Microglial Sp1 induced LRRK2 upregulation in response to manganese exposure, and 17ß-estradiol afforded protection against this manganese toxicity.

Chronic exposure to elevated levels of manganese (Mn) causes a neurological disorder referred to as manganism, presenting symptoms similar to those of Parkinson's disease (PD), yet the mechanisms by which Mn induces its neurotoxicity are not completely understood. 17ß-estradiol (E2) affords neuroprotection against Mn toxicity in various neural cell types including microglia. Our previous studies have shown that leucine-rich repeat kinase 2 (LRRK2) mediates Mn-induced inflammatory toxicity in microglia. The LRRK2 promoter sequences contain three putative binding sites of the transcription factor (TF), specificity protein 1 (Sp1), which increases LRRK2 promoter activity. In the present study, we tested if the Sp1-LRRK2 pathway plays a role in both Mn toxicity and the protection afforded by E2 against Mn toxicity in BV2 microglial cells. The results showed that Mn induced cytotoxicity, oxidative stress, and tumor necrosis factor-α production, which were attenuated by an LRRK2 inhibitor, GSK2578215A. The overexpression of Sp1 increased LRRK2 promoter activity, mRNA and protein levels, while inhibition of Sp1 with its pharmacological inhibitor, mithramycin A, attenuated the Mn-induced increases in LRRK2 expression. Furthermore, E2 attenuated the Mn-induced Sp1 expression by decreasing the expression of Sp1 via the promotion of the ubiquitin-dependent degradation pathway, which was accompanied by increased protein levels of RING finger protein 4, the E3-ligase of Sp1, Sp1 ubiquitination, and SUMOylation. Taken together, our novel findings suggest that Sp1 serves as a critical TF in Mn-induced LRRK2 expression as well as in the protection afforded by E2 against Mn toxicity through reduction of LRRK2 expression in microglia.

Role of Astrocytes in Parkinson's Disease Associated with Genetic Mutations and Neurotoxicants.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons and the aggregation of Lewy bodies in the basal ganglia, resulting in movement impairment referred to as parkinsonism. However, the etiology of PD is not well known, with genetic factors accounting only for 10-15% of all PD cases. The pathogenetic mechanism of PD is not completely understood, although several mechanisms, such as oxidative stress and inflammation, have been suggested. Understanding the mechanisms of PD pathogenesis is critical for developing highly efficacious therapeutics. In the PD brain, dopaminergic neurons degenerate mainly in the basal ganglia, but recently emerging evidence has shown that astrocytes also significantly contribute to dopaminergic neuronal death. In this review, we discuss the role of astrocytes in PD pathogenesis due to mutations in α-synuclein (PARK1), DJ-1 (PARK7), parkin (PARK2), leucine-rich repeat kinase 2 (LRRK2, PARK8), and PTEN-induced kinase 1 (PINK1, PARK6). We also discuss PD experimental models using neurotoxins, such as paraquat, rotenone, 6-hydroxydopamine, and MPTP/MPP+. A more precise and comprehensive understanding of astrocytes' modulatory roles in dopaminergic neurodegeneration in PD will help develop novel strategies for effective PD therapeutics.

Astrocytic Yin Yang 1 is critical for murine brain development and protection against apoptosis, oxidative stress, and inflammation.

The transcription factor Yin Yang 1 (YY1) is ubiquitously expressed in mammalian cells, regulating the expression of a variety of genes involved in proliferation, differentiation, and apoptosis in a context-dependent manner. While it is well-established that global YY1 knockout (KO) leads to embryonic death in mice and that YY1 deletion in neurons or oligodendrocytes induces impaired brain function, the role of astrocytic YY1 in the brain remains unknown. We investigated the role of astrocytic YY1 in the brain using a glial fibrillary acidic protein (GFAP)-specific YY1 conditional KO (YY1 cKO) mouse model to delete astrocytic YY1. Astrocytic YY1 cKO mice were tested for behavioral phenotypes, such as locomotor activity, coordination, and cognition, followed by an assessment of relevant biological pathways using RNA-sequencing analysis, immunoblotting, and immunohistochemistry in the cortex, midbrain, and cerebellum. YY1 cKO mice showed abnormal phenotypes, movement deficits, and cognitive dysfunction. At the molecular level, astrocytic YY1 deletion altered the expression of genes associated with proliferation and differentiation, p53/caspase apoptotic pathways, oxidative stress response, and inflammatory signaling including NF-κB, STAT, and IRF in all regions. Astrocytic YY1 deletion significantly increased the expression of GFAP as astrocytic activation and Iba1 as microglial activation, indicating astrocytic YY1 deletion activated microglia as well. Accordingly, multiple inflammatory cytokines and chemokines including TNF-α and CXCL10 were elevated. Combined, these novel findings suggest that astrocytic YY1 is a critical transcription factor for normal brain development and locomotor activity, motor coordination, and cognition. Astrocytic YY1 is also essential in preventing pathological oxidative stress, apoptosis, and inflammation.

Deletion of RE1-silencing transcription factor in striatal astrocytes exacerbates manganese-induced neurotoxicity in mice.

Chronic manganese (Mn) overexposure causes a neurological disorder, referred to as manganism, exhibiting symptoms similar to parkinsonism. Dysfunction of the repressor element-1 silencing transcription factor (REST) is associated with various neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and Mn-induced neurotoxicity, but its cellular and molecular mechanisms have yet to be fully characterized. Although neuronal REST is known to be neuroprotective, the role of astrocytic REST in neuroprotection remains to be established. We investigated if astrocytic REST in the striatal region of the mouse brain where Mn preferentially accumulates plays a role in Mn-induced neurotoxicity. Striatal astrocytic REST was deleted by infusion of adeno-associated viral vectors containing sequences of the glial fibrillary acidic protein promoter-driven Cre recombinase into the striatum of RESTflox/flox mice for 3 weeks, followed by Mn exposure (30 mg/kg, daily, intranasally) for another 3 weeks. Striatal astrocytic REST deletion exacerbated Mn-induced impairment of locomotor activity and cognitive function with further decrease in Mn-reduced protein levels of tyrosine hydroxylase and glutamate transporter 1 (GLT-1) in the striatum. Astrocytic REST deletion also exacerbated the Mn-induced proinflammatory mediator COX-2, as well as cytokines such as TNF-α, IL-1ß, and IL-6, in the striatum. Mn-induced detrimental astrocytic products such as proinflammatory cytokines on neuronal toxicity were attenuated by astrocytic REST overexpression, but exacerbated by REST inhibition in an in vitro model using primary human astrocytes and Lund human mesencephalic (LUHMES) neuronal culture. These findings indicate that astrocytic REST plays a critical role against Mn-induced neurotoxicity by modulating astrocytic proinflammatory factors and GLT-1.

Mechanisms of manganese-induced neurotoxicity and the pursuit of neurotherapeutic strategies.

Chronic exposure to elevated levels of manganese via occupational or environmental settings causes a neurological disorder known as manganism, resembling the symptoms of Parkinson's disease, such as motor deficits and cognitive impairment. Numerous studies have been conducted to characterize manganese's neurotoxicity mechanisms in search of effective therapeutics, including natural and synthetic compounds to treat manganese toxicity. Several potential molecular targets of manganese toxicity at the epigenetic and transcriptional levels have been identified recently, which may contribute to develop more precise and effective gene therapies. This review updates findings on manganese-induced neurotoxicity mechanisms on intracellular insults such as oxidative stress, inflammation, excitotoxicity, and mitophagy, as well as transcriptional dysregulations involving Yin Yang 1, RE1-silencing transcription factor, transcription factor EB, and nuclear factor erythroid 2-related factor 2 that could be targets of manganese neurotoxicity therapies. This review also features intracellular proteins such as PTEN-inducible kinase 1, parkin, sirtuins, leucine-rich repeat kinase 2, and α-synuclein, which are associated with manganese-induced dysregulation of autophagy/mitophagy. In addition, newer therapeutic approaches to treat manganese's neurotoxicity including natural and synthetic compounds modulating excitotoxicity, autophagy, and mitophagy, were reviewed. Taken together, in-depth mechanistic knowledge accompanied by advances in gene and drug delivery strategies will make significant progress in the development of reliable therapeutic interventions against manganese-induced neurotoxicity.

Astrocytic transcription factor REST upregulates glutamate transporter EAAT2, protecting dopaminergic neurons from manganese-induced excitotoxicity.

Chronic exposure to high levels of manganese (Mn) leads to manganism, a neurological disorder with similar symptoms to those inherent to Parkinson's disease. However, the underlying mechanisms of this pathological condition have yet to be established. Since the human excitatory amino acid transporter 2 (EAAT2) (glutamate transporter 1 in rodents) is predominantly expressed in astrocytes and its dysregulation is involved in Mn-induced excitotoxic neuronal injury, characterization of the mechanisms that mediate the Mn-induced impairment in EAAT2 function is crucial for the development of novel therapeutics against Mn neurotoxicity. Repressor element 1-silencing transcription factor (REST) exerts protective effects in many neurodegenerative diseases. But the effects of REST on EAAT2 expression and ensuing neuroprotection are unknown. Given that the EAAT2 promoter contains REST binding sites, the present study investigated the role of REST in EAAT2 expression at the transcriptional level in astrocytes and Mn-induced neurotoxicity in an astrocyte-neuron coculture system. The results reveal that astrocytic REST positively regulates EAAT2 expression with the recruitment of an epigenetic modifier, cAMP response element-binding protein-binding protein/p300, to its consensus binding sites in the EAAT2 promoter. Moreover, astrocytic overexpression of REST attenuates Mn-induced reduction in EAAT2 expression, leading to attenuation of glutamate-induced neurotoxicity in the astrocyte-neuron coculture system. Our findings demonstrate that astrocytic REST plays a critical role in protection against Mn-induced neurotoxicity by attenuating Mn-induced EAAT2 repression and the ensuing excitotoxic dopaminergic neuronal injury. This indicates that astrocytic REST could be a potential molecular target for the treatment of Mn toxicity and other neurological disorders associated with EAAT2 dysregulation.

Manganese-induced reactive oxygen species activate IκB kinase to upregulate YY1 and impair glutamate transporter EAAT2 function in human astrocytes in vitro.

Dysregulation of the astrocytic glutamate transporter excitatory amino acid transporter 2 (EAAT2) is associated with several neurological disorders, including Parkinson's disease, Alzheimer's disease, and manganism, the latter induced by chronic exposure to high levels of manganese (Mn). Mechanisms of Mn-induced neurotoxicity include impairment of EAAT2 function secondary to the activation of the transcription factor Yin Yang 1 (YY1) by nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). However, the upstream mechanisms by which Mn-induced NF-κB activates YY1 remain to be elucidated. In the present study, we used the H4 human astrocyte cell line to test if Mn activates YY1 through the canonical NF-κB signaling pathway, leading to EAAT2 repression. The results demonstrate that Mn exposure induced phosphorylation of the upstream kinase IκB kinase (IKK-ß), leading to NF-κB p65 translocation, increased YY1 promoter activity, mRNA/protein levels, and consequently repressed EAAT2. Results also demonstrated that Mn-induced oxidative stress and subsequent TNF-α production were upstream of IKK-ß activation, as antioxidants attenuated Mn-induced TNF-α production and IKK-ß activation. Moreover, TNF-α inhibition attenuated the Mn-induced activation of IKK-ß and YY1. Taken together, Mn-induced oxidative stress and TNF-α mediates activation of NF-κB signaling and YY1 upregulation, leading to repression of EAAT2. Thus, targeting reactive oxygen species (ROS), TNF-α and IKK-ß may attenuate Mn-induced YY1 activation and consequent EAAT2 repression.

Manganese Accumulation in the Brain via Various Transporters and Its Neurotoxicity Mechanisms.

Manganese (Mn) is an essential trace element, serving as a cofactor for several key enzymes, such as glutamine synthetase, arginase, pyruvate decarboxylase, and mitochondrial superoxide dismutase. However, its chronic overexposure can result in a neurological disorder referred to as manganism, presenting symptoms similar to those inherent to Parkinson's disease. The pathological symptoms of Mn-induced toxicity are well-known, but the underlying mechanisms of Mn transport to the brain and cellular toxicity leading to Mn's neurotoxicity are not completely understood. Mn's levels in the brain are regulated by multiple transporters responsible for its uptake and efflux, and thus, dysregulation of these transporters may result in Mn accumulation in the brain, causing neurotoxicity. Its distribution and subcellular localization in the brain and associated subcellular toxicity mechanisms have also been extensively studied. This review highlights the presently known Mn transporters and their roles in Mn-induced neurotoxicity, as well as subsequent molecular and cellular dysregulation upon its intracellular uptakes, such as oxidative stress, neuroinflammation, disruption of neurotransmission, α-synuclein aggregation, and amyloidogenesis.

Astrocyte-specific deletion of the transcription factor Yin Yang 1 in murine substantia nigra mitigates manganese-induced dopaminergic neurotoxicity.

Manganese (Mn)-induced neurotoxicity resembles Parkinson's disease (PD), but the mechanisms underpinning its effects remain unknown. Mn dysregulates astrocytic glutamate transporters, GLT-1 and GLAST, and dopaminergic function, including tyrosine hydroxylase (TH). Our previous in vitro studies have shown that Mn repressed GLAST and GLT-1 via activation of transcription factor Yin Yang 1 (YY1). Here, we investigated if in vivo astrocytic YY1 deletion mitigates Mn-induced dopaminergic neurotoxicity, attenuating Mn-induced reduction in GLAST/GLT-1 expression in murine substantia nigra (SN). AAV5-GFAP-Cre-GFP particles were infused into the SN of 8-week-old YY1 flox/flox mice to generate a region-specific astrocytic YY1 conditional knockout (cKO) mouse model. 3 weeks after adeno-associated viral (AAV) infusion, mice were exposed to 330 µg of Mn (MnCl2 30 mg/kg, intranasal instillation, daily) for 3 weeks. After Mn exposure, motor functions were determined in open-field and rotarod tests, followed by Western blotting, quantitative PCR, and immunohistochemistry to assess YY1, TH, GLAST, and GLT-1 levels. Infusion of AAV5-GFAP-Cre-GFP vectors into the SN resulted in region-specific astrocytic YY1 deletion and attenuation of Mn-induced impairment of motor functions, reduction of TH-expressing cells in SN, and TH mRNA/protein levels in midbrain/striatum. Astrocytic YY1 deletion also attenuated the Mn-induced decrease in GLAST/GLT-1 mRNA/protein levels in midbrain. Moreover, YY1 deletion abrogated its interaction with histone deacetylases in astrocytes. These results indicate that astrocytic YY1 plays a critical role in Mn-induced neurotoxicity in vivo, at least in part, by reducing astrocytic GLAST/GLT-1. Thus, YY1 might be a potential target for treatment of Mn toxicity and other neurological disorders associated with dysregulation of GLAST/GLT-1.