Involvement of mitochondria in Alzheimer's disease pathogenesis and their potential as targets for phytotherapeutics.

Alzheimer's disease (AD) is the leading cause of dementia around the globe. The disease's genesis is multifaceted, and its pathophysiology is complicated. Malfunction of mitochondria has been regarded as one of the intracellular events that are substantially damaged in the onset of AD and are likely a common trait of other neurodegenerative illnesses. Several mitochondrial characteristics begin to diminish with age, eventually reaching a state of significant functional failure concurrent with the beginning of neurodegenerative diseases, however, the exact timing of these processes is unknown. Mitochondrial malfunction has a multitude of negative repercussions, including reduced calcium buffering and secondary excitotoxicity contributing to synaptic dysfunction, also free radical production, and activation of the mitochondrial permeability transition. Hence mitochondria are considered a therapeutic target in neurodegenerative disorders such as Alzheimer's. Traditional medicinal systems practiced in different countries employing various medicinal plants postulated to have potential role in the therapy and management of memory impairment including amnesia, dementia as well as AD. Although, the preclinical and clinical studies using these medicinal plants or plant products have demonstrated the therapeutic efficacy for AD, the precise mechanism of action is still obscure. Therefore, this review discusses the contribution of mitochondria towards AD pathogenesis and considering phytotherapeutics as a potential therapeutic strategy.

Low-dose exposure to malathion and radiation results in the dysregulation of multiple neuronal processes, inducing neurotoxicity and neurodegeneration in mouse.

Neurodegenerative disorders are a debilitating and persistent threat to the global elderly population, carrying grim outcomes. Their genesis is often multifactorial, with a history of prior exposure to xenobiotics such as pesticides, heavy metals, enviornmental pollutants, ionizing radiation etc,. A holistic molecular insight into their mechanistic induction upon single or combinatorial exposure to different toxicants is still unclear. In the present study, one-month-old C57BL/6 male mice were administered orally with malathion (50 mg/kg body wt. for 14 days) and single whole-body radiation (0.5 Gy) on the 8th day. Post-treatment, behavioural assays for exploratory behaviour, memory, and learning were performed. After sacrifice, brains were collected for histology, biochemical assays, and transcriptomic analysis. Transcriptomic analysis revealed several altered processes like synaptic transmission and plasticity, neuronal survival, proliferation, and death. Signalling pathways like MAPK, PI3K-Akt, Apelin, NF-κB, cAMP, Notch etc., and pathways related to neurodegenerative diseases were altered. Increased astrogliosis was observed in the radiation and coexposure groups, with significant neuronal cell death and a reduction in the expression of NeuN. Sholl analysis, dendritic arborization and spine density studies revealed decreased total apical neuronal path length and dendritic spine density. Reduced levels of the antioxidants GST and GSH and acetylcholinesterase enzyme activity were also detected. However, no changes were seen in exploratory behaviour or learning and memory post-treatment. Thus, explicating the molecular mechanisms behind malathion and radiation can provide novel insights into external factor-driven neurotoxicity and neurodegenerative pathogenesis.

Pelvic irradiation induces behavioural and neuronal damage through gut dysbiosis in a rat model.

Radiation exposure can cause gut dysbiosis and there is a positive correlation between gut microbial imbalance and radiation-induced side effects in cancer patients. However, the influence of radiation on the gut-brain axis (GBA) and its neurological consequences are not well understood. Therefore, this study aimed to investigate the impact of pelvic irradiation on gut microbiota and the brain. Sprague Dawley rats were irradiated with a single dose of 6 Gy, and faecal samples were collected at different time points (7 and 12-days post-irradiation) for microbial analysis. Behavioural, histological, and gene expression analysis were performed to assess the effect of microbial dysbiosis on the brain. The findings indicated alterations in microbial diversity, disrupted intestinal morphology and integrity, neuronal death-related brain changes, neuroinflammation and reduced locomotor activity. Hippocampal gene expression analysis also showed a reduced expression of neural plasticity-related genes. Overall, this study demonstrated that pelvic irradiation affects gut microbiota, intestinal morphology, integrity, brain neuronal maturation, neural plasticity gene expression, and behaviour.

Revealing the Structural Organization of Gamma-irradiated Starch Granules Using Polarization-resolved Second Harmonic Generation Microscopy.

Starch is a semi-crystalline macromolecule with the presence of amorphous and crystalline components. The amorphous amylose and crystalline amylopectin regions in starch granules are susceptible to certain physical modifications, such as gamma irradiation. Polarization-resolved second harmonic generation (P-SHG) microscopy in conjunction with SHG-circular dichroism (CD) was used to assess the three-dimensional molecular order and inherent chirality of starch granules and their reaction to different dosages of gamma irradiation. For the first time, the relationship between starch achirality (χ21/χ16 and χ22/χ16) and chirality (χ14/χ16) determining susceptibility tensor ratios has been elucidated. The results showed that changes in the structure and orientation of long-chain amylopectin were supported by the decrease in the SHG anisotropy factor and the χ22/χ16 ratio. Furthermore, SHG-CD illustrated the molecular tilt angle by revealing the arrangement of amylopectin molecules pointing either upward or downward owing to molecular polarity.

MiR-4521 perturbs FOXM1-mediated DNA damage response in breast cancer.

Introduction: Forkhead (FOX) transcription factors are involved in cell cycle control, cellular differentiation, maintenance of tissues, and aging. Mutation or aberrant expression of FOX proteins is associated with developmental disorders and cancers. FOXM1, an oncogenic transcription factor, is a promoter of cell proliferation and accelerated development of breast adenocarcinomas, squamous carcinoma of the head, neck, and cervix, and nasopharyngeal carcinoma. High FOXM1 expression is correlated with chemoresistance in patients treated with doxorubicin and Epirubicin by enhancing the DNA repair in breast cancer cells. Method: miRNA-seq identified downregulation of miR-4521 in breast cancer cell lines. Stable miR-4521 overexpressing breast cancer cell lines (MCF-7, MDA-MB-468) were developed to identify miR-4521 target gene and function in breast cancer. Results: Here, we showed that FOXM1 is a direct target of miR-4521 in breast cancer. Overexpression of miR-4521 significantly downregulated FOXM1 expression in breast cancer cells. FOXM1 regulates cell cycle progression and DNA damage response in breast cancer. We showed that miR-4521 expression leads to increased ROS levels and DNA damage in breast cancer cells. FOXM1 plays a critical role in ROS scavenging and promotes stemness which contributes to drug resistance in breast cancer. We observed that breast cancer cells stably expressing miR-4521 lead to cell cycle arrest, impaired FOXM1 mediated DNA damage response leading to increased cell death in breast cancer cells. Additionally, miR-4521-mediated FOXM1 downregulation perturbs cell proliferation, invasion, cell cycle progression, and epithelial-to-mesenchymal progression (EMT) in breast cancer. Discussion: High FOXM1 expression has been associated with radio and chemoresistance contributing to poor patient survival in multiple cancers, including breast cancer. Our study showed that FOXM1 mediated DNA damage response could be targeted using miR-4521 mimics as a novel therapeutic for breast cancer.

Radioprotective potential of probiotics against gastrointestinal and neuronal toxicity: a preclinical study.

PURPOSE: Radiotherapy is a critical component of cancer treatment, along with surgery and chemotherapy. Approximately, 90% of cancer patients undergoing pelvic radiotherapy show gastrointestinal (GI) toxicity, including bloody diarrhea, and gastritis, most of which are associated with gut dysbiosis. In addition to the direct effect of radiation on the brain, pelvic irradiation can alter the gut microbiome, leading to inflammation and breakdown of the gut-blood barrier. This allows toxins and bacteria to enter the bloodstream and reach the brain. Probiotics have been proven to prevent GI toxicity by producing short-chain fatty acids and exopolysaccharides beneficial for protecting mucosal integrity and oxidative stress reduction in the intestine and also shown to be beneficial in brain health. Microbiota plays a significant role in maintaining gut and brain health, so it is important to study whether bacterial supplementation will help in maintaining the gut and brain structure after radiation exposure. METHODS: In the present study, male C57BL/6 mice were divided into control, radiation, probiotics, and probiotics + radiation groups. On the 7th day, animals in the radiation and probiotics + radiation groups received a single dose of 4 Gy to  whole-body. Posttreatment, mice were sacrificed, and the intestine and brain tissues were excised for histological analysis to assess GI and neuronal damage. RESULTS: Radiation-induced damage to the villi height and mucosal thickness was mitigated by the probiotic treatment significantly (p < 0.01). Further, radiation-induced pyknotic cell numbers in the DG, CA2, and CA3 areas were substantially reduced with bacterial supplementation (p < 0.001). Similarly, probiotics reduced neuronal inflammation induced by radiation in the cortex, CA2, and DG region (p < 0.01). Altogether, the probiotics treatment helps mitigate radiation-induced intestinal and neuronal damage. CONCLUSION: In conclusion, the probiotic formulation could attenuate the number of pyknotic cells in the hippocampal brain region and decrease neuroinflammation by reducing the number of microglial cells.

Gut microbiota-derived metabolites and their importance in neurological disorders.

Microbial-derived metabolites are the intermediate or end products of bacterial digestion. They are one of the most important molecules for the gut to connect with the brain. Depending on the levels of specific metabolites produced in the host, it can exert beneficial or detrimental effects on the brain and have been linked to several neurodegenerative and neuropsychiatric disorders. However, the underlying mechanisms remain largely unexplored. Insight into these mechanisms could reveal new pathways or targets, resulting in novel treatment approaches targeting neurodegenerative diseases. We have reviewed selected metabolites, including short-chain fatty acids, aromatic amino acids, trimethylamine-N-oxide,  urolithin A, anthocyanins, equols, imidazole, and propionate to highlight their mechanism of action, underlying role in maintaining intestinal homeostasis and regulating neuro-immunoendocrine function. Further discussed on  how altered metabolite levels can influence the gut-brain axis could lead to new prevention strategies or novel treatment approaches to neural disorders.

Pesticides Exposure-Induced Changes in Brain Metabolome: Implications in the Pathogenesis of Neurodegenerative Disorders.

Pesticides have been used in agriculture, public health programs, and pharmaceuticals for many decades. Though pesticides primarily target pests by affecting their nervous system and causing other lethal effects, these chemical entities also exert toxic effects in inadvertently exposed humans through inhalation or ingestion. Mounting pieces of evidence from cellular, animal, and clinical studies indicate that pesticide-exposed models display metabolite alterations of pathways involved in neurodegenerative diseases. Hence, identifying common key metabolites/metabolic pathways between pesticide-induced metabolic reprogramming and neurodegenerative diseases is necessary to understand the etiology of pesticides in the rise of neurodegenerative disorders. The present review provides an overview of specific metabolic pathways, including tryptophan metabolism, glutathione metabolism, dopamine metabolism, energy metabolism, mitochondrial dysfunction, fatty acids, and lipid metabolism that are specifically altered in response to pesticides. Furthermore, we discuss how these metabolite alterations are linked to the pathogenesis of neurodegenerative diseases and to identify novel biomarkers for targeted therapeutic approaches.

Modulation of CREB and its associated upstream signaling pathways in pesticide-induced neurotoxicity.

Human beings are exposed to various environmental xenobiotics throughout their life consisting of a broad range of physical and chemical agents that impart bodily harm. Among these, pesticide exposure that destroys insects mainly by damaging their central nervous system also exerts neurotoxic effects on humans and is implicated in the etiology of several degenerative disorders. The connectivity between CREB (cAMP Response Element Binding Protein) signaling activation and neuronal activity is of broad interest and has been thoroughly studied in various diseased states. Several genes, as well as protein kinases, are involved in the phosphorylation of CREB, including BDNF (Brain-derived neurotrophic factor), Pi3K (phosphoinositide 3-kinase), AKT (Protein kinase B), RAS (Rat Sarcoma), MEK (Mitogen-activated protein kinase), PLC (Phospholipase C), and PKC (Protein kinase C) that play an essential role in neuronal plasticity, long-term potentiation, neuronal survival, learning, and memory formation, cognitive function, synaptic transmission, and suppressing apoptosis. These elements, either singularly or in a cascade, can result in the modulation of CREB, making it a vulnerable target for various neurotoxic agents, including pesticides. This review provides insight into how these various intracellular signaling pathways converge to bring about CREB activation and how the activated or deactivated CREB levels can affect the gene expression of the upstream molecules. We also discuss the various target genes within the cascade vulnerable to different types of pesticides. Thus, this review will facilitate future investigations associated with pesticide neurotoxicity and identify valuable therapeutic targets.

Bi-Directional interactions between microbiota and ionizing radiation in head and neck and pelvic radiotherapy - clinical relevance.

Purpose: Rapid developments in high throughput screening technology for the detection and identification of the human microbiota have helped in understanding its influence on human health and disease. In the recent past, several seminal studies have demonstrated the influence of microbiota on outcomes of therapy-associated radiation exposure. In this review, we highlight the concepts related to the mechanisms by which radiation alters the microbiota composition linked with radiation-associated toxicity in head and neck and pelvic regions. We further discuss specific microbial changes that can be employed as a biomarker for radiation and tumor response.Conclusion: Knowledge of the influence of microbiota in radiation response and advances in microbiota manipulation techniques would help to design personalized treatment augmenting the efficacy of radiotherapy.

Pharmacological approach to increasing the retention of radiation-induced γ-H2AX foci using phosphatase inhibitors: significance in radiation biodosimetry.

In a scenario of accidental mass radiation exposure transportation and analysis of samples may take some time, resulting in loss of biomarker information over this period. The present study aims to use phosphatase inhibitors for longer retention of focal signals to adopt γ-H2AX as a biodosimetric biomarker for the management of early triage. Peripheral blood lymphocytes isolated from healthy individuals were irradiated in vitro with x-rays and γ-H2AX foci were analysed using fluorescent microscopy and flow cytometric methods. Further, the effect of protein phosphatase 2A inhibitors such as calyculin A, fostriecin and okadiac acid on the retention of foci was studied. Fluorescent microscopy was found to be a more sensitive method than flow cytometry. Calyculin A showed significant retention of focal signals at 6 h with 1.5-fold increased retention compared to radiation alone; this may prove beneficial in early triage management because of a better dose approximation.

Pharmacological approach to increase the retention of radiation induced gamma-H2AX foci using phosphatase inhibitors: significance in radiation biodosimetry.

In a scenario of accidental mass radiation exposure, transportation and analyzing samples may take its time resulting in loss of biomarker information over this period. The present study aims to use phosphatases inhibitors for longer retention of foci signals to adopt γ-H2AX as a biodosimetric biomarker for the management of early triage. Peripheral blood lymphocytes isolated from healthy individuals irradiated in vitro with X-rays, and γ-H2AX analysed using fluorescent microscopy and flow cytometric methods. Further, the effect of protein phosphatase 2A inhibitors like Calyculin A, Fostriecin and Okadiac acid on the retention of foci were studied. The fluorescent microscopy to be more sensitive method when compared to flow cytometry. Calyculin A showed significant retention of foci signals at 6h with 1.5 fold increased retention of foci signals, this may prove beneficial in early triage management, because of a better dose approximation.

Genetic Variants in CD44 and MAT1A Confer Susceptibility to Acute Skin Reaction in Breast Cancer Patients Undergoing Radiation Therapy.

PURPOSE: Heterogeneity in radiation therapy (RT)-induced normal tissue toxicity is observed in 10% of cancer patients, limiting the therapeutic outcomes. In addition to treatment-related factors, normal tissue adverse reactions also manifest from genetic alterations in distinct pathways majorly involving DNA damage-repair genes, inflammatory cytokine genes, cell cycle regulation, and antioxidant response. Therefore, the common sequence variants in these radioresponsive genes might modify the severity of normal tissue toxicity, and the identification of the same could have clinical relevance as a predictive biomarker. METHODS AND MATERIALS: The present study was conducted in a cohort of patients with breast cancer to evaluate the possible associations between genetic variants in radioresponsive genes described previously and the risk of developing RT-induced acute skin adverse reactions. We tested 22 genetic variants reported in 18 genes (ie, NFE2L2, OGG1, NEIL3, RAD17, PTTG1, REV3L, ALAD, CD44, RAD9A, TGFßR3, MAD2L2, MAP3K7, MAT1A, RPS6KB2, ZNF830, SH3GL1, BAX, and XRCC1) using TaqMan assay-based real-time polymerase chain reaction. At the end of RT, the severity of skin damage was scored, and the subjects were dichotomized as nonoverresponders (Radiation Therapy Oncology Group grade <2) and overresponders (Radiation Therapy Oncology Group grade ≥2) for analysis. RESULTS: Of the 22 single nucleotide polymorphisms studied, the rs8193 polymorphism lying in the micro-RNA binding site of 3'-UTR of CD44 was significantly (P=.0270) associated with RT-induced adverse skin reactions. Generalized multifactor dimensionality reduction analysis showed significant (P=.0107) gene-gene interactions between MAT1A and CD44. Furthermore, an increase in the total number of risk alleles was associated with increasing occurrence of overresponses (P=.0302). CONCLUSIONS: The genetic polymorphisms in radioresponsive genes act as genetic modifiers of acute normal tissue toxicity outcomes after RT by acting individually (rs8193), by gene-gene interactions (MAT1A and CD44), and/or by the additive effects of risk alleles.

Polymorphisms in double strand break repair related genes influence radiosensitivity phenotype in lymphocytes from healthy individuals.

BACKGROUND: A range of individual radiosensitivity observed in humans can influence individual's susceptibility toward cancer risk and radiotherapy outcome. Therefore, it is important to measure the variation in radiosensitivity and to identify the genetic factors influencing it. METHODS: By adopting a pathway specific genotype-phenotype design, we established the variability in cellular radiosensitivity by performing γ-H2AX foci assay in healthy individuals. Further, we genotyped ten selected SNPs in candidate genes XRCC3 (rs861539), XRCC4 (rs1805377), XRCC5 (rs3835), XRCC6 (rs2267437), ATM (rs3218698, rs1800057), LIG4 (rs1805388), NBN (rs1805794), RAD51 (rs1801320) and PRKDC (rs7003908), and analysed their influence on observed variation in radiosensitivity. RESULTS: The rs2267437 polymorphisms in XRCC6 was associated (P=0.0326) with increased DSB induction while rs1805388 in LIG4 (P=0.0240) was associated with increased radioresistance. Further, multiple risk alleles decreased the DSB repair capacity in an additive manner. Polymorphisms in candidate DSB repair genes can act individually or in combination to the efficacy of DSB repair process, resulting in variation of cellular radiosensitivity. CONCLUSIONS: Current study suggests that γ-H2AX assay may fulfil the role of a rapid and sensitive biomarker that can be used for epidemiological studies to measure variations in radiosensitivity. DSB repair gene polymorphisms can impact the formation and repair of DSBs. IMPACT: γ-H2AX foci analysis as well as DSBs repair gene polymorphisms can be used to assess cellular radiosensitivity, which will be useful in population risk assessment, disease prediction, individualization of radiotherapy and also in setting the radiation protection standards.

Polymorphisms in radio-responsive genes and its association with acute toxicity among head and neck cancer patients.

Cellular and molecular approaches are being explored to find a biomarker which can predict the development of radiation induced acute toxicity prior to radiation therapy. SNPs in radiation responsive genes may be considered as an approach to develop tools for finding the inherited basis of clinical radiosensitivity. The current study attempts to screen single nucleotide polymorphisms/deletions in DNA damage response, DNA repair, profibrotic cytokine as well as antioxidant response genes and its predictive potential with the normal tissue adverse reactions from 183 head and neck cancer patients undergoing platinum based chemoradiotherapy or radiotherapy alone. We analysed 22 polymorphisms in 17 genes having functional relevance to radiation response. Radiation therapy induced oral mucositis and skin erythema was considered as end point for clinical radiosensitivity. Direct correlation of heterozygous and mutant alleles with acute reactions as well as haplotype correlation revealed NBN variants to be of predictive significance in analysing oral mucositis prior to radiotherapy. In addition, genetic linkage disequilibrium existed in XRCC1 polymorphisms for >grade 2 oral mucositis and skin reaction indicating the complex inheritance pattern. The current study indicates an association for polymorphism in NBN with normal tissue radiosensitivity and further warrants the replication of such studies in a large set of samples.

Influence of double-strand break repair on radiation therapy-induced acute skin reactions in breast cancer patients.

PURPOSE: Curative radiation therapy (RT)-induced toxicity poses strong limitations for efficient RT and worsens the quality of life. The parameter that explains when and to what extent normal tissue toxicity in RT evolves would be of clinical relevance because of its predictive value and may provide an opportunity for personalized treatment approach. METHODS AND MATERIALS: DNA double-strand breaks and repair were analyzed by microscopic γ-H2AX foci analysis in peripheral lymphocytes from 38 healthy donors and 80 breast cancer patients before RT, a 2 Gy challenge dose of x-ray exposed in vitro. RESULTS: The actual damage (AD) at 0.25, 3, and 6 hours and percentage residual damage (PRD) at 3 and 6 hours were used as parameters to measure cellular radiosensitivity and correlated with RT-induced acute skin reactions in patients stratified as non-overresponders (NOR) (Radiation Therapy Oncology Group [RTOG] grade <2) and overresponders (OR) (RTOG grade ≥2). The results indicated that the basal and induced (at 0.25 and 3 hours) γ-H2AX foci numbers were nonsignificant (P>.05) between healthy control donors and the NOR and OR groups, whereas it was significant between ORs and healthy donors at 6 hours (P<.001). There was a significantly higher PRD in OR versus NOR (P<.05), OR versus healthy donors (P<.001) and NOR versus healthy donors (P<.01), supported further by the trend analysis (r=.2392; P=.0326 at 6 hours). CONCLUSIONS: Our findings strongly suggest that the measurement of PRD by performing γ-H2AX foci analysis has the potential to be developed into a clinically useful predictive assay.

DNA double-strand break analysis by γ-H2AX foci: a useful method for determining the overreactors to radiation-induced acute reactions among head-and-neck cancer patients.

PURPOSE: Interindividual variability in normal tissue toxicity during radiation therapy is a limiting factor for successful treatment. Predicting the risk of developing acute reactions before initiation of radiation therapy may have the benefit of opting for altered radiation therapy regimens to achieve minimal adverse effects with improved tumor cure. METHODS AND MATERIALS: DNA double-strand break (DSB) induction and its repair kinetics in lymphocytes of head-and-neck cancer patients undergoing chemoradiation therapy was analyzed by counting γ-H2AX foci, neutral comet assay, and a modified version of neutral filter elution assay. Acute normal tissue reactions were assessed by Radiation Therapy Oncology Group criteria. RESULTS: The correlation between residual DSBs and the severity of acute reactions demonstrated that residual γ-H2AX foci in head-and-neck cancer patients increased with the severity of oral mucositis and skin reaction. CONCLUSIONS: Our results suggest that γ-H2AX analysis may have predictive implications for identifying the overreactors to mucositis and skin reactions among head-and-neck cancer patients prior to initiation of radiation therapy.