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Protective effects of NAMPT or MAPK inhibitors and NaR on Wallerian degeneration of mammalian axons.

ABSTRACT: Wallerian degeneration (WD) is a conserved axonal self-destruction program implicated in several neurological diseases. WD is driven by the degradation of the NAD⁺ synthesizing enzyme NMNAT2, the buildup of its substrate NMN, and the activation of the NAD⁺ degrading SARM1, eventually leading to axonal fragmentation. The regulation and amenability of these events to therapeutic interventions remain unclear. Here we explored pharmacological strategies that modulate NMN and NAD⁺ metabolism, namely the inhibition of the NMN-synthesizing enzyme NAMPT, activation of the nicotinic acid riboside (NaR) salvage pathway and inhibition of the NMNAT2-degrading DLK MAPK pathway in an axotomy model in vitro. Results show that NAMPT and DLK inhibition cause a significant but time-dependent delay of WD. These time-dependent effects are related to NMNAT2 degradation and changes in NMN and NAD⁺ levels. Supplementation of NAMPT inhibition with NaR has an enhanced effect that does not depend on timing of intervention and leads to robust protection up to 4 days. Additional DLK inhibition extends this even further to 6 days. Metabolite analyses reveal complex effects indicating that NAMPT and MAPK inhibition act by reducing NMN levels, ameliorating NAD⁺ loss and suppressing SARM1 activity. Finally, the axonal NAD⁺/NMN ratio is highly predictive of cADPR levels, extending previous cell-free evidence on the allosteric regulation of SARM1. Our findings establish a window of axon protection extending several hours following injury. Moreover, we show prolonged protection by mixed treatments combining MAPK and NAMPT inhibition that proceed via complex effects on NAD⁺ metabolism and inhibition of SARM1.

SUBMITTER: Alexandris AS

PROVIDER: S-EPMC10621467 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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Protective effects of NAMPT or MAPK inhibitors and NaR on Wallerian degeneration of mammalian axons.

Alexandris Athanasios S AS Ryu Jiwon J Rajbhandari Labchan L Harlan Robert R McKenney James J Wang Yiqing Y Aja Susan S Graham David D Venkatesan Arun A Koliatsos Vassilis E VE

Neurobiology of disease 20220630

Wallerian degeneration (WD) is a conserved axonal self-destruction program implicated in several neurological diseases. WD is driven by the degradation of the NAD<sup>+</sup> synthesizing enzyme NMNAT2, the buildup of its substrate NMN, and the activation of the NAD<sup>+</sup> degrading SARM1, eventually leading to axonal fragmentation. The regulation and amenability of these events to therapeutic interventions remain unclear. Here we explored pharmacological strategies that modulate NMN and NA ...[more]

PMID: 35779777

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Project description:BackgroundDespite the widespread occurrence of axon and synaptic loss in the injured and diseased nervous system, the cellular and molecular mechanisms of these key degenerative processes remain incompletely understood. Wallerian degeneration (WD) is a tightly regulated form of axon loss after injury, which has been intensively studied in large myelinated fibre tracts of the spinal cord, optic nerve and peripheral nervous system (PNS). Fewer studies, however, have focused on WD in the complex neuronal circuits of the mammalian brain, and these were mainly based on conventional endpoint histological methods. Post-mortem analysis, however, cannot capture the exact sequence of events nor can it evaluate the influence of elaborated arborisation and synaptic architecture on the degeneration process, due to the non-synchronous and variable nature of WD across individual axons.ResultsTo gain a comprehensive picture of the spatiotemporal dynamics and synaptic mechanisms of WD in the nervous system, we identify the factors that regulate WD within the mouse cerebral cortex. We combined single-axon-resolution multiphoton imaging with laser microsurgery through a cranial window and a fluorescent membrane reporter. Longitudinal imaging of > 150 individually injured excitatory cortical axons revealed a threshold length below which injured axons consistently underwent a rapid-onset form of WD (roWD). roWD started on average 20 times earlier and was executed 3 times slower than WD described in other regions of the nervous system. Cortical axon WD and roWD were dependent on synaptic density, but independent of axon complexity. Finally, pharmacological and genetic manipulations showed that a nicotinamide adenine dinucleotide (NAD+)-dependent pathway could delay cortical roWD independent of transcription in the damaged neurons, demonstrating further conservation of the molecular mechanisms controlling WD in different areas of the mammalian nervous system.ConclusionsOur data illustrate how in vivo time-lapse imaging can provide new insights into the spatiotemporal dynamics and synaptic mechanisms of axon loss and assess therapeutic interventions in the injured mammalian brain.

Project description:BackgroundThe progressive nature of Wallerian degeneration has long been controversial. Conflicting reports that distal stumps of injured axons degenerate anterogradely, retrogradely, or simultaneously are based on statistical observations at discontinuous locations within the nerve, without observing any single axon at two distant points. As axon degeneration is asynchronous, there are clear advantages to longitudinal studies of individual degenerating axons. We recently validated the study of Wallerian degeneration using yellow fluorescent protein (YFP) in a small, representative population of axons, which greatly improves longitudinal imaging. Here, we apply this method to study the progressive nature of Wallerian degeneration in both wild-type and slow Wallerian degeneration (WldS) mutant mice.ResultsIn wild-type nerves, we directly observed partially fragmented axons (average 5.3%) among a majority of fully intact or degenerated axons 37-42 h after transection and 40-44 h after crush injury. Axons exist in this state only transiently, probably for less than one hour. Surprisingly, axons degenerated anterogradely after transection but retrogradely after a crush, but in both cases a sharp boundary separated intact and fragmented regions of individual axons, indicating that Wallerian degeneration progresses as a wave sequentially affecting adjacent regions of the axon. In contrast, most or all WldS axons were partially fragmented 15-25 days after nerve lesion, WldS axons degenerated anterogradely independent of lesion type, and signs of degeneration increased gradually along the nerve instead of abruptly. Furthermore, the first signs of degeneration were short constrictions, not complete breaks.ConclusionsWe conclude that Wallerian degeneration progresses rapidly along individual wild-type axons after a heterogeneous latent phase. The speed of progression and its ability to travel in either direction challenges earlier models in which clearance of trophic or regulatory factors by axonal transport triggers degeneration. WldS axons, once they finally degenerate, do so by a fundamentally different mechanism, indicated by differences in the rate, direction and abruptness of progression, and by different early morphological signs of degeneration. These observations suggest that WldS axons undergo a slow anterograde decay as axonal components are gradually depleted, and do not simply follow the degeneration pathway of wild-type axons at a slower rate.

Project description:Abstract: Wallerian degeneration (WD) is a process of autonomous distal degeneration of axons upon injury. Macrophages (MP) of the peripheral nervous system (PNS) are main cellular agent controlling this process. Some evidences suggest that resident PNS-MP along with MP of hematogenous origin may be involved but whether these two subsets exert distinct functions is unknown. Combining MP-designed fluorescent reporters mice, and coherent anti-stoke raman scattering (CARS) imaging of the sciatic nerve, we deciphered the spatio-temporal choreography of resident and recently recruited MP after injury and unveiled distinct functions of these subsets with recruited MP responsible of efficient myelin stripping and clearance while resident MP were involved in axonal regrowth. This work provides clues to tackle selectively cellular processes involved in neurodegenerative diseases. Methods: (relevant for this GEO dataset): RNAseq of sciatic nerve macrophages from mice after CCI: Sorted cells were lysed in 100µl of RA1/TCEP buffer (NucleoSpin RNA XS, Macherey-Nagel), snap frozen in liquid nitrogen and stored at -80C until RNA extraction. All samples were processed in parallel and RNA extraction (without Carrier RNA but with on-column DNase treatment) was performed according to manufacturer's instructions (NucleoSpin RNA XS, Macherey-Nagel). RNA was eluted in RNAse-free water. Preparation of cDNA libraries for RNAseq was done using the SmartSeq method according to manufacturer's instructions (SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing, Clontech/TaKaRa). Due to the low number of cells, the total amount of eluted RNA was used as starting material for reverse transcription, followed by 18 cycles of pre-amplification. 1ng of cDNA were used for RNAseq sequencing library preparation, according to manufacturer's instructions (Nextera XT DNA Library Preparation, Illumina). Final samples pooled library prep were sequenced on a Nextseq 500 ILLUMINA with MidOutPut cartridge (2x130Millions of 75 bases reads) with 2 runs (4plex and 5plex), corresponding to 2x30Millions of (paired-end) reads per sample after demultiplexing.

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Protective effects of NAMPT or MAPK inhibitors and NaR on Wallerian degeneration of mammalian axons.

Publications

Protective effects of NAMPT or MAPK inhibitors and NaR on Wallerian degeneration of mammalian axons.

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