A model of toxic neuropathy in Drosophila reveals a role for MORN4 in promoting axonal degeneration

Axonal degeneration is a molecular self-destruction cascade initiated following traumatic, toxic, and metabolic insults. Its mechanism underlies a number of disorders including hereditary and diabetic neuropathies and the neurotoxic side effects of chemotherapy drugs. Molecules that promote axonal degeneration could represent potential targets for therapy. To identify such molecules, we designed a screening platform based on intoxication of Drosophila larvae with paclitaxel (taxol), a chemotherapeutic agent that causes neuropathy in cancer patients. In Drosophila, taxol treatment causes swelling, fragmentation, and loss of axons in larval peripheral nerves. This axonal loss is not due to apoptosis of neurons. Taxol-induced axonal degeneration in Drosophila shares molecular execution mechanisms with vertebrates, including inhibition by both NMNAT (nicotinamide mononucleotide adenylyltransferase) expression and loss of wallenda/DLK (dual leucine zipper kinase). In a pilot RNAi-based screen we found that knockdown of retinophilin (rtp), which encodes a MORN (membrane occupation and recognition nexus) repeat-containing protein, protects axons from degeneration in the presence of taxol. Loss-of-function mutants of rtp replicate this axonal protection. Knockdown of rtp also delays axonal degeneration in severed olfactory axons. We demonstrate that the mouse ortholog of rtp, MORN4, promotes axonal degeneration in mouse sensory axons following axotomy, illustrating conservation of function. Hence, this new model can identify evolutionarily conserved genes that promote axonal degeneration, and so could identify candidate therapeutic targets for a wide-range of axonopathies

Expanded genetic screening in Caenorhabditis elegans identifies new regulators and an inhibitory role for NAD+ in axon regeneration.

The mechanisms underlying axon regeneration in mature neurons are relevant to the understanding of normal nervous system maintenance and for developing therapeutic strategies for injury. Here, we report novel pathways in axon regeneration, identified by extending our previous function-based screen using the C. elegans mechanosensory neuron axotomy model. We identify an unexpected role of the nicotinamide adenine dinucleotide (NAD+) synthesizing enzyme, NMAT-2/NMNAT, in axon regeneration. NMAT-2 inhibits axon regrowth via cell-autonomous and non-autonomous mechanisms. NMAT-2 enzymatic activity is required to repress regrowth. Further, we find differential requirements for proteins in membrane contact site, components and regulators of the extracellular matrix, membrane trafficking, microtubule and actin cytoskeleton, the conserved Kelch-domain protein IVNS-1, and the orphan transporter MFSD-6 in axon regrowth. Identification of these new pathways expands our understanding of the molecular basis of axonal injury response and regeneration

Nicotinamide adenine dinucleotide extends the lifespan of Caenorhabditis elegans mediated by sir-2.1 and daf-16

It is well understood that sir2 (sirtuin), an NAD-dependent deacetylase, is essential for the extension of lifespan under caloric restriction. However, the mechanism underlying activation of sir2 is unclear. Life extension through caloric restriction requires the sir2 ortholog sir-2.1 in nematodes but occurs independently of the forkhead-type transcription factor DAF-16. We aimed here to elucidate the correlation between life extension in nematodes and NAD-dependent activation of sirtuin by analyzing the relationship between NAD and DAF-16. Lifespan was extended when Caenorhabditis elegans were bred using medium containing NAD. An RNA interference experiment revealed that life extension by NAD was sir-2.1 dependent. However, life extension by NAD did not occur in daf-16-RNAi nematodes, suggesting that NAD-dependent longevity requires daf-16. This result suggested that different signaling pathways are involved in life extension resulting from caloric restriction and from NAD addition. Expression of sod-3, a target gene of daf-16, and increased oxidative-stress resistance and adiposity were observed in response to NAD addition, indicating that NAD activated daf-16 in each phenotype. These results suggest that NAD affected lifespan through the activation of SIR-2.1 and DAF-16 along a signaling pathway, namely insulin-like signalling pathway (at least parts of it), different from that associated with caloric restriction

Intracellular NAD+ levels are associated with LPS-induced TNF-α release in pro-inflammatory macrophages

Bacterial lipopolysaccharide induces changes in intracellular NAD+ levels in a pro-inflammatory, but not an anti-inflammatory, macrophage model that are correlated with the release of the pro-inflammatory cytokine tumour necrosis factor-α (TNF-α).</jats:p

Nmnat1 protects neuronal function without altering phospho-tau pathology in a mouse model of tauopathy

OBJECTIVE: The nicotinamide‐nucleotide adenylyltransferase protein Nmnat1 is a potent inhibitor of axonal degeneration in models of acute axonal injury. Hyperphosphorylation and aggregation of the microtubule‐associated protein Tau are associated with neurodegeneration in Alzheimer's Disease and other disorders. Previous studies have demonstrated that other Nmnat isoforms can act both as axonoprotective agents and have protein chaperone function, exerting protective effects in drosophila and mouse models of tauopathy. Nmnat1 targeted to the cytoplasm (cytNmnat1) is neuroprotective in a mouse model of neonatal hypoxia‐ischemia, but the effect of cytNmnat1 on tauopathy remains unknown. METHODS: We examined the impact of overexpression of cytNmnat1 on tau pathology, neurodegeneration, and brain functional connectivity in the P301S mouse model of chronic tauopathy. RESULTS: Overexpression of cytNmnat1 preserved cortical neuron functional connectivity in P301S mice in vivo. However, whereas Nmnat1 overexpression decreased the accumulation of detergent‐insoluble tau aggregates in the cerebral cortex, it exerted no effect on immunohistochemical evidence of pathologic tau phosphorylation and misfolding, hippocampal atrophy, or inflammatory markers in P301S mice. INTERPRETATION: Our results demonstrate that cytNmnat1 partially preserves neuronal function and decreases biochemically insoluble tau in a mouse model of chronic tauopathy without preventing tau phosphorylation, formation of soluble aggregates, or tau‐induced inflammation and atrophy. Nmnat1 might thus represent a therapeutic target for tauopathies

It takes two to tango: NAD+ and sirtuins in aging/longevity control

AbstractThe coupling of nicotinamide adenine dinucleotide (NAD+) breakdown and protein deacylation is a unique feature of the family of proteins called ‘sirtuins.’ This intimate connection between NAD+ and sirtuins has an ancient origin and provides a mechanistic foundation that translates the regulation of energy metabolism into aging and longevity control in diverse organisms. Although the field of sirtuin research went through intensive controversies, an increasing number of recent studies have put those controversies to rest and fully established the significance of sirtuins as an evolutionarily conserved aging/longevity regulator. The tight connection between NAD+ and sirtuins is regulated at several different levels, adding further complexity to their coordination in metabolic and aging/longevity control. Interestingly, it has been demonstrated that NAD+ availability decreases over age, reducing sirtuin activities and affecting the communication between the nucleus and mitochondria at a cellular level and also between the hypothalamus and adipose tissue at a systemic level. These dynamic cellular and systemic processes likely contribute to the development of age-associated functional decline and the pathogenesis of diseases of aging. To mitigate these age-associated problems, supplementation of key NAD+ intermediates is currently drawing significant attention. In this review article, we will summarize these important aspects of the intimate connection between NAD+ and sirtuins in aging/longevity control.</jats:p

NMNAT1 inhibits axon degeneration via blockade of SARM1-mediated NAD+ depletion

Overexpression of the NAD(+) biosynthetic enzyme NMNAT1 leads to preservation of injured axons. While increased NAD(+) or decreased NMN levels are thought to be critical to this process, the mechanism(s) of this axon protection remain obscure. Using steady-state and flux analysis of NAD(+) metabolites in healthy and injured mouse dorsal root ganglion axons, we find that rather than altering NAD(+) synthesis, NMNAT1 instead blocks the injury-induced, SARM1-dependent NAD(+) consumption that is central to axon degeneration. DOI: http://dx.doi.org/10.7554/eLife.19749.00

Targeting NMNAT1 to Axons and Synapses Transforms Its Neuroprotective Potency In Vivo

Axon and synapse degeneration are common components of many neurodegenerative diseases, and their rescue is essential for effective neuroprotection. The chimeric Wallerian degeneration slow protein (Wld(S)) protects axons dose dependently, but its mechanism is still elusive. We recently showed that Wld(S) acts at a non-nuclear location and is present in axons. This and other recent reports support a model in which Wld(S) protects by extranuclear redistribution of its nuclear NMNAT1 portion. However, it remains unclear whether cytoplasmic NMNAT1 acts locally in axons and synapses or at a non-nuclear site within cell bodies. The potency of axon protection by non-nuclear NMNAT1 relative to Wld(S) also needs to be established in vivo. Because the N-terminal portion of Wld(S) (N70) localized to axons, we hypothesized that it mediates the trafficking of the NMNAT1 portion. To test this, we substituted N70 with an axonal targeting peptide derived from amyloid precursor protein, and fused this to NMNAT1 with disrupted nuclear targeting. In transgenic mice, this transformed NMNAT1 from a molecule unable to inhibit Wallerian degeneration, even at high expression levels, into a protein more potent than Wld(S), able to preserve injured axons for several weeks at undetectable expression levels. Preventing NMNAT1 axonal delivery abolished its protective effect. Axonally targeted NMNAT1 localized to vesicular structures, colocalizing with extranuclear Wld(S), and was cotransported at least partially with mitochondria. We conclude that axonal targeting of NMNAT activity is both necessary and sufficient to delay Wallerian degeneration, and that promoting axonal and synaptic delivery greatly enhances the effectiveness

NMNAT1 Mutations Cause Leber Congenital Amaurosis

Leber congenital amaurosis (LCA) is an infantile-onset form of inherited retinal degeneration characterized by severe vision loss. Two-thirds of LCA cases are caused by mutations in 17 known disease genes (RetNet Retinal Information Network). Using exome sequencing, we identified a homozygous missense mutation (c.25G>A, p.Val9Met) in NMNAT1 as likely disease-causing in two siblings of a consanguineous Pakistani kindred affected by LCA. This mutation segregated with disease in their kindred, including in three other children with LCA. NMNAT1 resides in the previously identified LCA9 locus and encodes the nuclear isoform of nicotinamide mononucleotide adenylyltransferase, a rate-limiting enzyme in nicotinamide adenine dinucleotide $(NAD^+)$ biosynthesis. Functional studies showed the p.Val9Met mutation decreased NMNAT1 enzyme activity. Sequencing NMNAT1 in 284 unrelated LCA families identified 14 rare mutations in 13 additional affected individuals. These results are the first to link an NMNAT isoform to disease and indicate that NMNAT1 mutations cause LCA