Exploring block and permeation of N-methyl-D-Aspartate (NMDA) receptor channels for treatment of neurodegenerative disorders

N-methyl-D-aspartate receptors (NMDAR) are ionotropic glutamate receptors which can be blocked by Mg2+ in a voltage-dependent manner and are highly permeable to Ca2+, hence they represent a medically relevant target for neurodegenerative disorders caused by excitotoxicity. The two main objectives of this study were, (i) to determine the impact of Q/R/N, +1 and -8 sites modification in the M2 pore region of GluN2A NMDAR subunit on Mg2+ block and other open channel blockers; and (ii) to evaluate novel multi-target-directed ligands (MTDL) for Alzheimer’s disease therapy. The Xenopus laevis oocyte expression system was employed where NMDAR subunit cRNAs were injected into the oocytes and responses to NMDA/glycine and channel blockers were recorded using two-electrode voltage clamp (TEVC) electrophysiology. Pore region mutations to investigate the impact of Q/R/N and adjacent sites were characterized using Mg2+, memantine, MK-801, philanthotoxin analogues and an MTDL compound, CR18. NN at the Q/R/N and +1 sites in GluN2A subunits were mutated to GR and RR, while W at the -8 position (in relation to the Q/R/N site), was mutated to N. Wild type and mutated GluN2A were co-expressed with GluN1-1a in Xenopus oocytes and antagonistic responses by channel blockers were recorded with TEVC. At -75 mV, the RR mutation significantly increased IC50s of Mg2+, memantine and MK-801 by 27-, 42- and 325-fold respectively, compared to wild-type. As for the GR mutation, IC50s were also significantly increased for memantine and MK-801 by 5- and 132-fold respectively, compared to wild type. W to N mutation at the -8 position did not significantly affect blocking potencies for all channel blockers. Blocking potency for PhTX-343 was not significantly altered by any mutations. This study provided evidence that the presence of G and R at the Q/R/N and +1 sites are likely responsible for the changes in blocking sensitivity and play important roles in ion permeability. The fact that PhTX-343 remained potent despite the mutations suggest that this compound might have a different mode of action or different binding site other than the M2 region and should be further characterized. In the MTDL study, twenty one novel compounds were tested on GluN1-1a/GluN2A NMDAR subunits. Thirteen were memantine-derivatives (MAB) incorporated with antioxidant moieties, three were spermine-derived polyamines also incorporated with antioxidants, and five were combinatorial forms of donepezil and carvedilol. The antagonistic properties of the compounds were tested electrophysiologically at -60 mV and compared with Mg2+ and memantine. The MAB series were found to be weak NMDAR channel blockers suggesting the loss of memantine functionality due to attachment of the antioxidant structure to its amine group. Subsequently, modification of the linker point to memantine moieties to free its amine group eventually resulted in weaker NMDAR channel blockers with IC50s of more than 100 µM. The spermine-derived polyamines (CR compounds) were potent NMDAR blockers with IC50s (0.69 to 2.35 µM) comparable to memantine (2.28 µM) and significantly lower than Mg2+ (10.1 µM) and also exhibited voltage-dependence block. Our mutation study revealed that CR18, the most potent MTDL compound was less sensitive in NMDAR containing GR or RR mutation in GluN2A subunits. This is a favourable property of an NMDAR blocker for potential Alzheimer’s disease treatment since GluN3 subunits containing GR or RR at the Q/R/N and +1 sites are less permeable to Ca2+ influx and has been shown to exert neuroprotective effects

Neuroprotection Against NMDA-Induced Retinal Damage by Philanthotoxin-343 Involves Reduced Nitrosative Stress

N-methyl-D-aspartate receptor (NMDAR) overstimulation is known to mediate neurodegeneration, and hence represents a relevant therapeutic target for neurodegenerative disorders including glaucoma. This study examined the neuroprotective effects of philanthotoxin (PhTX)-343 against NMDA-induced retinal injury in rats. Male Sprague Dawley rats were divided into three groups; group 1 received phosphate buffer saline as the negative control, group 2 was injected with NMDA (160 nM) to induce retinal excitotoxic injury, and group 3 was pre-treated with PhTX-343 (160 nM) 24 h before NMDA exposure. All treatments were given intravitreally and bilaterally. Seven days post-treatment, rats were subjected to visual behaviour assessments using open field and colour recognition tests. Rats were then euthanized, and the retinas were harvested and subjected to haematoxylin and eosin (H&E) staining for morphometric analysis and 3-nitrotyrosine (3-NT) ELISA protocol as the nitrosative stress biomarker. PhTX-343 treatment prior to NMDA exposure improved the ability of rats to recognize visual cues and preserved visual functions (i.e., recognition of objects with different colours). Morphological examination of retinal tissues showed that the fractional ganglion cell layer thickness within the inner retina (IR) in the PhTX-343 treated group was greater by 1.28-fold as compared to NMDA-treated rats (p 0.05). Additionally, the number of retinal cell nuclei/100 μm2 in IR for the PhTX-343-treated group was greater by 1.82-fold compared to NMDA-treated rats (p 0.05). PhTX-343 also reduced the retinal 3-NT levels by 1.74-fold compared to NMDA-treated rats (p < 0.05). In conclusion, PhTX-343 pretreatment protects against NMDA-induced retinal morphological changes and visual impairment by suppressing nitrosative stress as reflected by the reduced retinal 3-NT level

Simple coordination and cognitive stimulation activities for cognitive function assessments using functional near-infrared spectroscopy

Functional near-infrared spectroscopy (fNIRS) is a non-invasive and high-density imaging device used for the evaluation of cognitive functions by measuring the oxygenated haemoglobin (HbO) and deoxygenated haemoglobin (HHb) levels in the prefrontal cortex. The present study determined the utilisation of fNIRS in detecting cerebral haemoglobin oxygenation level during coordination and simple cognitive stimulation activities in healthy young volunteers. Thirty subjects comprising equally of both genders were recruited. Subjects were tasked with coordination tests (plate tapping and block transfer tests) and cognitive stimulation activities (ruler drop test and MRAVLT) while the fNIRS system was attached to their prefrontal cortex area. The HbO and HHb levels were recorded and analysed using Repeated Measures ANOVA. The HbO levels during coordination tests differ significantly from resting state (p0.05), except channel 3. All cognitive stimulation activities increased HbO levels compared to resting state; this change was significant in channels 3 and 4 (p<0.05). In contrast, HHb levels during all cognitive tests were lower compared to the resting state and was found to be significant in channels 2 and 3 (p<0.05). These findings suggested that coordination and cognitive stimulation activities activate prefrontal cortex in healthy young adults and could be potentially utilised as a valid screening tool for cognitive function assessments via fNIRS

Exploring block and permeation of N-methyl-D-Aspartate (NMDA) receptor channels for treatment of neurodegenerative disorders

N-methyl-D-aspartate receptors (NMDAR) are ionotropic glutamate receptors which can be blocked by Mg2+ in a voltage-dependent manner and are highly permeable to Ca2+, hence they represent a medically relevant target for neurodegenerative disorders caused by excitotoxicity. The two main objectives of this study were, (i) to determine the impact of Q/R/N, +1 and -8 sites modification in the M2 pore region of GluN2A NMDAR subunit on Mg2+ block and other open channel blockers; and (ii) to evaluate novel multi-target-directed ligands (MTDL) for Alzheimer’s disease therapy. The Xenopus laevis oocyte expression system was employed where NMDAR subunit cRNAs were injected into the oocytes and responses to NMDA/glycine and channel blockers were recorded using two-electrode voltage clamp (TEVC) electrophysiology. Pore region mutations to investigate the impact of Q/R/N and adjacent sites were characterized using Mg2+, memantine, MK-801, philanthotoxin analogues and an MTDL compound, CR18. NN at the Q/R/N and +1 sites in GluN2A subunits were mutated to GR and RR, while W at the -8 position (in relation to the Q/R/N site), was mutated to N. Wild type and mutated GluN2A were co-expressed with GluN1-1a in Xenopus oocytes and antagonistic responses by channel blockers were recorded with TEVC. At -75 mV, the RR mutation significantly increased IC50s of Mg2+, memantine and MK-801 by 27-, 42- and 325-fold respectively, compared to wild-type. As for the GR mutation, IC50s were also significantly increased for memantine and MK-801 by 5- and 132-fold respectively, compared to wild type. W to N mutation at the -8 position did not significantly affect blocking potencies for all channel blockers. Blocking potency for PhTX-343 was not significantly altered by any mutations. This study provided evidence that the presence of G and R at the Q/R/N and +1 sites are likely responsible for the changes in blocking sensitivity and play important roles in ion permeability. The fact that PhTX-343 remained potent despite the mutations suggest that this compound might have a different mode of action or different binding site other than the M2 region and should be further characterized. In the MTDL study, twenty one novel compounds were tested on GluN1-1a/GluN2A NMDAR subunits. Thirteen were memantine-derivatives (MAB) incorporated with antioxidant moieties, three were spermine-derived polyamines also incorporated with antioxidants, and five were combinatorial forms of donepezil and carvedilol. The antagonistic properties of the compounds were tested electrophysiologically at -60 mV and compared with Mg2+ and memantine. The MAB series were found to be weak NMDAR channel blockers suggesting the loss of memantine functionality due to attachment of the antioxidant structure to its amine group. Subsequently, modification of the linker point to memantine moieties to free its amine group eventually resulted in weaker NMDAR channel blockers with IC50s of more than 100 µM. The spermine-derived polyamines (CR compounds) were potent NMDAR blockers with IC50s (0.69 to 2.35 µM) comparable to memantine (2.28 µM) and significantly lower than Mg2+ (10.1 µM) and also exhibited voltage-dependence block. Our mutation study revealed that CR18, the most potent MTDL compound was less sensitive in NMDAR containing GR or RR mutation in GluN2A subunits. This is a favourable property of an NMDAR blocker for potential Alzheimer’s disease treatment since GluN3 subunits containing GR or RR at the Q/R/N and +1 sites are less permeable to Ca2+ influx and has been shown to exert neuroprotective effects

Natural Products in Mitigation of Bisphenol A Toxicity: Future Therapeutic Use

Bisphenol A (BPA) is a ubiquitous environmental toxin with deleterious endocrine-disrupting effects. It is widely used in producing epoxy resins, polycarbonate plastics, and polyvinyl chloride plastics. Human beings are regularly exposed to BPA through inhalation, ingestion, and topical absorption routes. The prevalence of BPA exposure has considerably increased over the past decades. Previous research studies have found a plethora of evidence of BPA&rsquo;s harmful effects. Interestingly, even at a lower concentration, this industrial product was found to be harmful at cellular and tissue levels, affecting various body functions. A noble and possible treatment could be made plausible by using natural products (NPs). In this review, we highlight existing experimental evidence of NPs against BPA exposure-induced adverse effects, which involve the body&rsquo;s reproductive, neurological, hepatic, renal, cardiovascular, and endocrine systems. The review also focuses on the targeted signaling pathways of NPs involved in BPA-induced toxicity. Although potential molecular mechanisms underlying BPA-induced toxicity have been investigated, there is currently no specific targeted treatment for BPA-induced toxicity. Hence, natural products could be considered for future therapeutic use against adverse and harmful effects of BPA exposure

A Review of the Potential Health Benefits of <i>Nigella sativa</i> on Obesity and Its Associated Complications

Obesity has become a worldwide epidemic and its prevalence continues to increase at an alarming rate. It is considered a major risk factor for the development of several comorbidities, including type 2 diabetes, stroke, other cardiovascular diseases and even cancer. Conventional treatments for obesity, such as dietary interventions, exercise and pharmacotherapy, have proven to have limited effectiveness and are often associated with undesirable side effects. Therefore, there is a growing interest in exploring alternative therapeutic approaches. Nigella sativa (NS), a medicinal plant with multiple pharmacological properties, has gained attention due to its potential role in the treatment of obesity and its associated complications. The aim of this review is therefore to assess the effects of NS on obesity and its complications and to provide insights into the underlying mechanisms. From this review, NS appears to play a complementary or supportive role in the treatment of obesity and its complications. However, future studies are needed to verify the efficacy of NS in the treatment of obesity and its complications and to prove its safety so that it can be introduced in patients with obesity

Philanthotoxin-343 attenuates retinal and optic nerve injury, and protects visual function in rats with N-methyl-D-aspartate-induced excitotoxicity

Retinal ganglion cell (RGC) loss and optic neuropathy, both hallmarks of glaucoma, have been shown to involve N-methyl-D-aspartate receptor (NMDAR)-mediated excitotoxicity. This study investigated the neuroprotective effects of Philanthotoxin (PhTX)-343 in NMDA-induced retinal injury to alleviate ensuing visual impairments. Sprague-Dawley rats were divided into three; Group I was intravitreally injected with phosphate buffer saline as the control, Group II was injected with NMDA (160 nM) to induce retinal excitotoxic injury, while Group III was injected with PhTX-343 (160 nM) 24 h prior to excitotoxicity induction with NMDA. Rats were subjected to visual behaviour tests seven days post-treatment and subsequently euthanized. Rat retinas and optic nerves were subjected to H&E and toluidine blue staining, respectively. Histological assessments showed that NMDA exposure resulted in significant loss of retinal cell nuclei and thinning of ganglion cell layer (GCL). PhTX-343 pre-treatment prevented NMDA-induced changes where the RGC layer morphology is similar to the control. The numbers of nuclei in the NMDA group were markedly lower compared to the control (p[less than] 0.05). PhTX-343 group had significantly higher numbers of nuclei within 100 μm length and 100 μm2 area of GCL (2.9- and 1.7-fold, respectively) compared to NMDA group (p [less than] 0.05). PhTX-343 group also displayed lesser optic nerve fibres degeneration compared to NMDA group which showed vacuolation in all sections. In the visual behaviour test, the NMDA group recorded higher total distance travelled, and lower total immobile time and episodes compared to the control and PhTX-343 groups (p [less than] 0.05). Object recognition tests showed that the rats in PhTX-343 group could recognize objects better, whereas the same objects were identified as novel by NMDA rats despite multiple exposures (p [less than] 0.05). Visual performances in the PhTX-343 group were all comparable with the control (p[less than] 0.05). These findings suggested that PhTX-343 inhibit retinal cell loss, optic nerve damage, and visual impairments in NMDA-induced rats

Exploring the Associated Factors of Depression, Anxiety, and Stress among Healthcare Shift Workers during the COVID-19 Pandemic

Background: The recent pandemic of COVID-19 has had a tremendous impact on healthcare frontliners. This study sought to assess healthcare shift workers&rsquo; depression, anxiety, and stress and its associated factors. Methods: The sampling frame includes healthcare shift workers directly managing COVID-19 cases around Klang Valley, Malaysia. The participants&rsquo; mental health status was assessed using the Depression, Anxiety, Stress Scale-21 (DASS-21). The associated factors specified in this study include sleep quality, physical activities, and eating habits. Pearson&rsquo;s &chi;2 and simple and multivariable binary logistic regression models were constructed following the Hosmer&ndash;Lemeshow approach to determine the potential associated factors. Results: A total of 413 participants were recruited. Overall, 40.7% of participants had one or more symptoms of depression, anxiety, or stress. Poor sleep quality was significantly associated with all mental health outcomes of depression, anxiety, and stress. Inactivity was found to be strongly associated with symptoms of depression and anxiety. At the same time, eating habits were strongly associated with anxiety and stress. Conclusions: Sleep quality, inactivity, and eating habits that were found to be associated with the mental health status of healthcare shift workers are modifiable factors that must be addressed to curb mental health issues among this group of workers

Microarray Profiling of Differentially Expressed Genes in Coronary Artery Bypass Grafts of High-Risk Patients with Postoperative Cognitive Dysfunctions

Postoperative cognitive dysfunction (POCD) is cognitive decline after surgery. The authors hypothesized that gene-level changes could be involved in the pathogenesis of POCD. The present study evaluated the incidence of POCD and its associated differentially expressed genes. This was a prospective cohort study conducted on high-risk coronary artery bypass graft patients aged 40 to 75 years. POCD classification was based on a one standard deviation decline in the postoperative scores compared to the preoperative scores. The differentially expressed genes were identified using microarray analysis and validated using quantitative RT-PCR. Forty-six patients were recruited and completed the study. The incidence of POCD was identified using a set of neurocognitive assessments and found to be at 17% in these high-risk CABG patients. Six samples were selected for the gene expression analyses (3 non-POCD and 3 POCD samples). The findings showed five differentially expressed genes in the POCD group compared to the non-POCD group. The upregulated gene was ERFE, whereas the downregulated genes were KIR2DS2, KIR2DS3, KIR3DL2, and LIM2. According to the results, the gene expression profiles of POCD can be used to find potential proteins for POCD diagnostic and predictive biomarkers. Understanding the molecular mechanism of POCD development will further lead to early detection and intervention to reduce the severity of POCD, and hence, reduce the mortality and morbidity rate due to the condition