition to a role as well-recognized proinflammatory mediator, has been linked to inflammation, mitochondrial dysfunction, and eventual cellular apoptosis (Albensi, 2019). Thus, it can be interpreted that the attenuation of CISD2 owing to primary insults from neurotrauma or inflammation from CNS disease inhibits the protective effects against NFκB in cytoplasm and mitochondria leading to a remarkable inflammatory response and mitochondrial dysfunction. Furthermore, evidence has been obtained stating that calcium regulates the expression of TNF-α (Canellada et al., 2006) and modulates macrophage-driven inflammatory cascades (Racioppi et al., 2012). We posit that anti-inflammatory effects may be attributed to the regulatory role of CISD2 in calcium metabolism (Chang et al., 2012).

It has been suggested that CISD2 combines with BCL2 to antagonize the apoptotic mediator Beclin 1 (Chang et al., 2012). NFκB also regulates apoptotic effects via the combination or dissociation of Beclin 1/Bcl2 complex (Salminen et al., 2012). Moreover, elevated activity of NFκB transcription factors has also been demonstrated to modulate the expression of BCL2 (Chen et al., 2015). In this study, CISD2 was demonstrated to mediate the upstream regulation of NFκB, as observed by the DNA-binding activity of the NFκB p65 subunit. Collectively, the results indicate that CISD2 plays an upstream regulatory role in the functions of NFκB and BCL2. Advanced inflammation and mitochondrial dysfunction are highly likely to result in cell damage and negative cell survival. This may explain why CISD2 deficiency leads to potent inflammatory and apoptotic signaling.

As located in the outer membrane of mitochondria, CISD2 deficiency has been demonstrated to cause mitochondrial dysfunction and may undermine mitochondrial integrity. Note that mitochondrial dysfunction has been shown to inhibit M2 microglial polarization (Nakagawa and Chiba, 2014). It has been reported that M1 macrophages undergo aerobic glycolysis and pentose phosphate pathway (PPP) in obtaining a supply of energy. Conversely, M2 macrophages use acetyl-CoA and participate in the mitochondrial TCA (tricarboxylic acid) cycle (Culmsee et al., 2019). Thus, CISD2 deficiency-driven mitochondrial dysfunction contributes to a decrease in TCA cycle activities and augments aerobic glycolysis and the PPP pathway. In so doing, it leads to the promotion of the M1 phenotype along with the reduction of the M2 phenotype.

Some limitations have been attributed to this study. First, based on some previous novel findings involving non-immune cells (SH-SY5Y), we used the cell culture model developed with EOC microglial cell line to evaluate the mechanisms in this study. In future studies, it would be prudent to examine this putative mechanism in primary cultured microglia or via in vivo research using animal or human tissue. Moreover, the mechanism underlying the effect of CISD2 knockdown on M1/M2 microglia polarization will have to be verified using morphological analysis conducted in vitro or in vivo, such as staining for microglia M1/M2 profile marker. We demonstrated the involvement of CISD2 in the regulation of inflammatory genes under basal conditions in the present study using siCISD2 to attenuate CISD2 expression. Confirmation of this mechanism will be considered in further studies demonstrating the overexpression of CISD2 in microglia.

The results obtained in this study demonstrate that CISD2 exerts anti-inflammatory effects, which may be induced by the suppression of NFκB activation and the preservation of the protective M2 phenotype in non-stressed microglia. CISD2 deficiency in microglia could be one of the mechanisms underlying cell apoptosis, perhaps occurring via inflammation. CISD2-based anti-inflammatory therapy is a strong candidate for the treatment of neuroinflammation-driven aging and neurodegenerative diseases.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The study was reviewed and approved by the Animal Care and Ethics Committee of National Ilan University.

M-SL: writing—original draft, formal analysis, resources, methodology, funding acquisition, writing—review and editing, and conceptualization.

The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.