Relevance of biometals during neuronal differentiation and myelination: in vitro and in vivo studies.

Biometals are essential during the development of the central nervous system (CNS) since they participate in the organization and regulation of multiple processes related with the proper organization and functioning of brain structures. Neuronal differentiation is a specialized and complex process that occurs actively from embryonic development to the first years of life and is even maintained in specific areas of the mammalian adult brain. In this review, we focus on describing the cellular and molecular mechanisms of trace biometals such as iron (Fe), zinc (Zn), copper (Cu), and manganese (Mn) on neuronal specialization, comprising from brain uptake to effects on synaptogenesis, axonal outgrowth, myelination, and cellular and neurochemical phenotype determination. We highlight the relevance of biometals in the proper brain functioning by discussing some of the potentially detrimental effects when biometal dyshomeostasis occurs in the brain. Finally, future directions are proposed for exploring the relevance of biometals in brain function using pharmacological, molecular, and analytical approaches.

Oxidative Stress and Mitochondrial Complex I Dysfunction Correlate with Neurodegeneration in an α-Synucleinopathy Animal Model.

The α-synucleinopathies constitute a subset of neurodegenerative disorders, of which Parkinson's disease (PD) is the most common worldwide, characterized by the accumulation of misfolded α-synuclein in the cytoplasm of neurons, which spreads in a prion-like manner to anatomically interconnected brain areas. However, it is not clear how α-synucleinopathy triggers neurodegeneration. We recently developed a rat model through a single intranigral administration of the neurotoxic ß-sitosterol ß-D-glucoside (BSSG), which produces α-synucleinopathy. In this model, we aimed to evaluate the temporal pattern of levels in oxidative and nitrosative stress and mitochondrial complex I (CI) dysfunction and how these biochemical parameters are associated with neurodegeneration in different brain areas with α-synucleinopathy (Substantia nigra pars compacta, the striatum, in the hippocampus and the olfactory bulb, where α-syn aggregation spreads). Interestingly, an increase in oxidative stress and mitochondrial CI dysfunction accompanied neurodegeneration in those brain regions. Furthermore, in silico analysis suggests a high-affinity binding site for BSSG with peroxisome proliferator-activated receptors (PPAR) alpha (PPAR-α) and gamma (PPAR-γ). These findings will contribute to elucidating the pathophysiological mechanisms associated with α-synucleinopathies and lead to the identification of new early biomarkers and therapeutic targets.

Mechanistic Insight from Preclinical Models of Parkinson's Disease Could Help Redirect Clinical Trial Efforts in GDNF Therapy.

Parkinson's disease (PD) is characterized by four pathognomonic hallmarks: (1) motor and non-motor deficits; (2) neuroinflammation and oxidative stress; (3) pathological aggregates of the α-synuclein (α-syn) protein; (4) neurodegeneration of the nigrostriatal system. Recent evidence sustains that the aggregation of pathological α-syn occurs in the early stages of the disease, becoming the first trigger of neuroinflammation and subsequent neurodegeneration. Thus, a therapeutic line aims at striking back α-synucleinopathy and neuroinflammation to impede neurodegeneration. Another therapeutic line is restoring the compromised dopaminergic system using neurotrophic factors, particularly the glial cell-derived neurotrophic factor (GDNF). Preclinical studies with GDNF have provided encouraging results but often lack evaluation of anti-α-syn and anti-inflammatory effects. In contrast, clinical trials have yielded imprecise results and have reported the emergence of severe side effects. Here, we analyze the discrepancy between preclinical and clinical outcomes, review the mechanisms of the aggregation of pathological α-syn, including neuroinflammation, and evaluate the neurorestorative properties of GDNF, emphasizing its anti-α-syn and anti-inflammatory effects in preclinical and clinical trials.

Prophylactic Subacute Administration of Zinc Increases CCL2, CCR2, FGF2, and IGF-1 Expression and Prevents the Long-Term Memory Loss in a Rat Model of Cerebral Hypoxia-Ischemia.

Prophylactic subacute administration of zinc decreases lipoperoxidation and cell death following a transient cerebral hypoxia-ischemia, thus suggesting neuroprotective and preconditioning effects. Chemokines and growth factors are also involved in the neuroprotective effect in hypoxia-ischemia. We explored whether zinc prevents the cerebral cortex-hippocampus injury through regulation of CCL2, CCR2, FGF2, and IGF-1 expression following a 10 min of common carotid artery occlusion (CCAO). Male rats were grouped as follows: (1) Zn96h, rats injected with ZnCl2 (one dose every 24 h during four days); (2) Zn96h + CCAO, rats treated with ZnCl2 before CCAO; (3) CCAO, rats with CCAO only; (4) Sham group, rats with mock CCAO; and (5) untreated rats. The cerebral cortex-hippocampus was dissected at different times before and after CCAO. CCL2/CCR2, FGF2, and IGF-1 expression was assessed by RT-PCR and ELISA. Learning in Morris Water Maze was achieved by daily training during 5 days. Long-term memory was evaluated on day 7 after learning. Subacute administration of zinc increased expression of CCL2, CCR2, FGF2, and IGF-1 in the early and late phases of postreperfusion and prevented the CCAO-induced memory loss in the rat. These results might be explained by the induction of neural plasticity because of the expression of CCL2 and growth factors.

Prophylactic zinc and therapeutic selenium administration in adult rats prevents long-term cognitive and behavioral sequelae by a transient ischemic attack.

The transient hypoxic-ischemic attack, also known as a minor stroke, can result in long-term neurological issues such as memory loss, depression, and anxiety due to an increase in nitrosative stress. The individual or combined administration of chronic prophylactic zinc and therapeutic selenium is known to reduce nitrosative stress in the first seven days post-reperfusion and, due to an antioxidant effect, prevent cell death. Besides, zinc or selenium, individually administered, also causes antidepressant and anxiolytic effects. Therefore, this work evaluated whether combining zinc and selenium could prevent stroke-elicited cognition and behavior deficits after 30 days post-reperfusion. Accordingly, we assessed the expression of growth factors at 7 days post-reperfusion, a four-time course of memory (from 7 to 28 days post-learning test), and cell proliferation, depression, and anxiety-like behavior at 30 days post-reperfusion. Male Wistar rats with a weight between 190 and 240 g) were treated with chronic prophylactic zinc administration with a concentration of 0.2 mg/kg for 15 days before common carotid artery occlusion (10 min) and then with therapeutic selenium (6 µg/kg) for 7 days post-reperfusion. Compared with individual administrations, the administration combined of prophylactic zinc and therapeutic selenium decreased astrogliosis, increased growth factor expression, and improved cell proliferation and survival in two regions, the hippocampus, and cerebral cortex. These effects prevented memory loss, depression, and anxiety-like behaviors. In conclusion, these results demonstrate that the prophylactic zinc administration combined with therapeutic selenium can reduce the long-term sequelae caused by the transient ischemic attack. Significance statement. A minor stroke caused by a transient ischemic attack can result in psychomotor sequelae that affect not only the living conditions of patients and their families but also the economy. The incidence of these micro-events among young people has increased in the world. Nonetheless, there is no deep understanding of how this population group responds to regular treatments (Ekker and et al., 2018) [1]. On the basis that zinc and selenium have antioxidant, anti-inflammatory, and regenerative properties in stroke animal models, our work explored whether the chronic combined administration of prophylactic zinc and therapeutic selenium could prevent neurological sequelae in the long term in a stroke rat model of unilateral common carotid artery occlusion (CCAO) by 10-min. Our results showed that this combined treatment provided a long-term neuroprotective effect by decreasing astrogliosis, memory loss, anxiety, and depression-like behavior.

Prophylactic Zinc Administration Combined with Swimming Exercise Prevents Cognitive-Emotional Disturbances and Tissue Injury following a Transient Hypoxic-Ischemic Insult in the Rat.

Exercise performance and zinc administration individually yield a protective effect on various neurodegenerative models, including ischemic brain injury. Therefore, this work was aimed at evaluating the combined effect of subacute prophylactic zinc administration and swimming exercise in a transient cerebral ischemia model. The prophylactic zinc administration (2.5 mg/kg of body weight) was provided every 24 h for four days before a 30 min common carotid artery occlusion (CCAO), and 24 h after reperfusion, the rats were subjected to swimming exercise in the Morris Water Maze (MWM). Learning was evaluated daily for five days, and memory on day 12 postreperfusion; anxiety or depression-like behavior was measured by the elevated plus maze and the motor activity by open-field test. Nitrites, lipid peroxidation, and the activity of superoxide dismutase (SOD) and catalase (CAT) were assessed in the temporoparietal cortex and hippocampus. The three nitric oxide (NO) synthase isoforms, chemokines, and their receptor levels were measured by ELISA. Nissl staining evaluated hippocampus cytoarchitecture and Iba-1 immunohistochemistry activated the microglia. Swimming exercise alone could not prevent ischemic damage but, combined with prophylactic zinc administration, reversed the cognitive deficit, decreased NOS and chemokine levels, prevented tissue damage, and increased Iba-1 (+) cell number. These results suggest that the subacute prophylactic zinc administration combined with swimming exercise, but not the individual treatment, prevents the ischemic damage on day 12 postreperfusion in the transient ischemia model.

Taurine Increases Zinc Preconditioning-Induced Prevention of Nitrosative Stress, Metabolic Alterations, and Motor Deficits in Young Rats following Intrauterine Ischemia.

Oxygen deprivation in newborns leads to hypoxic-ischemic encephalopathy, whose hallmarks are oxidative/nitrosative stress, energetic metabolism alterations, nutrient deficiency, and motor behavior disability. Zinc and taurine are known to protect against hypoxic-ischemic brain damage in adults and neonates. However, the combined effect of prophylactic zinc administration and therapeutic taurine treatment on intrauterine ischemia- (IUI-) induced cerebral damage remains unknown. The present work evaluated this issue in male pups subjected to transient IUI (10 min) at E17 and whose mothers received zinc from E1 to E16 and taurine from E17 to postnatal day 15 (PND15) via drinking water. We assessed motor alterations, nitrosative stress, lipid peroxidation, and the antioxidant system comprised of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). Enzymes of neuronal energetic pathways, such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH), were also evaluated. The hierarchization score of the protective effect of pharmacological strategies (HSPEPS) was used to select the most effective treatment. Compared with the IUI group, zinc, alone or combined with taurine, improved motor behavior and reduced nitrosative stress by increasing SOD, CAT, and GPx activities and decreasing the GSSG/GSH ratio in the cerebral cortex and hippocampus. Taurine alone increased the AST/ALT, LDH/ALT, and AST/LDH ratios in the cerebral cortex, showing improvement of the neural bioenergetics system. This result suggests that taurine improves pyruvate, lactate, and glutamate metabolism, thus decreasing IUI-caused cerebral damage and relieving motor behavior impairment. Our results showed that taurine alone or in combination with zinc provides neuroprotection in the IUI rat model.

Elucidating the Neuropathologic Mechanisms of SARS-CoV-2 Infection.

The current pandemic caused by the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a public health emergency. To date, March 1, 2021, coronavirus disease 2019 (COVID-19) has caused about 114 million accumulated cases and 2.53 million deaths worldwide. Previous pieces of evidence suggest that SARS-CoV-2 may affect the central nervous system (CNS) and cause neurological symptoms in COVID-19 patients. It is also known that angiotensin-converting enzyme-2 (ACE2), the primary receptor for SARS-CoV-2 infection, is expressed in different brain areas and cell types. Thus, it is hypothesized that infection by this virus could generate or exacerbate neuropathological alterations. However, the molecular mechanisms that link COVID-19 disease and nerve damage are unclear. In this review, we describe the routes of SARS-CoV-2 invasion into the central nervous system. We also analyze the neuropathologic mechanisms underlying this viral infection, and their potential relationship with the neurological manifestations described in patients with COVID-19, and the appearance or exacerbation of some neurodegenerative diseases.

Unilateral intranigral administration of ß-sitosterol ß-D-glucoside triggers pathological α-synuclein spreading and bilateral nigrostriatal dopaminergic neurodegeneration in the rat.

The spreading and accumulation of α-synuclein and dopaminergic neurodegeneration, two hallmarks of Parkinson's disease (PD), have been faithfully reproduced in rodent brains by chronic, oral administration of ß-sitosterol ß-D-glucoside (BSSG). We investigated whether a single injection of BSSG (6 µg BSSG/µL DMSO) in the left substantia nigra of Wistar rats causes the same effects. Mock DMSO injections and untreated rats formed control groups. We performed immunostainings against the pathological α-synuclein, the dopaminergic marker tyrosine hydroxylase (TH), the neuroskeleton marker ß-III tubulin, the neurotensin receptor type 1 (NTSR1) as non-dopaminergic phenotype marker and Fluro-Jade C (F-J C) label for neurodegeneration. Using ß-galactosidase (ß-Gal) assay and active caspase-3 immunostaining, we assessed cell death mechanisms. Golgi-Cox staining was used to measure the density and types of dendritic spines of striatal medium spiny neurons. Motor and non-motor alterations were also evaluated. The study period comprised 15 to 120 days after the lesion. In the injured substantia nigra, BSSG caused a progressive α-synuclein aggregation and dopaminergic neurodegeneration caused by senescence and apoptosis. The α-synuclein immunoreactivity was also present within microglia cells. Decreased density of dopaminergic fibers and dendritic spines also occurred in the striatum. Remarkably, all the histopathological changes also appeared on the contralateral nigrostriatal system, and α-synuclein aggregates were present in other brain regions. Motor and non-motor behavioral alterations were progressive. Our data show that the stereotaxic BSSG administration reproduces PD α-synucleinopathy phenotype in the rat. This approach will aid in identifying the spread mechanism of α-synuclein pathology and validate anti-synucleinopathy therapies.

Prophylactic Zinc and Therapeutic Selenium Administration Increases the Antioxidant Enzyme Activity in the Rat Temporoparietal Cortex and Improves Memory after a Transient Hypoxia-Ischemia.

In the cerebral hypoxia-ischemia rat model, the prophylactic administration of zinc can cause either cytotoxicity or preconditioning effect, whereas the therapeutic administration of selenium decreases the ischemic damage. Herein, we aimed to explore whether supplementation of low doses of prophylactic zinc and therapeutic selenium could protect from a transient hypoxic-ischemic event. We administrated zinc (0.2 mg/kg of body weight; ip) daily for 14 days before a 10 min common carotid artery occlusion (CCAO). After CCAO, we administrated sodium selenite (6 µg/kg of body weight; ip) daily for 7 days. In the temporoparietal cerebral cortex, we determined nitrites by the Griess method and lipid peroxidation by the Gerard-Monnier assay. qPCR was used to measure mRNA of nitric oxide synthases, antioxidant enzymes, chemokines, and their receptors. We measured the enzymatic activity of SOD and GPx and protein levels of chemokines and their receptors by ELISA. We evaluated long-term memory using the Morris-Water maze test. Our results showed that prophylactic administration of zinc caused a preconditioning effect, decreasing nitrosative/oxidative stress and increasing GPx and SOD expression and activity, as well as eNOS expression. The therapeutic administration of selenium maintained this preconditioning effect up to the late phase of hypoxia-ischemia. Ccl2, Ccr2, Cxcl12, and Cxcr4 were upregulated, and long-term memory was improved. Pyknotic cells were decreased suggesting prevention of neuronal cell death. Our results show that the prophylactic zinc and therapeutic selenium administration induces effective neuroprotection in the early and late phases after CCAO.

Prophylactic Chronic Zinc Administration Increases Neuroinflammation in a Hypoxia-Ischemia Model.

Acute and subacute administration of zinc exert neuroprotective effects in hypoxia-ischemia animal models; yet the effect of chronic administration of zinc still remains unknown. We addressed this issue by injecting zinc at a tolerable dose (0.5 mg/kg weight, i.p.) for 14 days before common carotid artery occlusion (CCAO) in a rat. After CCAO, the level of zinc was measured by atomic absorption spectrophotometry, nitrites were determined by Griess method, lipoperoxidation was measured by Gerard-Monnier assay, and mRNA expression of 84 genes coding for cytokines, chemokines, and their receptors was measured by qRT-PCR, whereas nitrotyrosine, chemokines, and their receptors were assessed by ELISA and histopathological changes in the temporoparietal cortex-hippocampus at different time points. Long-term memory was evaluated using Morris water maze. Following CCAO, a significant increase in nitrosative stress, inflammatory chemokines/receptors, and cell death was observed after 8 h, and a 2.5-fold increase in zinc levels was detected after 7 days. Although CXCL12 and FGF2 protein levels were significantly increased, the long-term memory was impaired 12 days after reperfusion in the Zn+CCAO group. Our data suggest that the chronic administration of zinc at tolerable doses causes nitrosative stress, toxic zinc accumulation, and neuroinflammation, which might account for the neuronal death and cerebral dysfunction after CCAO.

Analysis of chemokines and receptors expression profile in the myelin mutant taiep rat.

Taiep rat has a failure in myelination and remyelination processes leading to a state of hypomyelination throughout its life. Chemokines, which are known to play a role in inflammation, are also involved in the remyelination process. We aimed to demonstrate that remyelination-stimulating factors are altered in the brainstem of 1- and 6-month-old taiep rats. We used a Rat RT(2) Profiler PCR Array to assess mRNA expression of 84 genes coding for cytokines, chemokines, and their receptors. We also evaluated protein levels of CCL2, CCR1, CCR2, CCL5, CCR5, CCR8, CXCL1, CXCR2, CXCR4, FGF2, and VEGFA by ELISA. Sprague-Dawley rats were used as a control. PCR Array procedure showed that proinflammatory cytokines were not upregulated in the taiep rat. In contrast, some mRNA levels of beta and alpha chemokines were upregulated in 1-month-old rats, but CXCR4 was downregulated at their 6 months of age. ELISA results showed that CXCL1, CCL2, CCR2, CCR5, CCR8, and CXCR4 protein levels were decreased in brainstem at the age of 6 months. These results suggest the presence of a chronic neuroinflammation process with deficiency of remyelination-stimulating factors (CXCL1, CXCR2, and CXCR4), which might account for the demyelination in the taiep rat.

Subacute zinc administration and L-NAME caused an increase of NO, zinc, lipoperoxidation, and caspase-3 during a cerebral hypoxia-ischemia process in the rat.

Zinc or L-NAME administration has been shown to be protector agents, decreasing oxidative stress and cell death. However, the treatment with zinc and L-NAME by intraperitoneal injection has not been studied. The aim of our work was to study the effect of zinc and L-NAME administration on nitrosative stress and cell death. Male Wistar rats were treated with ZnCl2 (2.5 mg/kg each 24 h, for 4 days) and N-ω-nitro-L-arginine-methyl ester (L-NAME, 10 mg/kg) on the day 5 (1 hour before a common carotid-artery occlusion (CCAO)). The temporoparietal cortex and hippocampus were dissected, and zinc, nitrites, and lipoperoxidation were assayed at different times. Cell death was assayed by histopathology using hematoxylin-eosin staining and caspase-3 active by immunostaining. The subacute administration of zinc before CCAO decreases the levels of zinc, nitrites, lipoperoxidation, and cell death in the late phase of the ischemia. L-NAME administration in the rats treated with zinc showed an increase of zinc levels in the early phase and increase of zinc, nitrites, and lipoperoxidation levels, cell death by necrosis, and the apoptosis in the late phase. These results suggest that the use of these two therapeutic strategies increased the injury caused by the CCAO, unlike the alone administration of zinc.