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Snyder Robinson Syndrome (SRS) is a rare disease associated with a defective spermine synthase gene and low intracellular spermine levels. In this study, a spermine replacement therapy was developed using a spermine prodrug that enters cells via the polyamine transport system. The prodrug was comprised of three components: a redox-sensitive quinone “trigger”, a “trimethyl lock (TML)” aryl “release mechanism”, and spermine. The presence of spermine in the design facilitated uptake by the polyamine transport system. The quinone–TML motifs provided a redox-sensitive agent, which upon intracellular reduction generated a hydroquinone, which underwent intramolecular cyclization to release free spermine and a lactone byproduct. Rewardingly, most SRS fibroblasts treated with the prodrug revealed a significant increase in intracellular spermine. Administering the spermine prodrug through feeding in a Drosophila model of SRS showed significant beneficial effects. In summary, a spermine prodrug is developed and provides a lead compound for future spermine replacement therapy experiments.

Polyamine dysregulation plays key roles in a broad range of human diseases from cancer to neurodegeneration. Snyder-Robinson syndrome (SRS) is the first known genetic disorder of the polyamine pathway, caused by X-linked recessive loss-of-function mutations in spermine synthase. In the Drosophila SRS model, altered spermidine/spermine balance has been associated with increased generation of ROS and aldehydes, consistent with elevated spermidine catabolism. These toxic byproducts cause mitochondrial and lysosomal dysfunction, which are also observed in cells from SRS patients. No efficient therapy is available. We explored the biochemical mechanism and discovered acetyl-CoA reduction and altered protein acetylation as potentially novel pathomechanisms of SRS. We repurposed the FDA-approved drug phenylbutyrate (PBA) to treat SRS using an in vivo Drosophila model and patient fibroblast cell models. PBA treatment significantly restored the function of mitochondria and autolysosomes and extended life span in vivo in the Drosophila SRS model. Treating fibroblasts of patients with SRS with PBA ameliorated autolysosome dysfunction. We further explored the mechanism of drug action and found that PBA downregulates the first and rate-limiting spermidine catabolic enzyme spermidine/spermine N1-acetyltransferase 1 (SAT1), reduces the production of toxic metabolites, and inhibits the reduction of the substrate acetyl-CoA. Taken together, we revealed PBA as a potential modulator of SAT1 and acetyl-CoA levels and propose PBA as a therapy for SRS and potentially other polyamine dysregulation-related diseases.

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.

We have identified a GRAP variant (c.311A>T; p.GIn104Leu) cose-gregating with autosomal recessive nonsyndromic deafness in two unrelated families. GRAP encodes a member of the highly conserved growth factor receptor-bound protein 2 (GRB2)/Sem-5/drk family of proteins, which are involved in Ras signaling; however, the function of the growth factor receptor-bound protein 2 (GRB2)-related adaptor protein (GRAP) in the auditory system is not known. Here, we show that, in mouse, Grap is expressed in the inner ear and the protein localizes to the neuronal fibers innervating cochlear and utricular auditory hair cells. Downstream of receptor kinase (drk), the Drosophila homolog of human GRAP, is expressed in Johnston's organ (JO), the fly hearing organ, and the loss of drk in JO causes scolopidium abnormalities. drk mutant flies present deficits in negative geotaxis behavior, which can be suppressed by human wild-type but not mutant GRAP. Furthermore, drk specifically colocalizes with synapsin at synapses, suggesting a potential role of such adaptor proteins in regulating actin cytoskeleton dynamics in the nervous system. Our findings establish a causative link between GRAP mutation and nonsyndromic deafness and suggest a function of GRAP/drk in hearing.

Tauopathies, including Alzheimer's disease, are a group of neurodegenerative diseases characterized by abnormal tau hyperphosphorylation that leads to formation of neurofibrillary tangles. Drosophila models of tauopathy display prominent features of the human disease including compromised lifespan, impairments of learning, memory and locomotor functions and age-dependent neurodegeneration visible as vacuolization. Here, we use a Drosophila model of frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), in order to study the neuroprotective capacity of a recently identified neuronal maintenance factor, nicotinamide mononucleotide (NAD) adenylyl transferase (NMNAT), a protein that has both NAD synthase and chaperone function. NMNAT is essential for maintaining neuronal integrity under normal conditions and has been shown to protect against several neurodegenerative conditions. However, its protective role in tauopathy has not been examined. Here, we show that overexpression of NMNAT significantly suppresses both behavioral and morphological deficits associated with tauopathy by means of reducing the levels of hyperphosphorylated tau oligomers. Importantly, the protective activity of NMNAT protein is independent of its NAD synthesis activity, indicating a role for direct protein-protein interaction. Next, we show that NMNAT interacts with phosphorylated tau in vivo and promotes the ubiquitination and clearance of toxic tau species. Consequently, apoptosis activation was significantly reduced in brains overexpressing NMNAT, and neurodegeneration was suppressed. Our report on the molecular basis of NMNAT-mediated neuroprotection in tauopathies opens future investigation of this factor in other protein foldopathies.

The neurotoxic non-protein amino acid, β-N-methylamino-L-alanine (BMAA), was first associated with the high incidence of Amyotrophic Lateral Sclerosis/Parkinsonism Dementia Complex (ALS/PDC) in Guam. Recently, BMAA has been implicated as a fierce environmental factor that contributes to the etiology of Alzheimer’s and Parkinson’s diseases, in addition to ALS. However, the toxicity of BMAA in vivo has not been clearly demonstrated. Here we report our investigation of the neurotoxicity of BMAA in Drosophila. We found that dietary intake of BMAA reduced life span, locomotor functions, and learning and memory abilities in flies. The severity of the alterations in phenotype is correlated with the concentration of BMAA detected in flies. Interestingly, developmental exposure to BMAA had limited impact on survival rate, but reduced fertility in females, and caused delayed neurological impairment in aged adults. Our studies indicate that BMAA exposure causes chronic neurotoxicity, and that Drosophila serves as a useful model in dissecting the pathogenesis of ALS/PDC.

We report the establishment of an in vivo model using the fruit fly Drosophila melanogaster to investigate the toxic effects of L-BMAA. We found that dietary intake of BMAA reduced the lifespan as well as the neurological functions of flies. Furthermore, we have developed an HPLC method to reliably detect both free and protein-bound BMAA in fly tissue extracts.

Nicotinamide mononucleotide adenylyltransferase (NMNAT) is a conserved enzyme in the NAD synthetic pathway. It has also been identified as an effective and versatile neuroprotective factor. However, it remains unclear how healthy neurons regulate the dual functions of NMNAT and achieve self-protection under stress. Here we show that Drosophila Nmnat (DmNmnat) is alternatively spliced into two mRNA variants, RA and RB, which translate to protein isoforms with divergent neuroprotective capacities against spinocerebellar ataxia 1-induced neurodegeneration. Isoform PA/PC translated from RA is nuclear-localized with minimal neuroprotective ability, and isoform PB/PD translated from RB is cytoplasmic and has robust neuroprotective capacity. Under stress, RB is preferably spliced in neurons to produce the neuroprotective PB/PD isoforms. Our results indicate that alternative splicing functions as a switch that regulates the expression of functionally distinct DmNmnat variants. Neurons respond to stress by driving the splicing switch to produce the neuroprotective variant and therefore achieve self-protection., Nicotinamide mononucleotide adenylyltransferase (NMNAT) acts in the NAD biosynthesis pathway and has neuroprotective activity. Ruan et al. show that the neuroprotective activity of NMNAT is restricted to a splice variant of the enzyme, and that this variant is preferentially spliced in response to stress.

Tauopathies, including Alzheimer disease (AD), are a group of neurodegenerative diseases characterized by abnormal hyperphosphorylation of microtubule-associated protein Tau that leads to the formation of neurofibrillary tangles. The understanding of the molecular mechanisms that underlie Tau pathophysiology is still incomplete. The fruit fly Drosophila melanogaster has emerged in the past decade as an excellent model for neurodegenerative diseases. Drosophila models of tauopathy display prominent features of the human disease, including compromised lifespan; impairments of learning, memory, and locomotor functions; and age-dependent neurodegeneration visible as vacuolization. Here, we provide an overview of the current understanding of tauopathy and the methodology of using Drosophila to model tauopathies. We discuss the findings from Drosophila models and the molecular insights into Tau toxicity that Drosophila studies have provided. We further present the value of Drosophila models in genetic and small-molecule screens to identify neuroprotective strategies against tauopathy.

SummaryThe exocyst is a complex of proteins originally identified in yeast that has been implicated in polarized secretion. Components of the exocyst have been implicated in neurite outgrowth, cell polarity, and cell viability. We have isolated an exocyst component, sec15, in a screen for genes required for synaptic specificity. Loss of sec15 causes a targeting defect of photoreceptors that coincides with mislocalization of specific cell adhesion and signaling molecules. Additionally, sec15 mutant neurons fail to localize other exocyst members like Sec5 and Sec8, but not Sec6, to neuronal terminals. However, loss of sec15 does not cause cell lethality in contrast to loss of sec5 or sec6. Our data suggest a role of Sec15 in an exocyst-like subcomplex for the targeting and subcellular distribution of specific proteins. The data also show that functions of other exocyst components persist in the absence of sec15, suggesting that different exocyst components have separable functions.

The isolation of chemically induced mutations in forward genetic screens is one of the hallmarks of Drosophila genetics. However, mapping the corresponding loci and identifying the molecular lesions associated with these mutations are often difficult and labor-intensive. Two mapping methods are most often used in flies: meiotic recombination mapping with marked chromosomes and deficiency mapping. The availability of the fly genome sequence allows the establishment and usage of molecular markers. Single-nucleotide polymorphisms have therefore recently been used to map several genes. Here we show that thousands of molecularly mapped P element insertions in fly strains that are publicly available provide a powerful alternative method to single-nucleotide polymorphism mapping. We present a strategy that allows mapping of lethal mutations, as well as viable mutations with visible phenotypes, with minimal resources. The most important unknown in using recombination rates to map at high resolution is how accurately recombination data correlate with molecular maps in small intervals. We therefore surveyed distortions of recombination rates in intervals P elements in Drosophila.

Axon degeneration is a common and often early feature of neurodegeneration that correlates with the clinical manifestations and progression of neurological disease. Nicotinamide mononucleotide adenylytransferase (NMNAT) is a neuroprotective factor that delays axon degeneration following injury and in models of neurodegenerative diseases suggesting a converging molecular pathway of axon self-destruction. The underlying mechanisms have been under intense investigation and recent reports suggest a central role for axonal mitochondria in both degeneration and NMNAT/WLD(S) (Wallerian degeneration slow)-mediated protection. We used dorsal root ganglia (DRG) explants and Drosophila larval motor neurons (MNs) as models to address the role of mitochondria in Wallerian degeneration (WD). We find that expression of Drosophila NMNAT delays WD in human DRG neurons demonstrating evolutionary conservation of NMNAT function. Morphological comparison of mitochondria from WLD(S)-protected axons demonstrates that mitochondria shrink post-axotomy, though analysis of complex IV activity suggests that they retain their functional capacity despite this morphological change. To determine whether mitochondria are a critical site of regulation for WD, we genetically ablated mitochondria from Drosophila MN axons via the mitochondria trafficking protein milton. Milton loss-of-function did not induce axon degeneration in Drosophila larval MNs, and when axotomized WD proceeded stereotypically in milton distal axons although with a mild, but significant delay. Remarkably, the protective effects of NMNAT/WLD(S) were also maintained in axons devoid of mitochondria. These experiments unveil an axon self-destruction cascade governing WD that is not initiated by axonal mitochondria and for the first time illuminate a mitochondria-independent mechanism(s) regulating WD and NMNAT/WLD(S)-mediated axon protection.

Active zones are specialized presynaptic structures critical for neurotransmission. We show that a neuronal maintenance factor, nicotinamide mononucleotide adenylyltransferase (NMNAT), is required for maintaining active zone structural integrity in Drosophila by interacting with the active zone protein, Bruchpilot (BRP), and shielding it from activity-induced ubiquitin–proteasome-mediated degradation. NMNAT localizes to the peri-active zone and interacts biochemically with BRP in an activity-dependent manner. Loss of NMNAT results in ubiquitination, mislocalization and aggregation of BRP, and subsequent active zone degeneration. We propose that, as a neuronal maintenance factor, NMNAT specifically maintains active zone structure by direct protein–protein interaction.

The selective degeneration of dendrites precedes neuronal cell death in hypoxia-ischemia (HI) and is a neuropathological hallmark of stroke. While it is clear that a number of different molecular pathways likely contribute to neuronal cell death in HI, the mechanisms that govern HI-induced dendrite degeneration are largely unknown. Here, we show that the NAD synthase nicotinamide mononucleotide adenylyltransferase (Nmnat) functions endogenously to protect Drosophila class IV dendritic arborization (da) sensory neurons against hypoxia-induced dendritic damage. Whereas dendrites of wild-type class IV neurons are largely resistant to morphological changes during prolonged periods of hypoxia (

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of these diseases, including Alzheimer's, Parkinson's and Huntington's disease are characterized by age-dependent deterioration in learning and memory functions and movement coordination2. Here we use behavioral assays, including the negative geotaxis assay3 and the aversive phototaxic suppression assay (APS assay)4,5, to show that some of the behavior characteristics associated with human neurodegeneration can be recapitulated in flies. In the negative geotaxis assay, the natural tendency of flies to move against gravity when agitated is utilized to study genes or conditions that may hinder locomotor capacities. In the APS assay, the learning and memory functions are tested in positively-phototactic flies trained to associate light with aversive bitter taste and hence avoid this otherwise natural tendency to move toward light. Testing these trained flies 6 hours post-training is used to assess memory functions. Using these assays, the contribution of any genetic or environmental factors toward developing neurodegeneration can be easily studied in flies.

Neurodegeneration can be triggered by genetic or environmental factors. Although the precise cause is often unknown, many neurodegenerative diseases share common features such as protein aggregation and age dependence. Recent studies in Drosophila have uncovered protective effects of NAD synthase nicotinamide mononucleotide adenylyltransferase (NMNAT) against activity-induced neurodegeneration and injury-induced axonal degeneration1,2. Here we show that NMNAT overexpression can also protect against spinocerebellar ataxia 1 (SCA1)-induced neurodegeneration, suggesting a general neuroprotective function of NMNAT. It protects against neurodegeneration partly through a proteasome-mediated pathway in a manner similar to heat-shock protein 70 (Hsp70). NMNAT displays chaperone function both in biochemical assays and cultured cells, and it shares significant structural similarity with known chaperones. Furthermore, it is upregulated in the brain upon overexpression of poly-glutamine expanded protein and recruited with the chaperone Hsp70 into protein aggregates. Our results implicate NMNAT as a stress-response protein that acts as a chaperone for neuronal maintenance and protection. Our studies provide an entry point for understanding how normal neurons maintain activity, and offer clues for the common mechanisms underlying different neurodegenerative conditions.

Wallerian degeneration refers to a loss of the distal part of an axon after nerve injury. Wallerian degeneration slow (Wlds) mice overexpress a chimeric protein containing the NAD synthase NMNAT (nicotinamide mononucleotide adenylyltransferase 1) and exhibit a delay in axonal degeneration. Currently, conflicting evidence raises questions as to whether NMNAT is the protecting factor and whether its enzymatic activity is required for such a possible function. Importantly, the link between nmnat and axon degeneration is at present solely based on overexpression studies of enzymatically active protein. Here we use the visual system of Drosophila as a model system to address these issues. We have isolated the first nmnat mutations in a multicellular organism in a forward genetic screen for synapse malfunction in Drosophila. Loss of nmnat causes a rapid and severe neurodegeneration that can be attenuated by blocking neuronal activity. Furthermore, in vivo neuronal expression of mutated nmnat shows that enzymatically inactive NMNAT protein retains strong neuroprotective effects and rescues the degeneration phenotype caused by loss of nmnat. Our data indicate an NAD-independent requirement of NMNAT for maintaining neuronal integrity that can be exploited to protect neurons from neuronal activity-induced degeneration by overexpression of the protein., The first mutant analysis of NMNAT (nicotinamide mononucleotide adenylyltransferase 1) reveals an essential neuronal protective role that functions independently of NMNAT's enzymatic activity. NMNAT can also be exploited to protect neurons against activity-induced neurodegeneration.

Summary Specifying synaptic partners and regulating synaptic numbers are at least partly activity-dependent processes during visual map formation in all systems investigated to date [1–5]. In Drosophila, six photoreceptors that view the same point in visual space have to be sorted into synaptic modules called cartridges in order to form a visuotopically correct map [6, 7]. Synapse numbers per photoreceptor terminal and cartridge are both precisely regulated [8–10]. However, it is unknown whether an activity-dependent mechanism or a genetically encoded developmental program regulates synapse numbers. We performed a large-scale quantitative ultrastructural analysis of photoreceptor synapses in mutants affecting the generation of electrical potentials ( norpA , trp;trpl ), neurotransmitter release ( hdc , syt ), vesicle endocytosis ( synj ), the trafficking of specific guidance molecules during photoreceptor targeting ( sec15 ), a specific guidance receptor required for visual map formation ( Dlar ), and 57 other novel synaptic mutants affecting 43 genes. Remarkably, in all these mutants, individual photoreceptors form the correct number of synapses per presynaptic terminal independently of cartridge composition. Hence, our data show that each photoreceptor forms a precise and constant number of afferent synapses independently of neuronal activity and partner accuracy. Our data suggest cell-autonomous control of synapse numbers as part of a developmental program of activity-independent steps that lead to a "hard-wired" visual map in the fly brain.