Bhupinder P.S. Vohra

Nicotinamide Mononucleotide Adenylyl Transferase-Mediated Axonal Protection Requires Enzymatic Activity But Not Increased Levels of Neuronal Nicotinamide Adenine Dinucleotide

Axonal degeneration is a hallmark of many neurological disorders. Studies in animal models of neurodegenerative diseases indicate that axonal degeneration is an early event in the disease process, and delaying this process can lead to decreased progression of the disease and survival extension. Overexpression of the Wallerian degeneration slow (Wlds) protein can delay axonal degeneration initiated via axotomy, chemotherapeutic agents, or genetic mutations. The Wlds protein consists of the N-terminal portion of the ubiquitination factor Ube4b fused to the nicotinamide adenine dinucleotide (NAD+) biosynthetic enzyme nicotinamide mononucleotide adenylyl transferase 1 (Nmnat1). We previously showed that the Nmnat1 portion of this fusion protein was the critical moiety for Wlds-mediated axonal protection. Here, we describe the development of an automated quantitative assay for assessing axonal degeneration. This method successfully showed that Nmnat1 enzymatic activity is important for axonal protection as mutants with reduced enzymatic activity lacked axon protective activity. We also found that Nmnat enzymes with diverse sequences and structures from various species, including Drosophila melanogaster, Saccharomyces cerevisiae, and archaebacterium Methanocaldococcus jannaschii, which encodes a protein with no homology to eukaryotic Nmnat enzymes, all mediate robust axonal protection after axotomy. Besides the importance of Nmnat enzymatic activity, we did not observe changes in the steady-state NAD+ level, and we found that inhibition of nicotinamide phosphoribosyltransferase (Nampt), which synthesizes substrate for Nmnat in mammalian cells, did not affect the protective activity of Nmnat1. These results provide the possibility of a role for new Nmnat enzymatic activity in axonal protection in addition to NAD+ synthesis.

Transgenic Mice Expressing the Nmnat1 Protein Manifest Robust Delay in Axonal Degeneration In Vivo

Axonal degeneration is a key component of a variety of neurological diseases. Studies using wlds mutant mice have demonstrated that delaying axonal degeneration slows disease course and prolongs survival in neurodegenerative disease models. The Wlds protein is normally localized to the nucleus, and contains the N terminus of ubiquitination factor Ube4b fused to full-length Nmnat1, an NAD biosynthetic enzyme. While Nmnat enzymatic activity is necessary for Wlds-mediated axonal protection, several important questions remain including whether the Ube4b component of Wlds also plays a role, and in which cellular compartment (nucleus vs cytosol) the axonal protective effects of Nmnat activity are mediated. While Nmnat alone is clearly sufficient to delay axonal degeneration in cultured neurons, we sought to determine whether it was also sufficient to promote axonal protection in vivo. Using cytNmnat1, an engineered mutant of Nmnat1 localized only to the cytoplasm and axon, that provides more potent axonal protection than that afforded by Wlds or Nmnat1, we generated transgenic mice using the prion protein promoter (PrP). The sciatic nerve of these cytNmnat1 transgenic mice was transected, and microscopic analysis of the distal nerve segment 7 d later revealed no evidence of axonal loss or myelin debris, indicating that Nmnat alone, without any other Wlds sequences, is all that is required to delay axonal degeneration in vivo. These results highlight the importance of understanding the mechanism of Nmnat-mediated axonal protection for the development of new treatment strategies for neurological disorders.

Amyloid Precursor Protein Cleavage-Dependent and -Independent Axonal Degeneration Programs Share a Common Nicotinamide Mononucleotide Adenylyltransferase 1-Sensitive Pathway

Axonal degeneration is a hallmark of many debilitating neurological disorders and is thought to be regulated by mechanisms distinct from those governing cell body death. Recently, caspase 6 activation via amyloid precursor protein (APP) cleavage and activation of DR6 was discovered to induce axon degeneration after NGF withdrawal. We tested whether this pathway is involved in axonal degeneration caused by withdrawal of other trophic support, axotomy or vincristine exposure. Neurturin deprivation, like NGF withdrawal activated this APP/DR6/caspase 6 pathway and resulted in axonal degeneration, however, APP cleavage and caspase 6 activation were not involved in axonal degeneration induced by mechanical or toxic insults. However, loss of surface APP (sAPP) and caspase 6 activation were observed during axonal degeneration induced by dynactin 1(Dctn1) dysfunction, which disrupts axonal transport. Mutations in Dctn1 are associated with motor neuron disease and frontal temporal dementia, thus suggesting that the APP/caspase 6 pathway could be important in specific types of disease-associated axonal degeneration. The NGF deprivation paradigm, with its defined molecular pathway, was used to examine the context of Nmnat-mediated axonal protection. We found that although Nmnat blocks axonal degeneration after trophic factor withdrawal, it did not prevent loss of axon sAPP or caspase 6 activation within the axon, suggesting it acts downstream of caspase 6. These results indicate that diverse insults induce axonal degeneration via multiple pathways and that these degeneration signals converge on a common, Nmnat-sensitive program that is uniquely involved in axonal, but not cell body, degeneration.

The timing and location of glial cell line-derived neurotrophic factor expression determine enteric nervous system structure and function.

Ret signaling is critical for formation of the enteric nervous system (ENS) because Ret activation promotes ENS precursor survival, proliferation, and migration and provides trophic support for mature enteric neurons. Although these roles are well established, we now provide evidence that increasing levels of the Ret ligand glial cell line-derived neurotrophic factor (GDNF) in mice causes alterations in ENS structure and function that are critically dependent on the time and location of increased GDNF availability. This is demonstrated using two different strains of transgenic mice and by injecting newborn mice with GDNF. Furthermore, because different subclasses of ENS precursors withdraw from the cell cycle at different times during development, increases in GDNF at specific times alter the ratio of neuronal subclasses in the mature ENS. In addition, we confirm that esophageal neurons are GDNF responsive and demonstrate that the location of GDNF production influences neuronal process projection for NADPH diaphorase-expressing, but not acetylcholinesterase-, choline acetyltransferase-, or tryptophan hydroxylase-expressing, small bowel myenteric neurons. We further demonstrate that changes in GDNF availability influence intestinal function in vitro and in vivo. Thus, changes in GDNF expression can create a wide variety of alterations in ENS structure and function and may in part contribute to human motility disorders.

Novel delta subunit mutation in slow-channel syndrome causes severe weakness by novel mechanisms

We investigated the basis for a novel form of the slow‐channel congenital myasthenic syndrome presenting in infancy in a single individual as progressive weakness and impaired neuromuscular transmission without overt degeneration of the motor endplate. Prolonged low‐amplitude synaptic currents in biopsied anconeus muscle at 9 years of age suggested a kinetic disorder of the muscle acetylcholine receptor. Ultrastructural studies at 16 months, at 9 years, and at 15 years of age showed none of the typical degenerative changes of the endplate associated with the slow‐channel congenital myasthenic syndrome, and acetylcholine receptor numbers were not significantly reduced. We identified a novel C‐to‐T substitution in exon 8 of the δ‐subunit that results in a serine to phenylalanine mutation in the region encoding the second transmembrane domain that lines the ion channel. Using Xenopus oocyte in vitro expression studies we confirmed that the δS268F mutation, as with other slow‐channel congenital myasthenic syndrome mutations, causes delayed closure of acetylcholine receptor ion channels. In addition, unlike other mutations in slow‐channel congenital myasthenic syndrome, this mutation also causes delayed opening of the channel, a finding that readily explains the marked congenital weakness in the absence of endplate degeneration. Finally, we used serial morphometric analysis of electron micrographs to explore the basis for the progressive weakness and decline of amplitude of endplate currents over a period of 14 years. We demonstrated a progressive widening and accumulation of debris in the synaptic cleft, resulting in loss of efficacy of released neurotransmitter and reduced safety factor. These studies demonstrate the role of previously unrecognized mechanisms of impairment of synaptic transmission caused by a novel mutation and show the importance of serial in vitro studies to elucidate novel disease mechanisms.

Transcriptional profiling after bile duct ligation identifies PAI‐1 as a contributor to cholestatic injury in mice

Extrahepatic cholestasis leads to complex injury and repair processes that result in bile infarct formation, neutrophil infiltration, cholangiocyte and hepatocyte proliferation, extracellular matrix remodeling, and fibrosis. To identify early molecular mechanisms of injury and repair after bile duct obstruction, microarray analysis was performed on liver tissue 24 hours after bile duct ligation (BDL) or sham surgery. The most upregulated gene identified encodes plasminogen activator inhibitor 1 (PAI‐1, Serpine 1), a protease inhibitor that blocks urokinase plasminogen activator (uPA) and tissue‐type plasminogen activator (tPA) activity. Because PAI‐1, uPA, and tPA influence growth factor and cytokine processing as well as extracellular matrix remodeling, we evaluated the role of PAI‐1 in cholestatic liver injury by comparing the injury and repair processes in wild‐type (WT) and PAI‐1–deficient (PAI‐1−/−) mice after BDL. PAI‐1−/− mice had fewer and smaller bile infarcts, less neutrophil infiltration, and higher levels of cholangiocyte and hepatocyte proliferation than WT animals after BDL. Furthermore, PAI‐1−/− mice had higher levels of tPA activation and mature hepatocyte growth factor (HGF) after BDL than WT mice, suggesting that PAI‐1 effects on HGF activation critically influence cholestatic liver injury. This was further supported by elevated levels of c‐Met and Akt phosphorylation in PAI‐1−/− mice after BDL. In conclusion, PAI‐1 deficiency reduces liver injury after BDL in mice. These data suggest that inhibiting PAI‐1 might attenuate liver injury in cholestatic liver diseases. (HEPATOLOGY 2005;42:1099–1108.)

Image-based Screening Identifies Novel Roles for IκB Kinase and Glycogen Synthase Kinase 3 in Axonal Degeneration

Axon degeneration is an active, evolutionarily conserved self-destruction program by which compromised axons fragment in response to varied insults. Unlike programmed cell death, axon degeneration is poorly understood. We have combined robotic liquid handling with automated microscopy and image analysis to create a robust screening platform to measure axon degeneration in mammalian primary neuronal cultures. Using this assay, we performed an unbiased screen of 480 bioactive compounds, identifying 11 that reproducibly delay fragmentation of severed axons in vitro, including two inhibitors of glycogen synthase kinase 3 and two inhibitors of IκB kinase. Knockdown of each of these targets by shRNA lentivirus also delays axon degeneration in vitro, further supporting their role in the axon degeneration program.

Organotypic specificity of key RET adaptor-docking sites in the pathogenesis of neurocristopathies and renal malformations in mice

The receptor tyrosine kinase ret protooncogene (RET) is implicated in the pathogenesis of several diseases and in several developmental defects, particularly those in neural crest-derived structures and the genitourinary system. In order to further elucidate RET-mediated mechanisms that contribute to these diseases and decipher the basis for specificity in the pleiotropic effects of RET, we characterized development of the enteric and autonomic nervous systems in mice expressing RET9 or RET51 isoforms harboring mutations in tyrosine residues that act as docking sites for the adaptors Plcgamma, Src, Shc, and Grb2. Using this approach, we found that development of the genitourinary system and the enteric and autonomic nervous systems is dependent on distinct RET-stimulated signaling pathways. Thus, mutation of RET51 at Y1062, a docking site for multiple adaptor proteins including Shc, caused distal colon aganglionosis reminiscent of Hirschsprung disease (HSCR). On the other hand, this mutation in RET9, which encodes an isoform that lacks the Grb2 docking site present in RET51, produced severe abnormalities in multiple organs. Mutations that abrogate RET-Plcgamma binding, previously shown to produce features reminiscent of congenital anomalies of kidneys or urinary tract (CAKUT) syndrome, produced only minor abnormalities in the nervous system. Abrogating RET51-Src binding produced no major defects in these systems. These studies provide insight into the basis of organotypic specificity and redundancy in RET signaling within these unique systems and in diseases such as HSCR and CAKUT.

Focal caspase activation underlies the endplate myopathy in slow-channel syndrome

Slow‐channel syndrome (SCS) is a progressive neuromuscular disorder caused by abnormal gating of mutant acetylcholine receptors (AChRs) in the neuromuscular junction (NMJ). The pathological hallmark is selective degeneration of the NMJ termed endplate myopathy. Endplate myopathy consists of a combination of ultrastructural abnormalities, including degenerating subsynaptic nuclei, mitochondria, and postsynaptic folds, caused by localized cation overload through mutant AChRs. Because some of these changes resemble those seen in programmed cell death, we evaluated SCS muscle for evidence of focal activation of apoptotic pathways. Using antisera specific for the activated forms of caspases, the family of cysteine proteases that underlies apoptosis, we demonstrated that active forms of initiator and effector caspases are selectively localized at the NMJ in SCS. In comparison with an electron microscopic assessment of the abnormalities seen in endplate myopathy, we found that activated caspases were present at between 15 and 57% of endplates, similar to the proportion of endplates with degenerating mitochondria or vacuoles. This greatly exceeds the number of NMJs exhibiting nuclear degeneration. These findings provide the first evidence supporting the view that caspase activation in human disease can play a prominent role in localized cellular degenerative processes without causing nuclear or cell death.

Protein Kinase Cζ and Glycogen Synthase Kinase-3β Control Neuronal Polarity in Developing Rodent Enteric Neurons, whereas SMAD Specific E3 Ubiquitin Protein Ligase 1 Promotes Neurite Growth But Does Not Influence Polarity

Enteric nervous system (ENS) precursors migrate extensively before differentiating to form uni-axonal or multi-axonal neurons. ENS precursor survival, neurite growth, and cell migration are all directed by Ret kinase, but downstream signaling pathways are incompletely understood. We now demonstrate that proteins regulating polarity in other cells including partitioning defective 3 (PAR3), PAR6, protein kinase Cζ (PKCζ), and glycogen synthase kinase 3β (GSK3β) are expressed in developing enteric neurons with a polarized distribution. Blocking PKCζ or GSK3β reduces ENS precursor migration and induces the formation of multi-axonal neurons. Axon elongation also depends on SMURF1 (SMAD specific E3 ubiquitin protein ligase 1), which promotes RhoA degradation and associates with polarity proteins. SMURF1 inhibition, however, does not increase the number of multi-axonal neurons in ENS precursors. These data link cell surface Ret activation with molecular machinery controlling cytoskeletal dynamics and suggest that polymorphisms influencing PKCζ or GSK3β might alter Hirschsprung disease penetrance or expressivity by affecting ENS precursor migration.