Effects of flanking sequences and cellular context on subcellular behavior and pathology of mutant HTT.

Huntington's disease (HD) is caused by an expansion of a poly glutamine (polyQ) stretch in the huntingtin protein (HTT) that is necessary to cause pathology and formation of HTT aggregates. Here we ask whether expanded polyQ is sufficient to cause pathology and aggregate formation. By addressing the sufficiency question, one can identify cellular processes and structural parameters that influence HD pathology and HTT subcellular behavior (i.e. aggregation state and subcellular location). Using Drosophila, we compare the effects of expressing mutant full-length human HTT (fl-mHTT) to the effects of mutant human HTTexon1 and to two commonly used synthetic fragments, HTT171 and shortstop (HTT118). Expanded polyQ alone is not sufficient to cause inclusion formation since full-length HTT and HTTex1 with expanded polyQ are both toxic although full-length HTT remains diffuse while HTTex1 forms inclusions. Further, inclusions are not sufficient to cause pathology since HTT171-120Q forms inclusions but is benign and co-expression of HTT171-120Q with non-aggregating pathogenic fl-mHTT recruits fl-mHTT to aggregates and rescues its pathogenicity. Additionally, the influence of sequences outside the expanded polyQ domain is revealed by finding that small modifications to the HTT118 or HTT171 fragments can dramatically alter their subcellular behavior and pathogenicity. Finally, mutant HTT subcellular behavior is strongly modified by different cell and tissue environments (e.g. fl-mHTT appears as diffuse nuclear in one tissue and diffuse cytoplasmic in another but toxic in both). These observations underscore the importance of cellular and structural context for the interpretation and comparison of experiments using different fragments and tissues to report the effects of expanded polyQ.

Progressive transcriptional changes in metabolic genes and altered fatbody homeostasis in Drosophila model of Huntington's disease.

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder marked by progressive neuronal atrophy, particularly in striatum and cerebral cortex. Although predominant manifestations of the disease include loss in the triad of motor, cognitive and behavioral capabilities, metabolic dysfunction in patients and HD models are being increasingly recognized. Patients display progressive body weight loss, which aggravates the disease and leads to cachexia in the terminal stages. Using the Drosophila model of HD, we have earlier reported that diseased flies exhibit an atypical pattern of lipid gain and loss with progression along with exhibiting extensive mitochondrial dysfunction, impaired calcium homeostasis and heightened apoptosis in the fatbody. Here, we first monitored the structural changes that abdominal fatbody undergoes with disease progression. Further, we checked the transcriptional changes of key metabolic genes in whole fly as well as genes regulating mitochondrial function, apoptosis, autophagy and calcium homeostasis in the abdominal fatbody. We found extensive alterations in whole-body and fatbody-specific transcriptional profile of the diseased flies, which was in consort with their stage-specific physiological state. Additionally, we also assessed lysosome-mediated autophagy in the fatbody of diseased flies in order to ascertain the mechanisms contributing to fatbody atrophy at the terminal stage. Interestingly, we found elevated autophagy in fatbody of flies throughout disease progression. This study provides new insights into the effect on peripheral metabolism due to degeneration of neurons in the neurodegenerative disease, thereby discerns novel mechanisms leading to cachexia in diseased flies and advocates for the need of managing metabolic dysfunctions in HD.

Metabolism in Huntington's disease: a major contributor to pathology.

Huntington's disease (HD) is a progressively debilitating neurodegenerative disease exhibiting autosomal-dominant inheritance. It is caused by an unstable expansion in the CAG repeat tract of HD gene, which transforms the disease-specific Huntingtin protein (HTT) to a mutant form (mHTT). The profound neuronal death in cortico-striatal circuits led to its identification and characterisation as a neurodegenerative disease. However, equally disturbing are the concomitant whole-body manifestations affecting nearly every organ of the diseased individuals, at varying extents. Altered central and peripheral metabolism of energy, proteins, nucleic acids, lipids and carbohydrates encompass the gross pathology of the disease. Intense fluctuation of body weight, glucose homeostasis and organ-specific subcellular abnormalities are being increasingly recognised in HD. Many of these metabolic abnormalities exist years before the neuropathological manifestations such as chorea, cognitive decline and behavioural abnormalities develop, and prove to be reliable predictors of the disease progression. In this review, we provide a consolidated overview of the central and peripheral metabolic abnormalities associated with HD, as evidenced from clinical and experimental studies. Additionally, we have discussed the potential of metabolic biomolecules to translate into efficient biomarkers for the disease onset as well as progression. Finally, we provide a brief outlook on the efficacy of existing therapies targeting metabolic remediation. While it is clear that components of altered metabolic pathways can mark many aspects of the disease, it is only conceivable that combinatorial therapies aiming for neuronal protection in consort with metabolic upliftment will prove to be more efficient than the existing symptomatic treatment options.

Combating silver nanoparticle-mediated toxicity in Drosophila melanogaster with curcumin.

Nanoscale materials display unique physical and chemical properties that enable their assimilation into a variety of industrial and consumer products. Amongst the widely used nanomaterials, silver nanoparticles (AgNPs) have gained tremendous recognition for various applications, owing to their extraordinary plasmonic and bactericidal properties. Despite of the extensive usage of AgNPs in various sectors, its impact on human health remains ambiguous. Several studies have established that higher doses of AgNPs are detrimental to organismal health. In order to attain the best from these versatile nanoparticles, a recent advent of green nanotechnology, that is, employment of metal nanoparticles synthesized using plant extracts, has emerged. Here, using Drosophila as a model system, we tested if adding curcumin, a biologically active polyphenolic compound present in turmeric, having multitudes of therapeutic properties, could mitigate AgNP-mediated biotoxicity. We found that co-administration of AgNPs with curcumin in the fly food could alleviate several harmful effects evoked by AgNPs ingestion in Drosophila model. Addition of curcumin superseded reduction in feeding, pupation, eclosion, pigmentation, and fertility caused by AgNPs ingestion. Interestingly, impairment in ovary development observed in flies reared on AgNPs-supplemented food was also partially restored by co-administration of AgNPs with curcumin. Furthermore, substantial alleviation of reactive oxygen species level and cell death was observed in larval tissues upon co-supplementation of AgNPs with curcumin. We therefore propose that curcumin, when administered with AgNPs, can abrogate the toxic manifestations of AgNPs ingestion and hence can be incorporated in various consumer products encompassing it.

Discriminatory alteration of carbohydrate homeostasis by gold nanoparticles ingestion in Drosophila.

With the extensive usage of gold nanoparticles (AuNPs) in various industrial sectors and biomedical applications, evaluation of their possible effects on human health becomes imperative. Therefore, the present study was aimed toward assessing the dose-dependent impact of AuNPs ingestion on metabolic homeostasis using Drosophila melanogaster as a model system. We found that larval ingestion of higher dose of AuNPs significantly reduced body weight. Further analysis of the crucial energy reservoir showed selective alteration in carbohydrate levels without any change in the lipid and protein levels. Transcriptional downregulation of glycogen synthase further supported impaired glycogen metabolism in flies supplemented with higher dose of AuNPs. Additionally, ingestion of higher dose of AuNPs in larvae results in significantly increased levels of reactive oxygen species (ROS) in the peripheral tissues, suggestive of stress condition. Our findings clearly imply that supplementing higher doses of AuNPs at an early developmental stage can potentially cause weight loss, impair glycogen metabolism, and elevate ROS production. Therefore, determination of a biologically effective dose is critical for the safety of mankind and vulnerable populations at the workplace.

Baseline Karnofsky performance status is independently predictive of death within 30 days of intracranial radiation therapy completion for metastatic disease.

INTRODUCTION: For patients with brain metastases, palliative radiation therapy (RT) has long been a standard of care for improving quality of life and optimizing intracranial disease control. The duration of time between completion of palliative RT and patient death has rarely been evaluated. METHODS: A compilation of two prospective institutional databases encompassing April 2015 through December 2018 was used to identify patients who received palliative intracranial radiation therapy. A multivariate logistic regression model characterized patients adjusting for age, sex, admission status (inpatient versus outpatient), Karnofsky Performance Status (KPS), and radiation therapy indication. RESULTS: 136 consecutive patients received intracranial palliative radiation therapy. Patients with baseline KPS <70 (OR = 2.2; 95%CI = 1.6-3.1; p < 0.0001) were significantly more likely to die within 30 days of treatment. Intracranial palliative radiation therapy was most commonly delivered to provide local control (66% of patients) or alleviate neurologic symptoms (32% of patients), and was most commonly delivered via whole brain radiation therapy in 10 fractions to 30 Gy (38% of patients). Of the 42 patients who died within 30 days of RT, 31 (74%) received at least 10 fractions. CONCLUSIONS: Our findings indicate that baseline KPS <70 is independently predictive of death within 30 days of palliative intracranial RT, and that a large majority of patients who died within 30 days received at least 10 fractions. These results indicate that for poor performance status patients requiring palliative intracranial radiation, hypofractionated RT courses should be strongly considered.

Radiosurgery dose reduction for brain metastases on immunotherapy (RADREMI): A prospective phase I study protocol.

INTRODUCTION: Up to 20% of patients with brain metastases treated with immune checkpoint inhibitor (ICI) therapy and concomitant stereotactic radiosurgery (SRS) suffer from symptomatic radiation necrosis. The goal of this study is to evaluate Radiosurgery Dose Reduction for Brain Metastases on Immunotherapy (RADREMI) on six-month symptomatic radiation necrosis rates. METHODS: This study is a prospective single arm Phase I pilot study which will recruit patients with brain metastases receiving ICI delivered within 30 days before SRS. All patients will be treated with RADREMI dosing, which involves SRS doses of 18 Gy for 0-2 cm lesions, 14 Gy for 2.1-3 cm lesions, and 12 Gy for 3.1-4 cm lesions. All patients will be monitored for six-month symptomatic radiation necrosis (defined as a six-month rate of clinical symptomatology requiring steroid administration and/or operative intervention concomitant with imaging findings consistent with radiation necrosis) and six-month local control. We expect that RADREMI dosing will significantly reduce the symptomatic radiation necrosis rate of concomitant SRS + ICI without significantly sacrificing the local control obtained by the present RTOG 90-05 SRS dosing schema. Local control will be defined according to the Response Assessment in Neuro-Oncology (RANO) criteria. DISCUSSION: This study is the first prospective trial to investigate the safety of dose-reduced SRS in treatment of brain metastases with concomitant ICI. The findings should provide fertile soil for future multi-institutional collaborative efficacy trials of RADREMI dosing for this patient population. TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT04047602 (registration date: July 25, 2019).

Comparative study of naturally occurring huntingtin fragments in Drosophila points to exon 1 as the most pathogenic species in Huntington's disease.

Although Huntington's disease is caused by the expansion of a CAG triplet repeat within the context of the 3144-amino acid huntingtin protein (HTT), studies reveal that N-terminal fragments of HTT containing the expanded PolyQ region can be produced by proteolytic processing and/or aberrant splicing. N-terminal HTT fragments are also prevalent in postmortem tissue, and expression of some of these fragments in model organisms can cause pathology. This has led to the hypothesis that N-terminal peptides may be critical modulators of disease pathology, raising the possibility that targeting aberrant splicing or proteolytic processing may present attractive therapeutic targets. However, many factors can contribute to pathology, including genetic background and differential expression of transgenes, in addition to intrinsic differences between fragments and their cellular effects. We have used Drosophila as a model system to determine the relative toxicities of different naturally occurring huntingtin fragments in a system in which genetic background, transgene expression levels and post-translational proteolytic processing can be controlled. These studies reveal that among the naturally occurring N-terminal HTT peptides, the exon 1 peptide is exceptionally pathogenic and exhibits unique structural and biophysical behaviors that do not appear to be incremental changes compared with other fragments. If this proves correct, efforts to specifically reduce the levels of exon 1 peptides or to target toxicity-influencing post-translational modifications that occur with the exon 1 context are likely to have the greatest impact on pathology.

Neuroprotective Potential of Eugenol in Polyglutamine-Mediated Neurodegenerative Disease Using Transgenic Drosophila Model.

Polyglutamine (PolyQ) diseases including Huntington's disease are devastating neurodegenerative disorders characterized by progressive neuronal loss and motor dysfunction. PolyQ pathology involves multiple cellular events and phytochemicals with multi-target mechanisms hold promise to treat these diseases with least side effects. One such promising phytochemical is Eugenol, which possesses antioxidant and anti-inflammatory properties, potentially targeting disrupted cellular pathways in PolyQ diseases. The present study investigated the effects of Eugenol on neurodegeneration and motor dysfunction in transgenic Drosophila models of PolyQ diseases. In this study, the robust pseudopupil assay was performed to analyze adult photoreceptor neuron degeneration, a marker of widespread degenerative events. Furthermore, the well-established crawling and climbing assays were conducted to evaluate progressive motor dysfunction in the PolyQ larvae and flies. This study found that Eugenol administration at disease onset or after progression reduced PolyQ disease phenotypes, particularly, neurodegeneration and motor dysfunction in a dose-dependent manner and with no side effects. Thus, this study suggests that Eugenol could be a viable candidate for developing treatments for PolyQ diseases, offering a multi-target approach with the potential for minimal or no side effects compared to conventional therapies.

Analysis of Retrospective Versus Prospective Peer Review in a Multisite Academic Radiation Department.

Purpose: Our multisite academic radiation department reviewed our experience with transitioning from weekly primarily retrospective to daily primarily prospective peer review to improve plan quality and decrease the rate of plan revisions after treatment start. Methods and Materials: This study was an institutional review board-approved prospective comparison of radiation treatment plan review outcomes of plans reviewed weekly (majority within 1 week after treatment start) versus plans reviewed daily (majority before treatment start, except brachytherapy, frame-based radiosurgery, and some emergent plans). Deviations were based on peer comments and considered major if plan revisions were recommended before the next fraction and minor if modifications were suggested but not required. Categorical variables were compared using χ2 distribution tests of independence; means were compared using independent t tests. Results: In all, 798 patients with 1124 plans were reviewed: 611 plans weekly and 513 plans daily. Overall, 76 deviations (6.8%) were noted. Rates of any deviation were increased in the daily era (8.6% vs 5.2%; P = .026), with higher rates of major deviations in the daily era (4.1% vs 1.6%; P = .012). Median working days between initial simulation and treatment was the same across eras (8 days). Deviations led to a plan revision at a higher rate in the daily era (84.1% vs 31.3%; P < .001). Conclusions: Daily prospective peer review is feasible in a multisite academic setting. Daily peer review with emphasis on prospective plan evaluation increased constructive plan feedback, plan revisions, and plan revisions being implemented before treatment start.

Preoperative stereotactic radiosurgery for patients with 1-4 brain metastases: A single-arm phase 2 trial outcome analysis (NCT03398694).

Background: Stereotactic radiosurgery (SRS) following surgical resection is the standard of care for patients with symptomatic oligo brain metastasis (BM), however, it is associated with 10-15% local failure. Targeting a resection cavity is imprecise, thus preoperative radiosurgery where the target is well-defined may be superior, however, the efficacy of preoperative SRS has not yet been tested in a clinical trial. Methods: We conducted a phase 2, single-arm trial of preoperative SRS followed by surgical resection in patients with 1-4 symptomatic oligo BMs (NCT03398694) with the primary objective of measuring 6-month local control (LC). SRS was delivered to all patients utilizing a gamma knife or linear accelerator as per RTOG-9005 dosing criteria [Shaw E, Scott C, Souhami L, et al. Single dose radiosurgical treatment of recurrent previously irradiated primary brain tumors and brain metastases: final report of RTOG protocol 90-05. Int J Radiat Oncol Biol Phys. 2000;47(2):291-298] based on tumor diameter with the exception that the largest lesion diameter treated was 5 cm with 15 Gy with all SRS treatment given in single fraction dosing. Results: The trial screened 50 patients, 48 patients were treated under the protocol and 32 patients completed the entire follow-up period. Of all the patients who completed the follow-up period, the primary endpoint of 6-month LC was 100% (95% CI: 0.891-1.000; P = .005). Secondary endpoints, presented as medians, were overall survival (17.6 months), progression-free survival (5.3 months), distant in-brain failure (40.8% at 1 year), leptomeningeal failure (4.8% at 1 year), and radiation necrosis (7.7% at 1 year). Conclusions: Our data confirms superior local control in patients who received preoperative SRS when compared to historical controls. Further study with a larger randomized cohort of patients is warranted to fully understand the benefits of preoperative SRS.

Genomic analysis of human brain metastases treated with stereotactic radiosurgery reveals unique signature based on treatment failure.

Stereotactic radiosurgery (SRS) has been shown to be efficacious for the treatment of limited brain metastasis (BM); however, the effects of SRS on human brain metastases have yet to be studied. We performed genomic analysis on resected brain metastases from patients whose resected lesion was previously treated with SRS. Our analyses demonstrated for the first time that patients possess a distinct genomic signature based on type of treatment failure including local failure, leptomeningeal spread, and radio-necrosis. Examination of the center and peripheral edge of the tumors treated with SRS indicated differential DNA damage distribution and an enrichment for tumor suppressor mutations and DNA damage repair pathways along the peripheral edge. Furthermore, the two clinical modalities used to deliver SRS, LINAC and GK, demonstrated differential effects on the tumor landscape even between controlled primary sites. Our study provides, in human, biological evidence of differential effects of SRS across BM's.

Methylene blue modulates huntingtin aggregation intermediates and is protective in Huntington's disease models.

Huntington's disease (HD) is a devastating neurodegenerative disorder with no disease-modifying treatments available. The disease is caused by expansion of a CAG trinucleotide repeat and manifests with progressive motor abnormalities, psychiatric symptoms, and cognitive decline. Expression of an expanded polyglutamine repeat within the Huntingtin (Htt) protein impacts numerous cellular processes, including protein folding and clearance. A hallmark of the disease is the progressive formation of inclusions that represent the culmination of a complex aggregation process. Methylene blue (MB), has been shown to modulate aggregation of amyloidogenic disease proteins. We investigated whether MB could impact mutant Htt-mediated aggregation and neurotoxicity. MB inhibited recombinant protein aggregation in vitro, even when added to preformed oligomers and fibrils. MB also decreased oligomer number and size and decreased accumulation of insoluble mutant Htt in cells. In functional assays, MB increased survival of primary cortical neurons transduced with mutant Htt, reduced neurodegeneration and aggregation in a Drosophila melanogaster model of HD, and reduced disease phenotypes in R6/2 HD modeled mice. Furthermore, MB treatment also promoted an increase in levels of BDNF RNA and protein in vivo. Thus, MB, which is well tolerated and used in humans, has therapeutic potential for HD.

Amendment of Altered Immune Response by Curcumin in Drosophila Model of Huntington's Disease.

BACKGROUND: Though primarily classified as a brain disorder, surplus studies direct Huntington's disease (HD) to be a multi-system disorder affecting various tissues and organs, thus affecting overall physiology of host. Recently, we have reported that neuronal expression of mutant huntingtin induces immune dysregulation in Drosophila and may pose chronic threat to challenged individuals. Therefore, we tested the polyphenolic compound curcumin to circumvent the impact of immune dysregulation in Drosophila model of HD. OBJECTIVE: The present study examined the molecular basis underlying immune derangements and immunomodulatory potential of curcumin in HD. METHODS: UAS-GAL4 system was used to imitate the HD symptoms in Drosophila, and the desired female progenies (elav > Httex1pQ25; control and elav > Httex1pQ93; diseased) were cultured on food mixed without and with 10 µM concentration of curcumin since early development. Effect of curcumin supplementation was investigated by monitoring the hemocytes' count and their functional abilities in diseased condition. Reactive oxygen species (ROS) level in cells was assessed by DHE staining and mitochondrial dysfunction was assessed by CMXros red dye. In addition, transcript levels of pro-inflammatory cytokines and anti-microbial peptides were monitored by qRT-PCR. RESULTS: We found that curcumin supplementation commendably reduced higher crystal cell count and phenoloxidase activity in diseased flies. Interestingly, curcumin significantly managed altered plasmatocytes count, improved their phagocytic activity by upregulating the expression of key phagocytic receptors in HD condition. Moreover, substantial alleviation of ROS levels and mitochondria dysfunction was observed in plasmatocytes of diseased flies upon curcumin supplementation. Furthermore, curcumin administration effectively attenuated transcriptional expression of pro-inflammatory cytokines and AMPs in diseased flies. CONCLUSIONS: Our results indicate that curcumin efficiently attenuates immune derangements in HD flies and may prove beneficial in alleviating complexities associated with HD.

Genomic Analysis of Human Brain Metastases Treated with Stereotactic Radiosurgery Under the Phase-II Clinical Trial (NCT03398694) Reveals DNA Damage Repair at the Peripheral Tumor Edge.

Stereotactic Radiosurgery (SRS) is one of the leading treatment modalities for oligo brain metastasis (BM), however no comprehensive genomic data assessing the effect of radiation on BM in humans exist. Leveraging a unique opportunity, as part of the clinical trial (NCT03398694), we collected post-SRS, delivered via Gamma-knife or LINAC, tumor samples from core and peripheral-edges of the resected tumor to characterize the genomic effects of overall SRS as well as the SRS delivery modality. Using these rare patient samples, we show that SRS results in significant genomic changes at DNA and RNA levels throughout the tumor. Mutations and expression profiles of peripheral tumor samples indicated interaction with surrounding brain tissue as well as elevated DNA damage repair. Central samples show GSEA enrichment for cellular apoptosis while peripheral samples carried an increase in tumor suppressor mutations. There are significant differences in the transcriptomic profile at the periphery between Gamma-knife vs LINAC.

Management of altered metabolic activity in Drosophila model of Huntington's disease by curcumin.

Huntington's disease (HD) is a devastating polyglutamine disorder characterized by extensive neurodegeneration and metabolic abnormalities at systemic, cellular and intracellular levels. Metabolic alterations in HD manifest as abnormal body weight, dysregulated biomolecule levels, impaired adipocyte functions, and defective energy state which exacerbate disease progression and pose acute threat to the health of challenged individuals in form of insulin resistance, cardiovascular disease, and energy crisis. To colossally mitigate disease symptoms, we tested the efficacy of curcumin in Drosophila model of HD. Curcumin is the bioactive component of turmeric (Curcuma longa Linn), well-known for its ability to modulate metabolic activities. We found that curcumin effectively managed abnormal body weight, dysregulated lipid content, and carbohydrate level in HD flies. In addition, curcumin administration lowered elevated reactive-oxygen-species levels in adult adipose tissue of diseased flies, and improved survival and locomotor function in HD flies at advanced disease stage. Altogether, these findings clearly suggest that curcumin efficiently attenuates metabolic derangements in HD flies and can prove beneficial in alleviating the complexities associated with HD.

Pan-neuronal expression of human mutant huntingtin protein in Drosophila impairs immune response of hemocytes.

Huntington's disease (HD) is a late-onset; progressive, dominantly inherited neurological disorder marked by an abnormal expansion of polyglutamine (poly Q) repeats in Huntingtin (HTT) protein. The pathological effects of mutant Huntingtin (mHTT) are not restricted to the nervous system but systemic abnormalities including immune dysregulation have been evidenced in clinical and experimental settings of HD. Indeed, mHTT is ubiquitously expressed and could induce cellular toxicity by directly acting on immune cells. However, it is still unclear if selective expression of mHTT exon1 in neurons could induce immune responses and hemocytes' function. In the present study, we intended to monitor perturbations in the hemocytes' population and their physiological functions in Drosophila, caused by pan-neuronal expression of mHTT protein. A measure of hemocyte count and their physiological activities caused by pan-neuronal expression of mHTT protein highlighted the extent of immune dysregulation occurring with disease progression. We found that pan-neuronal expression of mHTT significantly alters crystal cells and plasmatocyte count in larvae and adults with disease progression. Interestingly, plasmatocytes isolated from diseased conditions exhibit a gradual decline in phagocytic activity ex vivo at progressive stages of the disease as compared to age-matched control groups. In addition, diseased flies displayed elevated reactive oxygen species (ROS) in circulating plasmatocytes at the larval stage and in sessile plasmatocytes of hematopoietic pockets at terminal stages of disease. These findings strongly implicate that neuronal expression of mHTT alone is sufficient to induce non-cell-autonomous immune dysregulation in vivo.

An In Vitro and In Vivo Study of the Efficacy and Toxicity of Plant-Extract-Derived Silver Nanoparticles.

Silver nanoparticles (AgNPs) display unique plasmonic and antimicrobial properties, enabling them to be helpful in various industrial and consumer products. However, previous studies showed that the commercially acquired silver nanoparticles exhibit toxicity even in small doses. Hence, it was imperative to determine suitable synthesis techniques that are the most economical and least toxic to the environment and biological entities. Silver nanoparticles were synthesized using plant extracts and their physico-chemical properties were studied. A time-dependent in vitro study using HEK-293 cells and a dose-dependent in vivo study using a Drosophila model helped us to determine the correct synthesis routes. Through biological analyses, we found that silver nanoparticles' cytotoxicity and wound-healing capacity depended on size, shape, and colloidal stability. Interestingly, we observed that out of all the synthesized AgNPs, the ones derived from the turmeric extract displayed excellent wound-healing capacity in the in vitro study. Furthermore, the same NPs exhibited the least toxic effects in an in vivo study of ingestion of these NPs enriched food in Drosophila, which showed no climbing disability in flies, even at a very high dose (250 mg/L) for 10 days. We propose that stabilizing agents played a superior role in establishing the bio-interaction of nanoparticles. Our study reported here verified that turmeric-extract-derived AgNPs displayed biocompatibility while exhibiting the least cytotoxicity.

Deciphering the key mechanisms leading to alteration of lipid metabolism in Drosophila model of Huntington's disease.

Huntington's disease (HD) is an inherited, progressively debilitating disorder marked by prominent degeneration in striatal and cortical brain regions. HD is caused by (CAG)n repeat expansion in huntingtin (HTT) gene that translates into a mutant form of the ubiquitously present Huntingtin (HTT) protein. Extensive metabolic dysfunction coexisting with overt neuropathies has been evidenced in clinical and experimental settings of HD. Body weight loss despite normal to high caloric intake remains a critical determinant of the disease progression and a challenge for therapeutic interventions. In the present study, we intended to monitor the cellular and molecular perturbations in Drosophila, caused by pan-neuronal expression of mHTT (mutant Huntingtin) protein. We found aberrant transcription profile of key lipolytic and lipogenic genes in whole-body of the fly with disease progression. Interestingly, fatbody undergoes extensive alteration of vital cellular processes and eventually surrenders to increased apoptotic cell death in terminal stage of the disease. Extensive mitochondrial dysfunction from early disease stage along with calcium derangement at terminal stage were observed in fatbody, which contribute to its deteriorating integrity. All the mechanisms were monitored progressively, at different disease stages, and many alterations were documented in the early stage itself. Our study hence provides insight into the mechanisms through which neuronal expression of mHTT might be inflicting the profound systemic effects, specifically on lipid metabolism, and may open new therapeutic avenues for alleviation of the multidimensional disease.

Melatonin and curcumin reestablish disturbed circadian gene expressions and restore locomotion ability and eclosion behavior in Drosophila model of Huntington's disease.

Deficit in locomotion (motor) ability and disturbance of the circadian behavior and sleep-wake pattern characterize Huntington's disease (HD). Here, we examined the disturbance of circadian timing with the progression of HD pathogenesis, and tested the efficacy of melatonin and curcumin in preventing the motor deficit and disturbed eclosion behavior in the Drosophila model of HD. To examine circadian timing, we assayed mRNA expression of genes of the transcriptional feedback (TF) loop that generates the near 24-h rhythmicity. We performed qPCR of the Period, Timeless, Clock, Cycle, Clockwork, and Cryptochrome genes in transgenic fly heads from elav-Gal4 (pan neuronal) and PDF-Gal4 (PDF-specific neurons) driver lines through the progression of HD disease post-eclosion, from day 1 to its terminal stage on day 13. Cycle was arrhythmic from day 1, but Period and Timeless became arrhythmic on day 13 of the HD pathogenesis in elav, but not PDF, neurons. Twenty-four-hour mRNA rhythms showed alteration in the waveform properties (mesor and amplitude, not acrophase), but not in the persistence, in both elav-Gal4 and PDF-Gal4 HD flies; however, disturbance of the clock gene rhythm was delayed in PDF-Gal4 flies. To assess the preventive effects on HD pathogenesis, flies of both driver lines were provided with melatonin (50, 100, or 150 µg) or curcumin (10 µM) in the diet commencing from the larval stage. Both melatonin (100 µg) and curcumin reestablished the 24-h pattern in mRNA expression of Period and Timeless to normal (control) levels, and significantly improved both locomotion ability and eclosion behavior of HD flies. We suggest that the disturbance of circadian timekeeping progressively accelerated HD pathogenesis, possibly via modulation of the transcriptional state that resulted in the modification of the Huntington gene. These findings suggest melatonin and curcumin might be potential therapeutic agents for the treatment of HD in humans, although this needs specific investigation.