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

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.

Variability in RNA interference in neuroendocrine PC12 cell lines stably transfected with an shRNA plasmid.

RNA interference (RNAi) has quickly become a very powerful technique for specifically suppressing or knocking down the expression of any desired gene. Many fields of research, including neuroscience, have benefitted from RNAi methods. It has been well documented that different small interfering RNAs (siRNAs) and small hairpin RNAs (shRNAs) vary greatly in terms of their effectiveness, and much attention has been focused on guidelines and algorithms for the selection of effective siRNAs. However, it has not been widely appreciated that a single shRNA-expressing plasmid can also produce widely varying levels of knockdown in different stably transfected cell lines derived from the same transfection. Here we report that knockdown of three distinct target proteins varies from minimal to almost complete in independent, stably transfected PC12 cell lines. This variability in knockdown among cell lines emphasizes the importance of characterizing a number of cell lines when attempting to establish stable knockdown cell lines, but also offers the possibility of studying the effects of graded levels of protein expression.