Nmnat restores neuronal integrity by neutralizing mutant Huntingtin aggregate-induced progressive toxicity.

Accumulative aggregation of mutant Huntingtin (Htt) is a primary neuropathological hallmark of Huntington's disease (HD). Currently, mechanistic understanding of the cytotoxicity of mutant Htt aggregates remains limited, and neuroprotective strategies combating mutant Htt-induced neurodegeneration are lacking. Here, we show that in Drosophila models of HD, neuronal compartment-specific accumulation of mutant Htt aggregates causes neurodegenerative phenotypes. In addition to the increase in the number and size, we discovered an age-dependent acquisition of thioflavin S+, amyloid-like adhesive properties of mutant Htt aggregates and a concomitant progressive clustering of aggregates with mitochondria and synaptic proteins, indicating that the amyloid-like adhesive property underlies the neurotoxicity of mutant Htt aggregation. Importantly, nicotinamide mononucleotide adenylyltransferase (NMNAT), an evolutionarily conserved nicotinamide adenine dinucleotide (NAD+) synthase and neuroprotective factor, significantly mitigates mutant Htt-induced neurodegeneration by reducing mutant Htt aggregation through promoting autophagic clearance. Additionally, Nmnat overexpression reduces progressive accumulation of amyloid-like Htt aggregates, neutralizes adhesiveness, and inhibits the clustering of mutant Htt with mitochondria and synaptic proteins, thereby restoring neuronal function. Conversely, partial loss of endogenous Nmnat exacerbates mutant Htt-induced neurodegeneration through enhancing mutant Htt aggregation and adhesive property. Finally, conditional expression of Nmnat after the onset of degenerative phenotypes significantly delays the progression of neurodegeneration, revealing the therapeutic potential of Nmnat-mediated neuroprotection at advanced stages of HD. Our study uncovers essential mechanistic insights to the neurotoxicity of mutant Htt aggregation and describes the molecular basis of Nmnat-mediated neuroprotection in HD.

Dysfunction of GRAP, encoding the GRB2-related adaptor protein, is linked to sensorineural hearing loss.

We have identified a GRAP variant (c.311A>T; p.Gln104Leu) cosegregating 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.

Mislocalization of neuronal mitochondria reveals regulation of Wallerian degeneration and NMNAT/WLD(S)-mediated axon protection independent of axonal mitochondria.

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.

Nicotinamide mononucleotide adenylyltransferase maintains active zone structure by stabilizing Bruchpilot.

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.

NMNAT suppresses tau-induced neurodegeneration by promoting clearance of hyperphosphorylated tau oligomers in a Drosophila model of tauopathy.

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.

CREB-activity and nmnat2 transcription are down-regulated prior to neurodegeneration, while NMNAT2 over-expression is neuroprotective, in a mouse model of human tauopathy.

Tauopathies, characterized by neurofibrillary tangles (NFTs) of phosphorylated tau proteins, are a group of neurodegenerative diseases, including frontotemporal dementia and both sporadic and familial Alzheimer's disease. Forebrain-specific over-expression of human tau(P301L), a mutation associated with frontotemporal dementia with parkinsonism linked to chromosome 17, in rTg4510 mice results in the formation of NFTs, learning and memory impairment and massive neuronal death. Here, we show that the mRNA and protein levels of NMNAT2 (nicotinamide mononucleotide adenylyltransferase 2), a recently identified survival factor for maintaining neuronal health in peripheral nerves, are reduced in rTg4510 mice prior to the onset of neurodegeneration or cognitive deficits. Two functional cAMP-response elements (CREs) were identified in the nmnat2 promoter region. Both the total amount of phospho-CRE binding protein (CREB) and the pCREB bound to nmnat2 CRE sites in the cortex and the hippocampus of rTg4510 mice are significantly reduced, suggesting that NMNAT2 is a direct target of CREB under physiological conditions and that tau(P301L) overexpression down-regulates CREB-mediated transcription. We found that over-expressing NMNAT2 or its homolog NMNAT1, but not NMNAT3, in rTg4510 hippocampi from 6 weeks of age using recombinant adeno-associated viral vectors significantly reduced neurodegeneration caused by tau(P301L) over-expression at 5 months of age. In summary, our studies strongly support a protective role of NMNAT2 in the mammalian central nervous system. Decreased endogenous NMNAT2 function caused by reduced CREB signaling during pathological insults may be one of underlying mechanisms for neuronal death in tauopathies.

NAD synthase NMNAT acts as a chaperone to protect against neurodegeneration.

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 degeneration. 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.

The role of autophagy in Nmnat-mediated protection against hypoxia-induced dendrite degeneration.

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 (<1.0% O(2)), class IV neurons of nmnat heterozygous mutants exhibit significant dendrite loss and extensive fragmentation of the dendritic arbor under the same hypoxic conditions. Although basal levels of autophagy are required for neuronal survival, we demonstrate that autophagy is dispensable for maintaining the dendritic integrity of class IV neurons. However, we find that genetically blocking autophagy can suppress hypoxia-induced dendrite degeneration of nmnat heterozygous mutants in a cell-autonomous manner, suggestive of a self-destructive role for autophagy in this context. We further show that inducing autophagy by overexpression of the autophagy-specific kinase Atg1 is sufficient to cause dendrite degeneration of class IV neurons under hypoxia and that overexpression of Nmnat fails to protect class IV dendrites from the effects of Atg1 overexpression. Our studies reveal an essential neuroprotective role for endogenous Nmnat in hypoxia and demonstrate that Nmnat functions upstream of autophagy to mitigate the damage incurred by dendrites in neurons under hypoxic stress.

High-resolution, high-throughput analysis of Drosophila geotactic behavior.

Drosophila innate response to gravity, geotaxis, has been previously used to assess the impact of aging and disease on motor performance. Despite its rich history, fly geotaxis continues to be largely measured manually and assessed through simplistic metrics. The manual nature of this assay introduces substantial experimental variability while simplistic metrics provide limited analytic insights into the behavior. To address these shortcomings, we have constructed a fully automated, programable apparatus, and developed a multi-object tracking software capable of following sub-second movements of individual flies, thus allowing reproducible, detailed, and quantitative analysis of geotactic behavior. The apparatus triggers and monitors geotaxis of 10 fly cohorts simultaneously, with each cohort consisting of up to 7 flies. The tracking program isolates cohorts and records individual fly coordinate outputs allowing for simultaneous multi-group, multi-fly tracks per experiment, greatly improving throughput and resolution. The algorithm tracks individual flies during the entire run with ~97% accuracy, yielding detailed climbing curve, speed, and movement direction with 1/30 second resolution. Our tracking also allows the construction of multi-variable metrics and the detection of transitory movement phenotypes, such as slips and falls, which have thus far been neglected in geotaxis studies due to limited spatio-temporal resolution. Through a combination of automation and robust tracking, the platform is therefore poised to advance Drosophila geotaxis assay into a comprehensive assessment of locomotor behavior.

Copper enhances aggregational toxicity of mutant huntingtin in a Drosophila model of Huntington's Disease.

Huntington's disease (HD) is a progressive neurodegenerative disorder with clinical presentations of moderate to severe cognitive, motor, and psychiatric disturbances. HD is caused by the trinucleotide repeat expansion of CAG of the huntingtin (HTT) gene. The mutant HTT protein containing pathological polyglutamine (polyQ) extension is prone to misfolding and aggregation in the brain. It has previously been observed that copper and iron concentrations are increased in the striata of post-mortem human HD brains. Although it has been shown that the accumulation of mutant HTT protein can interact with copper, the underlying HD progressive phenotypes due to copper overload remains elusive. Here, in a Drosophila model of HD, we showed that copper induces dose-dependent aggregational toxicity and enhancement of Htt-induced neurodegeneration. Specifically, we found that copper increases mutant Htt aggregation, enhances the accumulation of Thioflavin S positive ß-amyloid structures within Htt aggregates, and consequently alters autophagy in the brain. Administration of copper chelator D-penicillamine (DPA) through feeding significantly decreases ß-amyloid aggregates in the HD pathological model. These findings reveal a direct role of copper in potentiating mutant Htt protein-induced aggregational toxicity, and further indicate the potential impact of environmental copper exposure in the disease onset and progression of HD.

Reduction of spermine synthase enhances autophagy to suppress Tau accumulation.

Precise polyamine metabolism regulation is vital for cells and organisms. Mutations in spermine synthase (SMS) cause Snyder-Robinson intellectual disability syndrome (SRS), characterized by significant spermidine accumulation and autophagy blockage in the nervous system. Emerging evidence connects polyamine metabolism with other autophagy-related diseases, such as Tauopathy, however, the functional intersection between polyamine metabolism and autophagy in the context of these diseases remains unclear. Here, we altered SMS expression level to investigate the regulation of autophagy by modulated polyamine metabolism in Tauopathy in Drosophila and human cellular models. Interestingly, while complete loss of Drosophila spermine synthase (dSms) impairs lysosomal function and blocks autophagic flux recapitulating SRS disease phenotype, partial loss of dSms enhanced autophagic flux, reduced Tau protein accumulation, and led to extended lifespan and improved climbing performance in Tauopathy flies. Measurement of polyamine levels detected a mild elevation of spermidine in flies with partial loss of dSms. Similarly, in human neuronal or glial cells, partial loss of SMS by siRNA-mediated knockdown upregulated autophagic flux and reduced Tau protein accumulation. Importantly, proteomics analysis of postmortem brain tissue from Alzheimer's disease (AD) patients showed a significant albeit modest elevation of SMS level. Taken together, our study uncovers a functional correlation between polyamine metabolism and autophagy in AD: SMS reduction upregulates autophagy, suppresses Tau accumulation, and ameliorates neurodegeneration and cell death. These findings provide a new potential therapeutic target for AD.

Protein aggregates are recruited to aggresome by histone deacetylase 6 via unanchored ubiquitin C termini.

The aggresome pathway is activated when proteasomal clearance of misfolded proteins is hindered. Misfolded polyubiquitinated protein aggregates are recruited and transported to the aggresome via the microtubule network by a protein complex consisting of histone deacetylase 6 (HDAC6) and the dynein motor complex. The current model suggests that HDAC6 recognizes protein aggregates by binding directly to polyubiquitinated proteins. Here, we show that there are substantial amounts of unanchored ubiquitin in protein aggregates with solvent-accessible C termini. The ubiquitin-binding domain (ZnF-UBP) of HDAC6 binds exclusively to the unanchored C-terminal diglycine motif of ubiquitin instead of conjugated polyubiquitin. The unanchored ubiquitin C termini in the aggregates are generated in situ by aggregate-associated deubiquitinase ataxin-3. These results provide structural and mechanistic bases for the role of HDAC6 in aggresome formation and further suggest a novel ubiquitin-mediated signaling pathway, where the exposure of ubiquitin C termini within protein aggregates enables HDAC6 recognition and transport to the aggresome.

The Architecture of the Presynaptic Release Site.

The architecture of the presynaptic release site is exquisitely designed to facilitate and regulate synaptic vesicle exocytosis. With the identification of some of the building blocks of the active zone and the advent of super resolution imaging techniques, we are beginning to understand the morphological and functional properties of synapses in great detail. Presynaptic release sites consist of the plasma membrane, the cytomatrix, and dense projections. These three components are morphologically distinct but intimately connected with each other and with postsynaptic specializations, ensuring the fidelity of synaptic vesicle tethering, docking, and fusion, as well as signal detection. Although the morphology and molecular compositions of active zones may vary among species, tissues, and cells, global architectural design of the release sites is highly conserved.

Presynaptic Cytomatrix Proteins.

The Cytomatrix Assembled at the active Zone (CAZ) of a presynaptic terminal displays electron-dense appearance and defines the center of the synaptic vesicle release. The protein constituents of CAZ are multiple-domain scaffolds that interact extensively with each other and also with an ensemble of synaptic vesicle proteins to ensure docking, fusion, and recycling. Reflecting the central roles of the active zone in synaptic transmission, CAZ proteins are highly conserved throughout evolution. As the nervous system increases complexity and diversity in types of neurons and synapses, CAZ proteins expand in the number of gene and protein isoforms and interacting partners. This chapter summarizes the discovery of the core CAZ proteins and current knowledge of their functions.

MicroRNA-Mediated Obstruction of Stem-loop Alternative Splicing (MIMOSAS): a global mechanism for the regulation of alternative splicing.

While RNA secondary structures are critical to regulate alternative splicing of long-range pre-mRNA, the factors that modulate RNA structure and interfere with the recognition of the splice sites are largely unknown. Previously, we identified a small, non-coding microRNA that sufficiently affects stable stem structure formation of Nmnat pre-mRNA to regulate the outcomes of alternative splicing. However, the fundamental question remains whether such microRNA-mediated interference with RNA secondary structures is a global molecular mechanism for regulating mRNA splicing. We designed and refined a bioinformatic pipeline to predict candidate microRNAs that potentially interfere with pre-mRNA stem-loop structures, and experimentally verified splicing predictions of three different long-range pre-mRNAs in the Drosophila model system. Specifically, we observed that microRNAs can either disrupt or stabilize stem-loop structures to influence splicing outcomes. Our study suggests that MicroRNA-Mediated Obstruction of Stem-loop Alternative Splicing (MIMOSAS) is a novel regulatory mechanism for the transcriptome-wide regulation of alternative splicing, increases the repertoire of microRNA function and further indicates cellular complexity of post-transcriptional regulation. One-Sentence Summary: MicroRNA-Mediated Obstruction of Stem-loop Alternative Splicing (MIMOSAS) is a novel regulatory mechanism for the transcriptome-wide regulation of alternative splicing.

Reduction of Spermine Synthase Suppresses Tau Accumulation Through Autophagy Modulation in Tauopathy.

Tauopathy, including Alzheimer Disease (AD), is characterized by Tau protein accumulation and autophagy dysregulation. Emerging evidence connects polyamine metabolism with the autophagy pathway, however the role of polyamines in Tauopathy remains unclear. In the present study we investigated the role of spermine synthase (SMS) in autophagy regulation and tau protein processing in Drosophila and human cellular models of Tauopathy. Our previous study showed that Drosophila spermine synthase (dSms) deficiency impairs lysosomal function and blocks autophagy flux. Interestingly, partial loss-of-function of SMS in heterozygous dSms flies extends lifespan and improves the climbing performance of flies with human Tau (hTau) overexpression. Mechanistic analysis showed that heterozygous loss-of-function mutation of dSms reduces hTau protein accumulation through enhancing autophagic flux. Measurement of polyamine levels detected a mild elevation of spermidine in flies with heterozygous loss of dSms. SMS knock-down in human neuronal or glial cells also upregulates autophagic flux and reduces Tau protein accumulation. Proteomics analysis of postmortem brain tissue from AD patients showed a significant albeit modest elevation of SMS protein level in AD-relevant brain regions compared to that of control brains consistently across several datasets. Taken together, our study uncovers a correlation between SMS protein level and AD pathogenesis and reveals that SMS reduction upregulates autophagy, promotes Tau clearance, and reduces Tau protein accumulation. These findings provide a new potential therapeutic target of Tauopathy.

Difluoromethylornithine rebalances aberrant polyamine ratios in Snyder-Robinson syndrome.

Snyder-Robinson syndrome (SRS) results from mutations in spermine synthase (SMS), which converts the polyamine spermidine into spermine. Affecting primarily males, common manifestations of SRS include intellectual disability, osteoporosis, hypotonia, and seizures. Symptom management is the only treatment. Reduced SMS activity causes spermidine accumulation while spermine levels are reduced. The resulting exaggerated spermidine:spermine ratio is a biochemical hallmark of SRS that tends to correlate with symptom severity. Our studies aim to pharmacologically manipulate polyamine metabolism to correct this imbalance as a therapeutic strategy for SRS. Here we report the repurposing of 2-difluoromethylornithine (DFMO), an FDA-approved inhibitor of polyamine biosynthesis, in rebalancing spermidine:spermine ratios in SRS patient cells. Mechanistic in vitro studies demonstrate that, while reducing spermidine biosynthesis, DFMO also stimulates the conversion of spermidine into spermine in hypomorphic SMS cells and induces uptake of exogenous spermine, altogether reducing the aberrant ratios. In a Drosophila SRS model characterized by reduced lifespan, DFMO improves longevity. As nearly all SRS patient mutations are hypomorphic, these studies form a strong foundation for translational studies with significant therapeutic potential.

Sorbitol reduction via govorestat ameliorates synaptic dysfunction and neurodegeneration in sorbitol dehydrogenase deficiency.

Sorbitol dehydrogenase (SORD) deficiency has been identified as the most frequent autosomal recessive form of hereditary neuropathy. Loss of SORD causes high sorbitol levels in tissues due to the inability to convert sorbitol to fructose in the 2-step polyol pathway, leading to degenerative neuropathy. The underlying mechanisms of sorbitol-induced degeneration have not been fully elucidated, and no current FDA-approved therapeutic options are available to reduce sorbitol levels in the nervous system. Here, in a Drosophila model of SORD deficiency, we showed synaptic degeneration in the brain, neurotransmission defect, locomotor impairment, and structural abnormalities in the neuromuscular junctions. In addition, we found reduced ATP production in the brain and ROS accumulation in the CNS and muscle, indicating mitochondrial dysfunction. Applied Therapeutics has developed a CNS-penetrant next-generation aldose reductase inhibitor (ARI), AT-007 (govorestat), which inhibits the conversion of glucose to sorbitol. AT-007 significantly reduced sorbitol levels in patient-derived fibroblasts, induced pluripotent stem cell-derived (iPSC-derived) motor neurons, and Drosophila brains. AT-007 feeding in Sord-deficient Drosophila mitigated synaptic degeneration and significantly improved synaptic transduction, locomotor activity, and mitochondrial function. Moreover, AT-007 treatment significantly reduced ROS accumulation in Drosophila CNS, muscle, and patient-derived fibroblasts. These findings uncover the molecular and cellular pathophysiology of SORD neuropathy and provide a potential treatment strategy for patients with SORD deficiency.

Nicotinamide mononucleotide adenylyltransferase is a stress response protein regulated by the heat shock factor/hypoxia-inducible factor 1alpha pathway.

Stress responses are cellular processes essential for maintenance of cellular integrity and defense against environmental and intracellular insults. Neurodegenerative conditions are linked with inadequate stress responses. Several stress-responsive genes encoding neuroprotective proteins have been identified, and among them, the heat shock proteins comprise an important group of molecular chaperones that have neuroprotective functions. However, evidence for other critical stress-responsive genes is lacking. Recent studies on the NAD synthesis enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT) have uncovered a novel neuronal maintenance and protective function against activity-, injury-, or misfolded protein-induced degeneration in Drosophila and in mammalian neurons. Here, we show that NMNAT is also a novel stress response protein required for thermotolerance and mitigation of oxidative stress-induced shortened lifespan. NMNAT is transcriptionally regulated during various stress conditions including heat shock and hypoxia through heat shock factor (HSF) and hypoxia-inducible factor 1α in vivo. HSF binds to nmnat promoter and induces NMNAT expression under heat shock. In contrast, under hypoxia, HIF1α up-regulates NMNAT indirectly through the induction of HSF. Our studies provide an in vivo mechanism for transcriptional regulation of NMNAT under stress and establish an essential role for this neuroprotective factor in cellular stress response.

Nmnat exerts neuroprotective effects in dendrites and axons.

Dendrites can be maintained for extended periods of time after they initially establish coverage of their receptive field. The long-term maintenance of dendrites underlies synaptic connectivity, but how neurons establish and then maintain their dendritic arborization patterns throughout development is not well understood. Here, we show that the NAD synthase Nicotinamide mononucleotide adenylyltransferase (Nmnat) is cell-autonomously required for maintaining type-specific dendritic coverage of Drosophila dendritic arborization (da) sensory neurons. In nmnat heterozygous mutants, dendritic arborization patterns of class IV da neurons are properly established before increased retraction and decreased growth of terminal branches lead to progressive defects in dendritic coverage during later stages of development. Although sensory axons are largely intact in nmnat heterozygotes, complete loss of nmnat function causes severe axonal degeneration, demonstrating differential requirements for nmnat dosage in the maintenance of dendritic arborization patterns and axonal integrity. Overexpression of Nmnat suppresses dendrite maintenance defects associated with loss of the tumor suppressor kinase Warts (Wts), providing evidence that Nmnat, in addition to its neuroprotective role in axons, can function as a protective factor against progressive dendritic loss. Moreover, motor neurons deficient for nmnat show progressive defects in both dendrites and axons. Our studies reveal an essential role for endogenous Nmnat function in the maintenance of both axonal and dendritic integrity and present evidence of a broad neuroprotective role for Nmnat in the central nervous system.