Monkeypox virus infection of human astrocytes causes gasdermin B cleavage and pyroptosis.

Monkeypox virus (MPXV) infections in humans cause neurological disorders while studies of MPXV-infected animals indicate that the virus penetrates the brain. Pyroptosis is an inflammatory type of regulated cell death, resulting from plasma membrane rupture (PMR) due to oligomerization of cleaved gasdermins to cause membrane pore formation. Herein, we investigated the human neural cell tropism of MPXV compared to another orthopoxvirus, vaccinia virus (VACV), as well as its effects on immune responses and cell death. Astrocytes were most permissive to MPXV (and VACV) infections, followed by microglia and oligodendrocytes, with minimal infection of neurons based on plaque assays. Aberrant morphological changes were evident in MPXV-infected astrocytes that were accompanied with viral protein (I3) immunolabelling and detection of over 125 MPXV-encoded proteins in cell lysates by mass spectrometry. MPXV- and VACV-infected astrocytes showed increased expression of immune gene transcripts (IL12, IRF3, IL1B, TNFA, CASP1, and GSDMB). However, MPXV infection of astrocytes specifically induced proteolytic cleavage of gasdermin B (GSDMB) (50 kDa), evident by the appearance of cleaved N-terminal-GSDMB (30 kDa) and C-terminal- GSDMB (18 kDa) fragments. GSDMB cleavage was associated with release of lactate dehydrogenase and increased cellular nucleic acid staining, indicative of PMR. Pre-treatment with dimethyl fumarate reduced cleavage of GSDMB and associated PMR in MPXV-infected astrocytes. Human astrocytes support productive MPXV infection, resulting in inflammatory gene induction with accompanying GSDMB-mediated pyroptosis. These findings clarify the recently recognized neuropathogenic effects of MPXV in humans while also offering potential therapeutic options.

Low-Field Magnetic Stimulation Alleviates MPTP-Induced Alterations in Motor Function and Dopaminergic Neurons in Male Mice.

Recent studies show that repetitive transcranial magnetic stimulation (rTMS) improves cognitive and motor functions in patients with Parkinson's Disease (PD). Gamma rhythm low-field magnetic stimulation (LFMS) is a new non-invasive rTMS technique that generates diffused and low-intensity magnetic stimulation to the deep cortical and subcortical areas. To investigate the potential therapeutic effects of LFMS in PD, we subjected an experimental mouse model to LFMS (as an early treatment). We examined the LFMS effect on motor functions as well as neuronal and glial activities in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated male C57BL/6J mice. Mice received MPTP injection (30 mg/kg, i.p., once daily for 5 days) followed by LFMS treatment, 20 min each day for 7 days. LFMS treatment improved motor functions compared with the sham-treated MPTP mice. Further, LFMS significantly improved tyrosine hydroxylase (TH) and decreased glial fibrillary acidic protein (GFAP) levels in substantia nigra pars compacta (SNpc) and non-significantly in striatal (ST) regions. LFMS treatment improved neuronal nuclei (NeuN) levels in SNpc. Our findings suggest that early LFMS treatment improves neuronal survival and, in turn, motor functions in MPTP-treated mice. Further investigation is required to clearly define the molecular mechanisms by which LFMS improves motor and cognitive function in PD patients.

Concussion/Mild Traumatic Brain Injury (TBI) Induces Brain Insulin Resistance: A Positron Emission Tomography (PET) Scanning Study.

Brain injury/concussion is a growing epidemic throughout the world. Although evidence supports association between traumatic brain injury (TBI) and disturbance in brain glucose metabolism, the underlying molecular mechanisms are not well established. Previously, we reported the release of cellular prion protein (PrPc) from the brain to circulation following TBI. The PrPc level was also found to be decreased in insulin-resistant rat brains. In the present study, we investigated the molecular link between PrPc and brain insulin resistance in a single and repeated mild TBI-induced mouse model. Mild TBI was induced in mice by dropping a weight (~95 g at 1 m high) on the right side of the head. The procedure was performed once and thrice (once daily) for single (SI) and repeated induction (RI), respectively. Micro PET/CT imaging revealed that RI mice showed significant reduction in cortical, hippocampal and cerebellum glucose uptake compared to SI and control. Mice that received RI also showed significant motor and cognitive deficits. In co-immunoprecipitation, the interaction between PrPc, flotillin and Cbl-associated protein (CAP) observed in the control mice brains was disrupted by RI. Lipid raft isolation showed decreased levels of PrPc, flotillin and CAP in the RI mice brains. Based on observation, it is clear that PrPc has an interaction with CAP and the dislodgment of PrPc from cell membranes may lead to brain insulin resistance in a mild TBI mouse model. The present study generated a new insight into the pathogenesis of brain injury, which may result in the development of novel therapy.

Cannabinoid receptors distribution in mouse cortical plasma membrane compartments.

The type 1 and type 2 cannabinoid receptors (CB1 and CB2 receptors) are class A G protein-coupled receptors (GPCRs) that are activated by endogenous lipids called endocannabinoids to modulate neuronal excitability and synaptic transmission in neurons throughout the central nervous system (CNS), and inflammatory processes throughout the body. CB1 receptor is one of the most abundant GPCRs in the CNS and is involved in many physiological and pathophysiological processes, including mood, appetite, and nociception. CB2 receptor is primarily found on immunomodulatory cells of both the CNS and the peripheral immune system. In this study, we isolated lipid raft and non-lipid raft fractions of plasma membrane (PM) from mouse cortical tissue by using cold non-ionic detergent and sucrose gradient centrifugation to study the localization of CB1 receptor and CB2 receptor. Lipid raft and non-lipid raft fractions were confirmed by flotillin-1, caveolin-1 and transferrin receptor as their protein biomarkers. Both CB1 receptor and CB2 receptor were found in non-raft compartments that is inconsistent with previous findings in cultured cell lines. This study demonstrates compartmentalization of both CB1 receptor and CB2 receptor in cortical tissue and warrants further investigation of CB1 receptor and CB2 receptor compartmental distribution in various brain regions and cell types.

Cellular Prion Protein (PrPc): Putative Interacting Partners and Consequences of the Interaction.

Cellular prion protein (PrPc) is a small glycosylphosphatidylinositol (GPI) anchored protein most abundantly found in the outer leaflet of the plasma membrane (PM) in the central nervous system (CNS). PrPc misfolding causes neurodegenerative prion diseases in the CNS. PrPc interacts with a wide range of protein partners because of the intrinsically disordered nature of the protein's N-terminus. Numerous studies have attempted to decipher the physiological role of the prion protein by searching for proteins which interact with PrPc. Biochemical characteristics and biological functions both appear to be affected by interacting protein partners. The key challenge in identifying a potential interacting partner is to demonstrate that binding to a specific ligand is necessary for cellular physiological function or malfunction. In this review, we have summarized the intracellular and extracellular interacting partners of PrPc and potential consequences of their binding. We also briefly describe prion disease-related mutations at the end of this review.

The Concept of an Epilepsy Brain Bank.

Epilepsy comprises more than 40 clinical syndromes affecting millions of patients and families worldwide. To decode the molecular and pathological framework of epilepsy researchers, need reliable human epilepsy and control brain samples. Brain bank organizations collecting and supplying well-documented clinically and pathophysiologically tissue specimens are important for high-quality neurophysiology and neuropharmacology studies for epilepsy and other neurological diseases. New development in molecular mechanism and new treatment methods for neurological disorders have evoked increased demands for human brain tissue. An epilepsy brain bank is a storage source for both the frozen samples as well as the formaldehyde fixed paraffin embedded (FFPE) tissue from epilepsy surgery resections. In 2014, the University of Saskatchewan have started collecting human epilepsy brain tissues for the first time in Canada. This review highlights the necessity and importance of Epilepsy Brain bank that provides unique access for research to valuable source of brain tissue and blood samples from epilepsy patients.

Low-Field Magnetic Stimulation Restores Cognitive and Motor Functions in the Mouse Model of Repeated Traumatic Brain Injury: Role of Cellular Prion Protein.

Traumatic brain injury (TBI)/concussion is a growing epidemic throughout the world. Memory and neurobehavioral dysfunctions are among the sequelae of TBI. Dislodgement of cellular prion protein (PrPc) and disruption of circadian rhythm have been linked to TBI. Low-field magnetic stimulation (LFMS) is a new noninvasive repetitive transcranial magnetic stimulation (rTMS) technique that generates diffused and low-intensity magnetic stimulation to deep cortical and subcortical areas. The role of LFMS on PrPc, proteins related to the circadian rhythm, and behavior alterations in a repeated TBI mouse model were studied in the present study. TBI was induced to the mice (right hemisphere) using weight-drop method, once daily for 3 days. LFMS treatment was given for 20 min once daily for 4 days (immediately after each TBI induction). The results showed that LFMS-treated TBI mice significantly improved cognitive and motor function as evidenced by open field exploration, rotarod, and novel location recognition tasks. In addition, a significant increase in PrPc and decreased glial fibrillary acidic protein levels were observed in cortical and hippocampal regions of LFMS-treated TBI mice brain compared with sham-treated TBI mice, while neuronal nuclei level was significantly increased in cortical region. In LFMS-treated mice, a decrease in proteins related to circadian rhythm were observed, compared with sham-treated TBI mice. The results obtained from the study demonstrated the neuroprotective effect of LFMS, which may be through regulating PrPc and/or proteins related to circadian rhythm. Thus, the present study suggests that LFMS may improve the subject's neurological condition following TBI.

Analysis of the T354P mutation of the sodium/iodide cotransporter gene in children with congenital hypothyroidism due to dyshormonogenesis.

BACKGROUND: Congenital hypothyroidism (CH) due to the thyroid dyshormonogenesis is more prevalent in Iran in comparison to other countries. Sodium iodide symporter (NIS) is one of the plasma membrane glycoproteins that is located on the basolateral side of thyroid follicular cells and mediates active I(-) trapping into these cells. Playing a prominent role in thyroid hormone synthesis, NIS gene mutations can be a cause of permanent CH with the etiology of dyshormonogenesis. The aim of this study was to investigate the occurrence of T354P mutation of the NIS gene, in a group of children affected with permanent CH in Isfahan. MATERIALS AND METHODS: Thirty-five patients with the etiology of dyshormonogenesis, and 35 healthy children, collected between 2002 and 2011 in Isfahan Endocrine and Metabolism Research Center, were examined for T354P mutation of the NIS gene by direct polymerase chain reaction-sequencing method. RESULTS: No T354P mutation was detected in any of the studied children. CONCLUSIONS: More subjects with confirmed iodide transport defects should be screened for detecting the frequency of different reported NIS gene mutations in our population.

Association Study Between Metabolic Syndrome and rs8066560 Polymorphism in the Promoter Region of Sterol Regulatory Element-binding Transcription Factor 1 Gene in Iranian Children and Adolescents.

BACKGROUND: Metabolic syndrome (MetS) is a prevalent disorder in pediatric age groups, described by a combination of genetic and environmental factors. Sterol regulatory element-binding transcription factor 1 (SREBF-1) induces the expression of a family of genes involved in fatty acid synthesis. Moreover, dysregulation of miR-33b, which is located within the intron 17 of the SREBF-1 gene, disrupts fatty acid oxidation and insulin signaling, thus leading to MetS. The aim of the present study was to investigate the association between SREBF-1 rs8066560 polymorphism and MetS in Iranian children and adolescents. METHODS: This study includes 100 MetS and 100 normal individuals aged 9-19 years. Anthropological and biochemical indexes were measured. The -1099G > A polymorphism was genotyped by TaqMan real-time polymerase chain reaction. RESULTS: Significant differences were observed in anthropometric measurements and lipid profiles between MetS and normal children. There were no differences in the genotype frequencies or allele distribution for -1099G > A polymorphism between MetS and control groups. High-density lipoprotein cholesterol levels were significantly higher in the MetS GG group than in the A allele carrier group. The genotype AA controls had significantly increased cholesterol and low-density lipoprotein cholesterol levels than AG genotypes. By logistic regression using different genetic models, no significant association was observed between SREBF-1 rs8066560 polymorphism and the risk of MetS. CONCLUSIONS: We conclude that the -1099G > A variant on SREBF-1 gene associated with serum lipid profiles, however, it may not be a major risk factor for the MetS in Iranian children and adolescents.

Comparison of TaqMan Real-Time and Tetra-Primer ARMS PCR Techniques for Genotyping of Rs 8066560 Variant in Children and Adolescents with Metabolic Syndrome.

BACKGROUND: Single nucleotide polymorphisms (SNPs) are major contributors to susceptibility or resistance to various human diseases. Metabolic syndrome (MetS) is a collection of risk factors, including abdominal obesity, dyslipidemia and increased blood pressure. MetS is more common among Iranian children and adolescents in comparison to other ethnicities. Sterol regulatory element binding factors (SREBFs) involve in the regulation of carbohydrate and lipid metabolism. The tetra-primer amplification refractory mutation system PCR (tetra-primer ARMS-PCR/T-ARMS-PCR) is a fast and economical means of assaying SNPs, requiring only PCR amplification and subsequent electrophoresis for the determination of genotypes. OBJECTIVES: This study aims to optimize and compare the results of tetra-primer ARMS-PCR for genotyping of rs8066560 in Iranian children and adolescents being afflicted with metabolic syndrome with the TaqMan assay. MATERIAL AND METHODS: In this study, a total of 50 individuals with 9-19 years of age, including 25 healthy subjects and 25 MetS cases were studied. The tetra-primer ARMS-PCR was used to genotype the rs8066560. RESULTS: After PCR optimization, we could successfully detect the rs8066560 polymorphism in all the studied subjects. Furthermore, we observed complete concordance between tetra-primer ARMS-PCR assay and TaqMan method`s results. CONCLUSIONS: Tetra-primer ARMS-PCR can be utilized as a cost-effective, rapid and reproducible method for SNP genotyping especially while performing large-scale epidemiological/association at studies.

Association of rs8066560 variant in the sterol regulatory element-binding protein 1 (SREBP-1) and miR-33b genes with hyperglycemia and insulin resistance.

OBJECTIVE: This study aims to evaluate the association of miR-33b/SREBP-1 rs8066560 with hyperglycemia and insulin resistance in Iranian children and adolescents. METHODS: This case-control study comprised 200 participants with 9-19 years of age, consisting of 100 MetS and 100 healthy controls. Anthropometric and biochemical parameters were measured, and the polymorphism was genotyped by TaqMan real-time PCR method. RESULTS: Logistic regression analysis of genotype distribution of rs8066560 did not show a significant association with MetS neither in boys (OR, 1.745; 95% CI, 0.695-4.382; p=0.236) nor in girls (OR, 1.075; 95% CI, 0.420-2.752; p=0.880). Participants with GG/AG genotypes had significant higher LDL-C levels than those with the AA genotype. No statistically significant difference was documented in fasting blood glucose and insulin levels between the three rs8066560 genotypes. CONCLUSIONS: We found that rs8066560 variant on SREBP-1 and miR-33b genes is associated with LDL-C levels; however, it may not be an important determinant for the MetS, hyperglycemia, and insulin resistance in children and adolescents.

The G395R Mutation of the Sodium/Iodide Symporter (NIS) Gene in Patients with Dyshormonogenetic Congenital Hypothyroidism.

BACKGROUND: Considering the high prevalence of congenital hypothyroidism (CH) in Isfahan and its different etiologies in comparison with other countries, the high rate of parental consanguinity, and the role of NIS gene in permanent CH due to dyshormonogenesis, the aim of this study was to investigate the G395R mutation of the NIS gene in patients with permanent CH due to dyshormonogenesis METHODS: In this case-control study, patients diagnosed with permanent CH due to dyshormonogenesis during CH screening program were selected. Venous blood sample was obtained to determine the G395R mutations of NIS gene using polymerase chain reaction (PCR) sequencing method. RESULTS: In this study, 35 CH patients with permanent CH due to dyshormonogenesis and 35 neonates with normal screening results as a control group were studied. We did not find any changes of the mentioned mutation of NIS gene in the patients' group. CONCLUSION: Considering the findings of the current study, it seems that further studies with larger sample size and with consideration of other gene mutations such as pendrin and thyroglobulin are needed for more accurate conclusion.