Nicotinamide mononucleotide adenylyl transferase 1 protects against acute neurodegeneration in developing CNS by inhibiting excitotoxic-necrotic cell death

Abstract

Hypoxic-ischemic (H-I) injury to the developing brain is a significant cause of morbidity and mortality in humans. Other than hypothermia, there is no effective treatment to prevent or lessen the consequences of neonatal H-I. Increased expression of the NAD synthesizing enzyme nicotinamide mononucleotide adenylyl transferase 1 (Nmnat1) has been shown to be neuroprotective against axonal injury in the peripheral nervous system. To investigate the neuroprotective role of Nmnat1 against acute neurodegeneration in the developing CNS, we exposed wild-type mice and mice overexpressing Nmnat1 in the cytoplasm (cytNmnat1-Tg mice) to a well-characterized model of neonatal H-I brain injury. As early as 6 h after H-I, cytNmnat1-Tg mice had strikingly less injury detected by MRI. CytNmnat1-Tg mice had markedly less injury in hippocampus, cortex, and striatum than wild-type mice as assessed by loss of tissue volume 7 d days after H-I. The dramatic protection mediated by cytNmnat1 is not mediated through modulating caspase3-dependent cell death in cytNmnat1-Tg brains. CytNmnat1 protected neuronal cell bodies and processes against NMDA-induced excitotoxicity, whereas caspase inhibition or B-cell lymphoma-extra large (Bcl-XL) protein overexpression had no protective effects in cultured cortical neurons. These results suggest that cytNmnat1 protects against neonatal HI-induced CNS injury by inhibiting excitotoxicity-induced, caspase-independent injury to neuronal processes and cell bodies. As such, the Nmnat1 protective pathway could be a useful therapeutic target for acute and chronic neurodegenerative insults mediated by excitotoxicity.

NMNAT3 is protective against the effects of neonatal cerebral hypoxia‐ischemia

“…Indeed, immunohistochemical localization of cerebral NMNAT3 appeared to be primarily present in neuronal cell bodies of the cerebral cortex and hippocampus (Fig. 1), and previous research has shown that overexpression of cytoplasmic NMNAT1 following neonatal cerebral H‐I resulted in a more global protective effect similar to the one found with NMNAT3 in this study 11. In addition, we find that endogenous NMNAT3 levels are increased during the first 72 h following neonatal H‐I.…”

“…Yet, the various NMNAT isoforms may individually differ in the cellular mechanisms that they employ to protect the injured brain. For example, in this work we found that NMNAT3 overexpression following H‐I decreased caspase‐3 activation, whereas this apoptotic enzyme appeared not to be affected following HI in neonatal mice expressing high levels of cytoplasmic NMNAT1 11. These differences further contribute to newer evidence showing that the neuroprotective effects of NMNATs are not exclusively related to their NAD+ synthetic activity 53.…”

“…A set of coronal brain sections (spaced 50 μ mol/L apart) beginning at the genu of the corpus extending to the mid body of the dorsal hippocampus were mounted in DAPI mounting medium (Vector Labs) and imaged via Nanozoomer (Hamamatsu Photonics). Percent tissue loss was calculated by comparing areas of distinct brain regions (cortex, striatum, and hippocampus) in the injured hemisphere (left) and uninjured hemisphere (right) using the Nanozoomer imaging software as previously described 11, 17. Three coronal sections per quantification area per animal were used for each of the brain regions studied.…”

“…At 24 h postexcitotoxic injury with Ibotenic acid or prior to paraformaldehyde fixation, the media of cortical and hippocampal neuron cultures were collected and assayed for lactate dehydrogenase (LDH) using a cytotoxicity detection kit (Roche) as previously described 11. Briefly, 100 μ L of culture medium per well/sample in duplicate was incubated with gentle shaking with the kit reagents for 30 min in the dark, after which the product was measured at 490 nm utilizing a standard 96‐well microplate reader (Bio‐Tek Instruments).…”

“…Although the neuroprotective actions of NMNATs in the mammalian brain remain largely unexplored, recent evidence suggests that NMNATs are also strongly protective against axonal and neuronal CNS degeneration. For example, increases in the expression of NMNAT1 and NMNAT3 decrease mouse retinal ganglion cell degeneration following ischemia,9, 10 and overexpression of cytoplasmic NMNAT1 protects the term‐equivalent neonatal mouse brain against the degenerative effects of cerebral H‐I via inhibition of glutamate‐dependent excitotoxic cell injury 11. In contrast, depletion of NMNAT2, the NMNAT isoform most abundant in the brain, restricts the outgrowth of retinal and cortical fibers in vitro 12.…”