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Gliomas are highly malignant brain tumors with poor prognosis and short survival. NAD+ has been shown to impact multiple processes that are dysregulated in cancer; however, anti-cancer therapies targeting NAD+ synthesis have had limited success due to insufficient mechanistic understanding. Here, we adapted a Drosophila glial neoplasia model and discovered the genetic requirement for NAD+ synthase nicotinamide mononucleotide adenylyltransferase (NMNAT) in glioma progression in vivo and in human glioma cells. Overexpressing enzymatically active NMNAT significantly promotes glial neoplasia growth and reduces animal viability. Mechanistic analysis suggests that NMNAT interferes with DNA damage-p53-caspase-3 apoptosis signaling pathway by enhancing NAD+-dependent posttranslational modifications (PTMs) poly(ADP-ribosyl)ation (PARylation) and deacetylation of p53. Since PARylation and deacetylation reduce p53 pro-apoptotic activity, modulating p53 PTMs could be a key mechanism by which NMNAT promotes glioma growth. Our findings reveal a novel tumorigenic mechanism involving protein complex formation of p53 with NAD+ synthetic enzyme NMNAT and NAD+-dependent PTM enzymes that regulates glioma growth., eLife digest One of the most common types of brain cancer, glioma, emerges when harmful mutations take place in the ‘glial’ cells tasked with supporting neurons. When these genetically damaged cells are not fixed or eliminated, they can go on to multiply uncontrollability. A protein known as p53 can help to repress emerging tumors by stopping mutated cells in their tracks. Glioma is a highly deadly cancer, and treatments are often ineffective. Some of these approaches have focused on a protein involved in the creation of the coenzyme NAD+, which is essential to the life processes of all cells. However, these drugs have had poor outcomes. Instead, Liu et al. focused on NMNAT, the enzyme that participates in the final stage of the creation of NAD+. NMNAT is known to protect neurons, but it is unclear how it involved in cancer. Experiments in fruit flies which were then validated in human glioma cells showed that increased NMNAT activity allowed glial cells with harmful mutations to survive and multiply. Detailed molecular analysis showed that NMNAT orchestrates chemical modifications that inactivate p53. It does so by working with other molecular actors to direct NAD+ to add and remove chemical groups that control the activity of p53. Taken together, these results show how NMNAT can participate in the emergence of brain cancers. They also highlight the need for further research on whether drugs that inhibit this enzyme could help to suppress tumors before they become deadly.

Harmful algal blooms (HABs) are a rising health and environmental concern in the United States, particularly in South Florida. Skin contact and the ingestion of contaminated water or fish and other seafood have been proven to have severe toxicity to humans in some cases. However, the impact of aerosolized HAB toxins is poorly understood. In particular, knowledge regarding either the immediate or long-term effects of exposure to aerosolized cyanotoxins produced by freshwater blue-green algae does not exist. The aim of this study was to probe the toxicity of aerosolized cyanobacterial blooms using Drosophila melanogaster as an animal model. The exposure of aerosolized HABs at an early age leads to the most severe long-term impact on health and longevity among all age groups. Young groups and old males showed a strong acute response to HAB exposure. In addition, brain morphological analysis using fluorescence imaging reveals significant indications of brain degeneration in females exposed to aerosolized HABs in early or late stages. These results indicate that one-time exposure to aerosolized HAB particles causes a significant health risk, both immediately and in the long-term. Interestingly, age at the time of exposure plays an important role in the specific nature of the impact of aerosol HABs. As BMAA and microcystin have been found to be the significant toxins in cyanobacteria, the concentration of both toxins in the water and aerosols was examined. BMAA and microcystin are consistently detected in HAB waters, although their concentrations do not always correlate with the severity of the health impact, suggesting the potential contribution from additional toxins present in the aerosolized HAB. This study demonstrates, for the first time, the health risk of exposure to aerosolized HAB, and further highlights the critical need and importance of understanding the toxicity of aerosolized cyanobacteria HAB particles and determining the immediate and long-term health impacts of HAB exposure.

Polyamines are tightly regulated polycations that are essential for life. Loss-of-function mutations in spermine synthase (SMS), a polyamine biosynthesis enzyme, cause Snyder-Robinson syndrome (SRS), an X-linked intellectual disability syndrome; however, little is known about the neuropathogenesis of the disease. Here we show that loss of dSms in Drosophila recapitulates the pathological polyamine imbalance of SRS and causes survival defects and synaptic degeneration. SMS deficiency leads to excessive spermidine catabolism, which generates toxic metabolites that cause lysosomal defects and oxidative stress. Consequently, autophagy–lysosome flux and mitochondrial function are compromised in the Drosophila nervous system and SRS patient cells. Importantly, oxidative stress caused by loss of SMS is suppressed by genetically or pharmacologically enhanced antioxidant activity. Our findings uncover some of the mechanisms underlying the pathological consequences of abnormal polyamine metabolism in the nervous system and may provide potential therapeutic targets for treating SRS and other polyamine-associated neurological disorders. Mutations in spermine synthase lead to Snyder-Robinson syndrome, a form of intellectual disability syndrome. Here the authors develop a Drosophila model of this disease, and show that lysosomal dysfunction and oxidative stress contribute to the morphological phenotype in these flies, as well as to cellular deficits in cells derived from patients.

Polyamines are tightly regulated polycations that are essential for life. Loss-of-function mutations in spermine synthase (SMS), a polyamine biosynthesis enzyme, cause Snyder-Robinson syndrome (SRS), an X-linked intellectual disability syndrome; however, little is known about the neuropathogenesis of the disease. Here we show that loss of dSms in Drosophila recapitulates the pathological polyamine imbalance of SRS and causes survival defects and synaptic degeneration. SMS deficiency leads to excessive spermidine catabolism, which generates toxic metabolites that cause lysosomal defects and oxidative stress. Consequently, autophagy–lysosome flux and mitochondrial function are compromised in the Drosophila nervous system and SRS patient cells. Importantly, oxidative stress caused by loss of SMS is suppressed by genetically or pharmacologically enhanced antioxidant activity. Our findings uncover some of the mechanisms underlying the pathological consequences of abnormal polyamine metabolism in the nervous system and may provide potential therapeutic targets for treating SRS and other polyamine-associated neurological disorders., Mutations in spermine synthase lead to Snyder-Robinson syndrome, a form of intellectual disability syndrome. Here the authors develop a Drosophila model of this disease, and show that lysosomal dysfunction and oxidative stress contribute to the morphological phenotype in these flies, as well as to cellular deficits in cells derived from patients.

The R21C substitution in cardiac troponin I (cTnI) is the only identified mutation within its unique N-terminal extension that is associated with hypertrophic cardiomyopathy (HCM) in man. Particularly, this mutation is located in the consensus sequence for β-adrenergic-activated protein kinase A (PKA)-mediated phosphorylation. The mechanisms by which this mutation leads to heart disease are still unclear. Therefore, we generated cTnI knock-in mouse models carrying an R21C mutation to evaluate the resultant functional consequences. Measuring the in vivo levels of incorporated mutant and WT cTnI, and their basal phosphorylation levels by top-down mass spectrometry demonstrated: 1) a dominant-negative effect such that, the R21C+/- hearts incorporated 24.9% of the mutant cTnI within the myofilament; and 2) the R21C mutation abolished the in vivo phosphorylation of Ser(23)/Ser(24) in the mutant cTnI. Adult heterozygous (R21C+/-) and homozygous (R21C+/+) mutant mice activated the fetal gene program and developed a remarkable degree of cardiac hypertrophy and fibrosis. Investigation of cardiac skinned fibers isolated from WT and heterozygous mice revealed that the WT cTnI was completely phosphorylated at Ser(23)/Ser(24) unless the mice were pre-treated with propranolol. After propranolol treatment (-PKA), the pCa-tension relationships of all three mice (i.e. WT, R21C+/-, and R21C+/+) were essentially the same. However, after treatment with propranolol and PKA, the R21C cTnI mutation reduced (R21C+/-) or abolished (R21C+/+) the well known decrease in the Ca(2+) sensitivity of tension that accompanies Ser(23)/Ser(24) cTnI phosphorylation. Altogether, the combined effects of the R21C mutation appear to contribute toward the development of HCM and suggest that another physiological role for the phosphorylation of Ser(23)/Ser(24) in cTnI is to prevent cardiac hypertrophy.

In skeletal muscle, the myosin molecule contains two sets of noncovalently attached low molecular weight proteins, the regulatory (RLC) and essential (ELC) light chains. To assess the functional and developmental significance of the fast skeletal isoform of the RLC (RLC-f), the murine fast skeletal RLC gene (Mylpf) was disrupted by homologous recombination. Heterozygotes containing an intronic neo cassette (RLC-/+) had approximately one-half of the amount of the RLC-f mRNA compared to wild-type (WT) mice but their muscles were histologically normal in both adults and neonates. In contrast, homozygous mice (RLC-/-) had no RLC-f mRNA or protein and completely lacked both fast and slow skeletal muscle. This was likely due to interference with mRNA processing in the presence of the neo cassette. These RLC-f null mice died immediately after birth, presumably due to respiratory failure since their diaphragms lacked skeletal muscle. The body weight of newborn RLC-f null mice was decreased 30% compared to heterozygous or WT newborn mice. The lack of skeletal muscle formation in the null mice did not affect the development of other organs including the heart. In addition, we found that WT mice did not express the ventricular/slow skeletal RLC isoform (RLC-v/s) until after birth, while it was expressed normally in the embryonic heart. The lack of skeletal muscle formation observed in RLC-f null mice indicates the total dependence of skeletal muscle development on the presence of RLC-f during embryogenesis. This observation, along with the normal function of the RLC-v/s in the heart, implicates a coupled, diverse pathway for RLC-v/s and RLC-f during embryogenesis, where RLC-v/s is responsible for heart development and RLC-f is necessary for skeletal muscle formation. In conclusion, in this study we demonstrate that the Mylpf gene is critically important for fast and slow skeletal muscle development.

Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant disease caused by mutations in all of the major sarcomeric proteins, including the ventricular myosin regulatory light-chain (RLC). The E22K-RLC mutation has been associated with a rare variant of cardiac hypertrophy defined by mid-left ventricular obstruction due to papillary muscle hypertrophy. This mutation was later found to cause ventricular and septal hypertrophy. We have generated transgenic (Tg) mouse lines of myc-WT (wild type) and myc-E22K mutant of human ventricular RLC and have examined the functional consequences of this FHC mutation in skinned cardiac-muscle preparations. In longitudinal sections of whole mouse hearts stained with hematoxylin and eosin, the E22K-mutant hearts of 13-month-old animals showed signs of inter-ventricular septal hypertrophy and enlarged papillary muscles with no filament disarray. Echo examination did not reveal evidence of cardiac hypertrophy in Tg-E22K mice compared to Tg-WT or Non-Tg hearts. Physiological studies utilizing skinned cardiac-muscle preparations showed an increase by ΔpCa50≥0.1 in Ca2+ sensitivity of myofibrillar ATPase activity and force development in Tg-E22K mice compared with Tg-WT or Non-Tg littermates. Our results suggest that E22K-linked FHC is mediated through Ca2+-dependent events. The FHC-mediated structural perturbations in RLC that affect Ca2+ binding properties of the mutated myocardium are responsible for triggering the abnormal function of the heart that in turn might initiate a hypertrophic process and lead to heart failure.

R21C is the only FHC-associated mutation located in the N-terminal domain of cardiac TnI (cTnI) and is within the consensus sequence for PKA phosphorylation (RR21RSS). We have developed an R21C cTnI knock-in (KI) mouse model and have evaluated the mouse hearts for biochemical, biophysical, structural and functional changes. Our results show that the R21C KI mice (heterozygous;R21C+/- and homozygous;R21C+/+) developed the FHC phenotype as evidenced by the presence of hypertrophy and fibrosis. Some hypertrophic markers such as, ANP, BNP and β-MHC were found elevated at a late age (18 months). The R21C+/- and R21C+/+ mice had decreased phosphorylation at Ser23/24 (∼18% and 90%, respectively) compared to WT mice. Top down mass spectrometry of cTnI from the R21C+/- mice demonstrated a molar ratio of 1:4 R21C:WT in the hearts. Using three different methods to sacrifice the WT and mutant mice, we did not find any significant decrease in the Ca2+ sensitivity of force upon PKA treatment. Western blot analysis of these mice showed that the endogenous cTnI in the WT and R21C+/- mice is completely phosphorylated at Ser23/24. However, when mice were treated with propanolol (β-adrenergic receptor antagonist) before sacrifice, the Ca2+ sensitivity was decreased after PKA treatment of the WT (0.25 pCa) and R21C+/- mice (0.14 pCa). In contrast the R21C+/+ mice did not show a significant decrease in Ca2+ sensitivity after PKA treatment. No significant changes were found in the maximal force in all three mice, before and after PKA treatment. Our results suggest that the primary mechanism for producing hypertrophy in the R21C mice results from the impaired ability of the myofilament to respond to the desensitizing effects of PKA phosphorylation. Supported by NIH grant HL042325

Clinical studies have revealed that mutations in the ventricular myosin regulatory light chain (RLC) lead to the development of familial hypertrophic cardiomyopathy (FHC), an autosomal dominant disease characterized by left ventricular hypertrophy, myofibrillar disarray and sudden cardiac death. While mutations in other contractile proteins have been studied widely by others, there is no report elucidating the mechanism(s) associated with FHC-linked RLC mutations. In this study, we have assessed the functional consequences of two RLC mutations, R58Q and N47K, in transgenic mice. Clinical phenotypes associated with these mutations included inter-ventricular hypertrophy, abnormal ECG findings and the R58Q mutation caused multiple cases of premature sudden cardiac death. Simultaneous measurements of the ATPase and force in transgenic skinned papillary muscle fibers from mutated versus control mice showed an increase in the Ca(2+) sensitivity of ATPase and steady-state force only in R58Q fibers. The calculated energy cost or rate of dissociation of force generating myosin cross-bridges (ATPase/force ratio) plotted as a function of activation state was the same in all groups of fibers. Both mutations caused prolonged [Ca(2+)] transients in electrically stimulated intact papillary muscles; however, the R58Q mutation also resulted in a significantly prolonged force transient. Our results suggest that the phenotypes of FHC observed in patients harboring these RLC mutations correlate with the extent of physiological changes monitored in transgenic fibers. Cardiac hypertrophy observed in patients is most likely caused by the activation of compensatory mechanisms ensuing from higher workloads due to incomplete relaxation as evidenced by prolonged [Ca(2+)] transients for both N47K and R58Q fibers. Furthermore, the poor prognosis of the R58Q patients may be associated with more severe diastolic dysfunction due to the slower off-rate of Ca(2+) from troponin C leading to longer force and [Ca(2+)] transients and increased Ca(2+) sensitivity of ATPase and force.