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Axon degeneration is a prominent event in many neurodegenerative disorders. Axon injury stimulates an intrinsic self-destruction program that culminates in activation of the prodegeneration factor SARM1 and local dismantling of damaged axon segments. In healthy axons, SARM1 activity is restrained by constant delivery of the axon survival factor NMNAT2. Elevating NMNAT2 is neuroprotective, while loss of NMNAT2 evokes SARM1-dependent axon degeneration. As a gatekeeper of axon survival, NMNAT2 abundance is an important regulatory node in neuronal health, highlighting the need to understand the mechanisms behind NMNAT2 protein homeostasis. We demonstrate that pharmacological inhibition of the MAP3Ks dual leucine zipper kinase (DLK) and leucine zipper kinase (LZK) elevates NMNAT2 abundance and strongly protects axons from injury-induced degeneration. We discover that MAPK signaling selectively promotes degradation of palmitoylated NMNAT2, as well as palmitoylated SCG10. Conversely, nonpalmitoylated NMNAT2 is degraded by the Phr1/Skp1a/Fbxo45 ligase complex. Combined inactivation of both pathways leads to synergistic accumulation of NMNAT2 in axons and dramatically enhanced protection against pathological axon degeneration. Hence, the subcellular localization of distinct pools of NMNAT2 enables differential regulation of NMNAT2 abundance to control axon survival.

Axon injury in response to trauma or disease stimulates a self-destruction program that promotes the localized clearance of damaged axon segments. Sterile alpha and Toll/interleukin receptor (TIR) motif-containing protein 1 (SARM1) is an evolutionarily conserved executioner of this degeneration cascade, also known as Wallerian degeneration; however, the mechanism of SARM1-dependent neuronal destruction is still obscure. SARM1 possesses a TIR domain that is necessary for SARM1 activity. In other proteins, dimerized TIR domains serve as scaffolds for innate immune signaling. In contrast, dimerization of the SARM1 TIR domain promotes consumption of the essential metabolite NAD⁺ and induces neuronal destruction. This activity is unique to the SARM1 TIR domain, yet the structural elements that enable this activity are unknown. In this study, we identify fundamental properties of the SARM1 TIR domain that promote NAD⁺ loss and axon degeneration. Dimerization of the TIR domain from the Caenorhabditis elegans SARM1 ortholog TIR-1 leads to NAD⁺ loss and neuronal death, indicating these activities are an evolutionarily conserved feature of SARM1 function. Detailed analysis of sequence homology identifies canonical TIR motifs as well as a SARM1-specific (SS) loop that are required for NAD⁺ loss and axon degeneration. Furthermore, we identify a residue in the SARM1 BB loop that is dispensable for TIR activity yet required for injury-induced activation of full-length SARM1, suggesting that SARM1 function requires multidomain interactions. Indeed, we identify a physical interaction between the autoinhibitory N terminus and the TIR domain of SARM1, revealing a previously unrecognized direct connection between these domains that we propose mediates autoinhibition and activation upon injury.

Injury-induced (Wallerian) axonal degeneration is regulated via the opposing actions of pro-degenerative factors such as SARM1 and a MAPK signal and pro-survival factors, the most important of which is the NAD+ biosynthetic enzyme NMNAT2 that inhibits activation of the SARM1 pathway. Here we investigate the mechanism by which MAPK signaling facilitates axonal degeneration. We show that MAPK signaling promotes the turnover of the axonal survival factor NMNAT2 in cultured mammalian neurons as well as the Drosophila ortholog dNMNAT in motoneurons. The increased levels of NMNAT2 are required for the axonal protection caused by loss of MAPK signaling. Regulation of NMNAT2 by MAPK signaling does not require SARM1, and so cannot be downstream of SARM1. Hence, pro-degenerative MAPK signaling functions upstream of SARM1 by limiting the levels of the essential axonal survival factor NMNAT2 to promote injury-dependent SARM1 activation. These findings are consistent with a linear molecular pathway for the axonal degeneration program.

Mechanisms for cooperation between the cytosolic Hsp70 system and the ubiquitin proteasome system during protein triage are not clear. Herein, we identify new mechanisms for selection of misfolded cytosolic proteins for degradation via defining functional interactions between specific cytosolic Hsp70/Hsp40 pairs and quality control ubiquitin ligases. These studies revolved around the use of S. cerevisiae to elucidate the degradation pathway of a terminally misfolded reporter protein, short-lived GFP (slGFP). The Type I Hsp40 Ydj1 acts with Hsp70 to suppress slGFP aggregation. In contrast, the Type II Hsp40 Sis1 is required for proteasomal degradation of slGFP. Sis1 and Hsp70 operate sequentially with the quality control E3 ubiquitin ligase Ubr1 to target slGFP for degradation. Compromise of Sis1 or Ubr1 function leads slGFP to accumulate in a Triton X-100-soluble state with slGFP degradation intermediates being concentrated into perinuclear and peripheral puncta. Interestingly, when Sis1 activity is low the slGFP that is concentrated into puncta can be liberated from puncta and subsequently degraded. Conversely, in the absence of Ubr1, slGFP and the puncta that contain slGFP are relatively stable. Ubr1 mediates proteasomal degradation of slGFP that is released from cytosolic protein handling centers. Pathways for proteasomal degradation of misfolded cytosolic proteins involve functional interplay between Type II Hsp40/Hsp70 chaperone pairs, PQC E3 ligases, and storage depots for misfolded proteins.

Glaucoma is one of the leading causes of irreversible blindness worldwide and vision loss in the disease results from the deterioration of retinal ganglion cells (RGC) and their axons. Metabolic dysfunction of RGC plays a significant role in the onset and progression of the disease in both human patients and rodent models, highlighting the need to better define the mechanisms regulating cellular energy metabolism in glaucoma. This study sought to determine if Sarm1 , a gene involved in axonal degeneration and NAD+ metabolism, contributes to glaucomatous RGC loss in a mouse model with chronic elevated intraocular pressure (IOP). Our data demonstrate that after 16 weeks of elevated IOP, Sarm1 knockout (KO) mice retain significantly more RGC than control animals. Sarm1 KO mice also performed significantly better when compared to control mice during optomotor testing, indicating that visual function is preserved in this group. Our findings also indicate that Sarm1 KO mice display mild ocular developmental abnormalities, including reduced optic nerve axon diameter and lower visual acuity than controls. Finally, we present data to indicate that SARM1 expression in the optic nerve is most prominently associated with oligodendrocytes. Taken together, these data suggest that attenuating Sarm1 activity through gene therapy, pharmacologic inhibition, or NAD+ supplementation, may be a novel therapeutic approach for patients with glaucoma.

Transcription errors occur in all living cells; however, it is unknown how these errors affect cellular health. To answer this question, we monitor yeast cells that are genetically engineered to display error-prone transcription. We discover that these cells suffer from a profound loss in proteostasis, which sensitizes them to the expression of genes that are associated with protein-folding diseases in humans; thus, transcription errors represent a new molecular mechanism by which cells can acquire disease phenotypes. We further find that the error rate of transcription increases as cells age, suggesting that transcription errors affect proteostasis particularly in aging cells. Accordingly, transcription errors accelerate the aggregation of a peptide that is implicated in Alzheimer's disease, and shorten the lifespan of cells. These experiments reveal a previously unappreciated role for transcriptional fidelity in cellular health and aging. Transcription, like DNA replication, is an error-prone process. Vermulst et al. show that transcription errors increase with age in yeast, and find that prematurely increasing the error rate overwhelms the proteotoxic stress response, allowing aggregation-prone proteins to escape protein quality control.

Axon degeneration is a prominent component of many neurological disorders. Identifying cellular pathways that contribute to axon vulnerability may identify new therapeutic strategies for maintenance of neural circuits. Dual leucine zipper kinase (DLK) is an axonal stress response MAP3K that is chronically activated in several neurodegenerative diseases. Activated DLK transmits an axon injury signal to the neuronal cell body to provoke transcriptional adaptations. However, the consequence of enhanced DLK signaling to axon vulnerability is unknown. We find that stimulating DLK activity predisposes axons to SARM1-dependent degeneration. Activating DLK reduces levels of the axon survival factors NMNAT2 and SCG10, accelerating their loss from severed axons. Moreover, mitochondrial dysfunction independently decreases the levels of NMNAT2 and SCG10 in axons, and in conjunction with DLK activation, leads to a dramatic loss of axonal NMNAT2 and SCG10 and evokes spontaneous axon degeneration. Hence, enhanced DLK activity reduces axon survival factor abundance and renders axons more susceptible to trauma and metabolic insult.

Protein aggregation is a hallmark of a large and diverse number of conformational diseases. Molecular chaperones of the Hsp40 family (Escherichia coli DnaJ homologs) recognize misfolded disease proteins and suppress the accumulation of toxic protein species. Type I Hsp40s are very potent at suppressing protein aggregation and facilitating the refolding of damaged proteins. Yet, the molecular mechanism for the recognition of nonnative polypeptides by Type I Hsp40s such as yeast Ydj1 is not clear. Here we computationally identify a unique motif that is selectively recognized by Ydj1p. The motif is characterized by the consensus sequence GX[LMQ]{P}X{P}{CIMPVW}, where [XY] denotes either X or Y and {XY} denotes neither X nor Y. We further verify the validity of the motif by site-directed mutagenesis and show that substrate binding by Ydj1 requires recognition of this motif. A yeast proteome screen revealed that many proteins contain more than one stretch of residues that contain the motif and are separated by varying numbers of amino acids. In light of our results, we propose a 2-site peptide-binding model and a plausible mechanism of peptide presentation by Ydj1p to the chaperones of the Hsp70 family. Based on our results, and given that Ydj1p and its human ortholog Hdj2 are functionally interchangeable, we hypothesize that our results can be extended to understanding human diseases.

Charcot-Marie-Tooth disease (CMT) is an inherited peripheral neuropathy characterized by sensory dysfunction and muscle weakness, manifesting in the most distal limbs first and progressing more proximal. Over a hundred genes are currently linked to CMT with enrichment for activities in myelination, axon transport, and protein synthesis. Mutations in tRNA synthetases cause dominantly inherited forms of CMT and animal models with CMT-linked mutations in these enzymes display defects in neuronal protein synthesis. Rescuing protein synthesis in CMT mutant neurons could offer exciting therapeutic options beyond symptom management. To address this need, we expressed CMT-linked variants in tyrosyl tRNA synthetase (YARS-CMT) in primary sensory neurons and evaluated impacts on protein synthesis and cell viability. YARS-CMT expression reduced protein synthesis in these neurons prior to the onset of caspase-dependent axon degeneration and cell death. To determine how YARS-CMT expression affects axon outgrowth, we dissociated and replated these neurons to stimulate axon regeneration. To our surprise, axonal regrowth occurred normally in replated YARS-CMT neurons. Moreover, replating YARS-CMT neurons rescued protein synthesis. Inhibiting mTOR suppressed rescue of protein synthesis after replating, consistent with its significant role in protein synthesis during axon regeneration. These discoveries identify new avenues for augmenting protein synthesis in diseased neurons and restoring protein synthesis in CMT or other neurological disorders.

Neurotrophic factors are critical for establishing functional connectivity in the nervous system and sustaining neuronal survival through adulthood. As the first neurotrophic factor purified, nerve growth factor (NGF) is extensively studied for its prolific role in axon outgrowth, pruning, and survival. Applying NGF to diseased neuronal tissue is an exciting therapeutic option and understanding how NGF regulates local axon susceptibility to pathological degeneration is critical for exploiting its full potential. Our study identifies surprising connections between NGF signaling and proteostasis of axon maintenance factors. NGF deprivation increases Nmnat2 and Stmn2 protein levels in axon segments with a corresponding delay in Wallerian degeneration. Conversely, acute NGF stimulation reduces local abundance of these axon maintenance factors and accelerates Wallerian degeneration. Pharmacological studies implicate phospholipase C as the key effector in TrkA activation, which drives degradation of palmitoylated Stmn2. While seemingly opposed to neuroprotective activities well-documented for NGF, downregulating Nmnat2 and Stmn2 favors axonal outgrowth over transient hyper-susceptibility to Sarm1-dependent degeneration. This new facet of NGF biology has important implications for axonal remodeling during development and sustained integrity through adulthood.