RACK1 and IRE1 participate in the translational quality control of amyloid precursor protein in Drosophila models of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by dysregulation of the expression and processing of the amyloid precursor protein (APP). Protein quality control systems are dedicated to remove faulty and deleterious proteins to maintain cellular protein homeostasis (proteostasis). Identidying mechanisms underlying APP protein regulation is crucial for understanding AD pathogenesis. However, the factors and associated molecular mechanisms regulating APP protein quality control remain poorly defined. In this study, we show that mutant APP with its mitochondrial-targeting sequence ablated exhibited predominant endoplasmic reticulum (ER) distribution and led to aberrant ER morphology, deficits in locomotor activity, and shortened lifespan. We searched for regulators that could counteract the toxicity caused by the ectopic expression of this mutant APP. Genetic removal of the ribosome-associated quality control (RQC) factor RACK1 resulted in reduced levels of ectopically expressed mutant APP. By contrast, gain of RACK1 function increased mutant APP level. Additionally, overexpression of the ER stress regulator (IRE1) resulted in reduced levels of ectopically expressed mutant APP. Mechanistically, the RQC related ATPase VCP/p97 and the E3 ubiquitin ligase Hrd1 were required for the reduction of mutant APP level by IRE1. These factors also regulated the expression and toxicity of ectopically expressed wild type APP, supporting their relevance to APP biology. Our results reveal functions of RACK1 and IRE1 in regulating the quality control of APP homeostasis and mitigating its pathogenic effects, with implications for the understanding and treatment of AD.

Stalled translation by mitochondrial stress upregulates a CNOT4-ZNF598 ribosomal quality control pathway important for tissue homeostasis.

Translational control exerts immediate effect on the composition, abundance, and integrity of the proteome. Ribosome-associated quality control (RQC) handles ribosomes stalled at the elongation and termination steps of translation, with ZNF598 in mammals and Hel2 in yeast serving as key sensors of translation stalling and coordinators of downstream resolution of collided ribosomes, termination of stalled translation, and removal of faulty translation products. The physiological regulation of RQC in general and ZNF598 in particular in multicellular settings is underexplored. Here we show that ZNF598 undergoes regulatory K63-linked ubiquitination in a CNOT4-dependent manner and is upregulated upon mitochondrial stresses in mammalian cells and Drosophila. ZNF598 promotes resolution of stalled ribosomes and protects against mitochondrial stress in a ubiquitination-dependent fashion. In Drosophila models of neurodegenerative diseases and patient cells, ZNF598 overexpression aborts stalled translation of mitochondrial outer membrane-associated mRNAs, removes faulty translation products causal of disease, and improves mitochondrial and tissue health. These results shed lights on the regulation of ZNF598 and its functional role in mitochondrial and tissue homeostasis.

Role of Mentzer Index for Differential Diagnosis of Iron Deficiency Anaemia and Beta Thalassemia Trait.

BACKGROUND: The most common differential diagnoses of microcytic hypochromic anaemia are iron deficiency anaemia and beta thalassemia. Globally, thalassemia affects approximately 4.4 out of every 10,000 live births whereas iron deficiency anaemia comprises half of all anaemia worldwide as per world health organization. The definitive diagnosis of beta thalassemia trait and iron deficiency anaemia requires haemoglobin analysis and iron studies respectively, which are not possible to perform in all suspected cases especially in resource limited settings. The study aims to evaluate the reliability of mentzer index in differentiating beta thalassemia trait from iron deficiency anaemia. METHODS: This was a cross sectional, observational study done on 59 patients each of beta thalassemia trait and iron deficiency anaemia from August 2019 to July 2020. Patients who were found to be having iron deficiency anaemia diagnosed by iron studies and beta thalassemia trait diagnosed by Hb electrophoresis were enrolled in the study using simple random sampling technique. RESULTS: Mentzer index correctly identified 95.76% of overall patients. Area under receiver operating characteristic curve was 0.993 (95% CI, 0.985-1.002, p<0.001). For beta thalassemia trait, mentzer index showed a sensitivity of 93.2%, specificity of 98.3%, positive predictive value of 98.2%, negative predictive value of 93.5%; while for iron deficiency anaemia, sensitivity of 98.3%, specificity of 93.2%, positive predictive value of 93.5% and negative predictive value of 98.2%. Youden's index was 91.5. CONCLUSIONS: The findings of the present study make mentzer index a reliable screening method, especially in a resource poor setting, like Nepal. Further confirmation by gold standard tests is recommended.

Prevalence and clinical impact of anemia in patients diagnosed with chronic obstructive pulmonary disease: A cross-sectional study.

Background and Aims: Unlike classically described polycythemia, anemia is found to be more prevalent in patients with chronic obstructive pulmonary disease (COPD). Anemia increases the cost of hospital stay and causes an increased risk of adverse outcomes including death in COPD patients. This study was done to find the prevalence of anemia in COPD patients, the factors associated, and the outcomes of anemic COPD. Methods: It was a quantitative, descriptive-analytical, and cross-sectional study conducted in Tribhuvan University Teaching Hospital's medical wards and the Emergency Room from September 2019 to September 2020. A simple random sampling method was used. Clinical information was obtained, and patients were followed up 3 months after discharge to document the number of exacerbations and deaths if present. Results: The patients in our study had a mean age of 70.80 ± 11.16 years. Most were female. Most (85.5%) had a history of exposure to firewood smoke. Twenty-three percent of the patients had anemia and these patients had significantly greater mortality 3 months postdischarge. Middle-old and old were more likely to have anemia with odds ratio (OR) of 2.55 (confidence interval [CI]: 0.48-13.5) and 13.6 (CI: 1.12-24.2), respectively. Current smokers had less likelihood of having anemia (OR: 0.05, CI: 0.006-0.49). Multivariate analysis showed that age, sex, and smoking status were significant determinants of anemia in COPD. There was no association between anemia and duration of hospital stay. However, mortality was higher at 3 months in COPD patients with anemia (p < 0.001). Conclusion: In COPD patients, anemia is prevalent comorbidity that is significantly linked to higher mortality but not to exacerbations. It is unknown, though, if treating anemia in COPD patients will affect the patient's outcome. Additional research in this area may be possible.

The mTORC2/AKT/VCP axis is associated with quality control of the stalled translation of poly(GR) dipeptide repeats in C9-ALS/FTD.

Expansion of G4C2 hexanucleotide repeats in the chromosome 9 ORF 72 (C9ORF72) gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) with frontotemporal dementia (C9-ALS/FTD). Dipeptide repeats generated by unconventional translation, especially the R-containing poly(GR), have been implicated in C9-ALS/FTD pathogenesis. Mutations in other genes, including TAR DNA-binding protein 43 KD (TDP-43), fused in sarcoma (FUS), and valosin-containing protein, have also been linked to ALS/FTD, and upregulation of amyloid precursor protein (APP) is observed at the early stage of ALS and FTD. Fundamental questions remain as to the relationships between these ALS/FTD genes and whether they converge on similar cellular pathways. Here, using biochemical, cell biological, and genetic analyses in Drosophila disease models, patient-derived fibroblasts, and mammalian cell culture, we show that mechanistic target of rapamycin complex 2 (mTORC2)/AKT signaling is activated by APP, TDP-43, and FUS and that mTORC2/AKT and its downstream target valosin-containing protein mediate the effect of APP, TDP-43, and FUS on the quality control of C9-ALS/FTD-associated poly(GR) translation. We also find that poly(GR) expression results in reduction of global translation and that the coexpression of APP, TDP-43, and FUS results in further reduction of global translation, presumably through the GCN2/eIF2α-integrated stress response pathway. Together, our results implicate mTORC2/AKT signaling and GCN2/eIF2α-integrated stress response as common signaling pathways underlying ALS/FTD pathogenesis.

Reverse electron transfer is activated during aging and contributes to aging and age-related disease.

Mechanisms underlying the depletion of NAD+ and accumulation of reactive oxygen species (ROS) in aging and age-related disorders remain poorly defined. We show that reverse electron transfer (RET) at mitochondrial complex I, which causes increased ROS production and NAD+ to NADH conversion and thus lowered NAD+ /NADH ratio, is active during aging. Genetic or pharmacological inhibition of RET decreases ROS production and increases NAD+ /NADH ratio, extending the lifespan of normal flies. The lifespan-extending effect of RET inhibition is dependent on NAD+ -dependent Sirtuin, highlighting the importance of NAD+ /NADH rebalance, and on longevity-associated Foxo and autophagy pathways. RET and RET-induced ROS and NAD+ /NADH ratio changes are prominent in human induced pluripotent stem cell (iPSC) model and fly models of Alzheimer's disease (AD). Genetic or pharmacological inhibition of RET prevents the accumulation of faulty translation products resulting from inadequate ribosome-mediated quality control, rescues relevant disease phenotypes, and extends the lifespan of Drosophila and mouse AD models. Deregulated RET is therefore a conserved feature of aging, and inhibition of RET may open new therapeutic opportunities in the context of aging and age-related diseases including AD.

Prevention of ribosome collision-induced neuromuscular degeneration by SARS CoV-2-encoded Nsp1.

An overarching goal of aging and age-related neurodegenerative disease research is to discover effective therapeutic strategies applicable to a broad spectrum of neurodegenerative diseases. Little is known about the extent to which targetable pathogenic mechanisms are shared among these seemingly diverse diseases. Translational control is critical for maintaining proteostasis during aging. Gaining control of the translation machinery is also crucial in the battle between viruses and their hosts. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing COVID-19 pandemic. Here, we show that overexpression of SARS-CoV-2-encoded nonstructural protein 1 (Nsp1) robustly rescued neuromuscular degeneration and behavioral phenotypes in Drosophila models of Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. These diseases share a common mechanism: the accumulation of aberrant protein species due to the stalling and collision of translating ribosomes, leading to proteostasis failure. Our genetic and biochemical analyses revealed that Nsp1 acted in a multipronged manner to resolve collided ribosomes, abort stalled translation, and remove faulty translation products causative of disease in these models, at least in part through the ribosome recycling factor ABCE1, ribosome-associated quality-control factors, autophagy, and AKT signaling. Nsp1 exhibited exquisite specificity in its action, as it did not modify other neurodegenerative conditions not known to be associated with ribosome stalling. These findings uncover a previously unrecognized mechanism of Nsp1 in manipulating host translation, which can be leveraged for combating age-related neurodegenerative diseases that are affecting millions of people worldwide and currently without effective treatment.

Delayed Visual Loss in a Patient with Snake Bite: Case Report of an Unusual Neuro-Ophthalmic Presentation.

Neurotoxin-related optic neuritis (ON) after snake bite is uncommon. Here, we present a case of a 70-year-old female who developed bilateral painless loss of vision after she received treatment with anti-snake venom (ASV). She had only perception of light on assessment of visual acuity on admission which then improved drastically after administration of intravenous methylprednisolone (MP) after making the provisional diagnosis of ON on the basis of history and clinical findings of the patient. Imaging and visual-evoked potential could not be done initially, and they were done after the administration of intravenous MP which had normal findings. ASV, though being a lifesaving treatment, has been sometimes associated with ON.

Cushing Syndrome in a Pediatric Patient with Topical Steroid Overuse.

Cushing syndrome is a state of hypercortisolism from exogenous or endogenous exposure to glucocorticoids resulting in various clinical manifestations. In this case report, we present a case of a 15-month-old child who presented with cushingoid facies due to over-the-counter misuse of a very potent topical steroid (clobetasol 0.05%) for suspected scabies. Laboratory measurement of urinary free cortisol level was low, and 8 : 00 am basal cortisol level was measured, which was decreased, which confirmed the diagnosis of Cushing syndrome due to exogenous source. Over-the-counter topical steroids should not be used, and one should always consult a registered medical practitioner before using such products. Physicians when prescribing topical steroids should warn patients about the potential side effects of prolonged use of topical steroids.

An Unusual Case of Eosinophilia with Systemic Lupus Erythematosus: A Case Report and Review of Literature.

Eosinophilia can be caused by various conditions, parasitic infection being the most common cause. Here, we present a case of a 17-year male who presented with multisystem involvement and eosinophilia. He was later diagnosed to have systemic lupus erythematosus with eosinophilia which is a rare combination. Despite being a diagnostic challenge, these patients can be well managed with immunosuppressive therapy if recognized in time.

Regulation of reverse electron transfer at mitochondrial complex I by unconventional Notch action in cancer stem cells.

Metabolic flexibility is a hallmark of many cancers where mitochondrial respiration is critically involved, but the molecular underpinning of mitochondrial control of cancer metabolic reprogramming is poorly understood. Here, we show that reverse electron transfer (RET) through respiratory chain complex I (RC-I) is particularly active in brain cancer stem cells (CSCs). Although RET generates ROS, NAD+/NADH ratio turns out to be key in mediating RET effect on CSC proliferation, in part through the NAD+-dependent Sirtuin. Mechanistically, Notch acts in an unconventional manner to regulate RET by interacting with specific RC-I proteins containing electron-transporting Fe-S clusters and NAD(H)-binding sites. Genetic and pharmacological interference of Notch-mediated RET inhibited CSC growth in Drosophila brain tumor and mouse glioblastoma multiforme (GBM) models. Our results identify Notch as a regulator of RET and RET-induced NAD+/NADH balance, a critical mechanism of metabolic reprogramming and a metabolic vulnerability of cancer that may be exploited for therapeutic purposes.

Inefficient quality control of ribosome stalling during APP synthesis generates CAT-tailed species that precipitate hallmarks of Alzheimer's disease.

Amyloid precursor protein (APP) metabolism is central to Alzheimer's disease (AD) pathogenesis, but the key etiological driver remains elusive. Recent failures of clinical trials targeting amyloid-ß (Aß) peptides, the proteolytic fragments of amyloid precursor protein (APP) that are the main component of amyloid plaques, suggest that the proteostasis-disrupting, key pathogenic species remain to be identified. Previous studies suggest that APP C-terminal fragment (APP.C99) can cause disease in an Aß-independent manner. The mechanism of APP.C99 pathogenesis is incompletely understood. We used Drosophila models expressing APP.C99 with the native ER-targeting signal of human APP, expressing full-length human APP only, or co-expressing full-length human APP and ß-secretase (BACE), to investigate mechanisms of APP.C99 pathogenesis. Key findings are validated in mammalian cell culture models, mouse 5xFAD model, and postmortem AD patient brain materials. We find that ribosomes stall at the ER membrane during co-translational translocation of APP.C99, activating ribosome-associated quality control (RQC) to resolve ribosome collision and stalled translation. Stalled APP.C99 species with C-terminal extensions (CAT-tails) resulting from inadequate RQC are prone to aggregation, causing endolysosomal and autophagy defects and seeding the aggregation of amyloid ß peptides, the main component of amyloid plaques. Genetically removing stalled and CAT-tailed APP.C99 rescued proteostasis failure, endolysosomal/autophagy dysfunction, neuromuscular degeneration, and cognitive deficits in AD models. Our finding of RQC factor deposition at the core of amyloid plaques from AD brains further supports the central role of defective RQC of ribosome collision and stalled translation in AD pathogenesis. These findings demonstrate that amyloid plaque formation is the consequence and manifestation of a deeper level proteostasis failure caused by inadequate RQC of translational stalling and the resultant aberrantly modified APP.C99 species, previously unrecognized etiological drivers of AD and newly discovered therapeutic targets.

Critical enzymes for biosynthesis of cucurbitacin derivatives in watermelon and their biological significance.

Various cucurbitacins have been isolated, and their structures have been elucidated. Owing to their economic potential and importance as active pharmacological compounds, their cytotoxicity in various cancer cells has been assessed. Here, we mined several candidate genes with potential involvement in cucurbitacin biosynthesis in watermelon (Citrullus lanatus) and performed in vitro enzymatic assays and instrumental analyses using various substrates to identify cucurbitacin functions and products. Enzymatic activities of two acetyltransferases (ACTs) and one UDP-glucosyltransferase (UGT) against cucurbitacins were confirmed, resulting in the synthesis of novel cucurbitacins in vivo and/or in vitro to our knowledge. As ACTs and UGT are involved in the dynamic conversion of cucurbitacins by catalyzing acetylation and glucosylation at moieties in the cucurbitacins skeleton, these findings improve our knowledge on how these genes contribute to the diversity of cucurbitacins.

Cucurbitacin B Activates Bitter-Sensing Gustatory Receptor Neurons via Gustatory Receptor 33a in Drosophila melanogaster.

The Gustatory system enables animals to detect toxic bitter chemicals, which is critical for insects to survive food induced toxicity. Cucurbitacin is widely present in plants such as cucumber and gourds that acts as an anti-herbivore chemical and an insecticide. Cucurbitacin has a harmful effect on insect larvae as well. Although various beneficial effects of cucurbitacin such as alleviating hyperglycemia have also been documented, it is not clear what kinds of molecular sensors are required to detect cucurbitacin in nature. Cucurbitacin B, a major bitter component of bitter melon, was applied to induce action potentials from sensilla of a mouth part of the fly, labellum. Here we identify that only Gr33a is required for activating bitter-sensing gustatory receptor neurons by cucurbitacin B among available 26 Grs, 23 Irs, 11 Trp mutants, and 26 Gr-RNAi lines. We further investigated the difference between control and Gr33a mutant by analyzing binary food choice assay. We also measured toxic effect of Cucurbitacin B over 0.01 mM range. Our findings uncover the molecular sensor of cucurbitacin B in Drosophila melanogaster. We propose that the discarded shell of Cucurbitaceae can be developed to make a new insecticide.

Molecular sensor of nicotine in taste of Drosophila melanogaster.

Nicotine is an alkaloid and potent parasympathomimetic stimulant found in the leaves of many plants including Nicotiana tabacum, which functions as an anti-herbivore chemical and an insecticide. Chemoreceptors embedded in the gustatory receptor neurons (GRNs) enable animals to judge the quality of bitter compounds and respond to them. Various taste receptors such as gustatory receptors (GRs), ionotropic receptors (IRs), transient receptor potential channels (TRPs), and pickpocket channels (PPKs) have been shown to have important roles in taste sensation. However, the mechanism underlying nicotine taste sensation has not been resolved in the insect model. Here we identify molecular receptors to detect the taste of nicotine and provide electrophysiological and behavioral evidence that gustatory receptors are required for avoiding nicotine-laced foods. Our results demonstrate that gustatory receptors are reasonable targets to develop new pesticides that maximize the insecticidal effects of nicotine.

Mechanism of Acetic Acid Gustatory Repulsion in Drosophila.

The decision to consume or reject a food based on the degree of acidity is critical for animal survival. However, the gustatory receptors that detect sour compounds and influence feeding behavior have been elusive. Here, using the fly, Drosophila melanogaster, we reveal that a member of the ionotropic receptor family, IR7a, is essential for rejecting foods laced with high levels of acetic acid. IR7a is dispensable for repulsion of other acidic compounds, indicating that the gustatory sensation of acids occurs through a repertoire rather than a single receptor. The fly's main taste organ, the labellum, is decorated with bristles that house dendrites of gustatory receptor neurons (GRNs). IR7a is expressed in a subset of bitter GRNs rather than GRNs dedicated to sour taste. Our findings indicate that flies taste acids through a repertoire of receptors, enabling them to discriminate foods on the basis of acid composition rather than just pH.

Gustatory receptor 28b is necessary for avoiding saponin in Drosophila melanogaster.

Saponins function as a natural self-defense mechanism for plants to deter various insects due to their unpleasant taste and their toxicity. Here, we provide evidence that saponin from Quillaja saponaria functions as an antifeedant as well as an insecticide to ward off insects in both the larval and the adult stages. Using a behavioral screen of 26 mutant fly lines, we show that the Gr28b gene cluster plays a role in saponin avoidance in the labellum. The Gr28b mutant does not avoid saponin and exhibits increased lethality when fed saponin-mixed food. Tissue-specific rescue experiments with five different Gr28b isoforms revealed that only the Gr28b.c isoform is required for saponin sensation. We propose that in contrast to sensing many other bitter compounds, saponin sensing does not require the function of core taste receptors, such as GR32a, GR33a, and GR66a. Our results reveal a novel role for GR28b in taste. In addition, the ability of saponin to act as insecticides as well as antifeedants suggests its potential application in controlling insect pests.