The Translocase of the Outer Mitochondrial Membrane (TOM40) is required for mitochondrial dynamics and neuronal integrity in Dorsal Root Ganglion Neurons.

Polymorphisms and altered expression of the Translocase of the Outer Mitochondrial Membrane - 40 kD (Tom40) are observed in neurodegenerative disease subjects. We utilized in vitro cultured dorsal root ganglion (DRG) neurons to investigate the association of TOM40 depletion to neurodegeneration, and to unravel the mechanism of neurodegeneration induced by decreased levels of TOM40 protein. We provide evidence that severity of neurodegeneration induced in the TOM40 depleted neurons increases with the increase in the depletion of TOM40 and is exacerbated by an increase in the duration of TOM40 depletion. We also demonstrate that TOM40 depletion causes a surge in neuronal calcium levels, decreases mitochondrial motility, increases mitochondrial fission, and decreases neuronal ATP levels. We observed that alterations in the neuronal calcium homeostasis and mitochondrial dynamics precede BCL-xl and NMNAT1 dependent neurodegenerative pathways in the TOM40 depleted neurons. This data also suggests that manipulation of BCL-xl and NMNAT1 may be of therapeutic value in TOM40 associated neurodegenerative disorders.

Enteric Neurodegeneration is Mediated Through Independent Neuritic and Somal Mechanisms in Rotenone and MPP+ Toxicity.

Gut motility malfunction and pathological changes in the enteric nervous system (ENS) are observed in the early stages of Parkinson's disease (PD). In many cases disturbances in the autonomous functions such as gut motility precedes the observed loss of central motor functions in PD. However, the mechanism by which ENS degeneration occurs in PD is unknown. We show that parkinsonian mimetics rotenone and MPP+ induce neurite degeneration that precedes cell death in primary enteric neurons cultured in vitro. If the neuronal death signals originate from degenerating neurites, neuronal death should be prevented by inhibiting neurite degeneration. Our data demonstrate that overexpression of cytNmnat1, an axon protector, maintains healthy neurites in enteric neurons treated with either of the parkinsonian mimetics, but cannot protect the soma. We also demonstrate that neurite protection via cytNmnat1 is independent of mitochondrial dynamics or ATP levels. Overexpression of Bcl-xl, an anti-apoptotic factor, protects both the neuronal cell body and the neurites in both rotenone and MPP+ treated enteric neurons. Our data reveals that Bcl-xl and cytNmnat1 act through separate mechanisms to protect enteric neurites. Our findings suggest that neurite protection alone is not sufficient to inhibit enteric neuronal degeneration in rotenone or MPP+ toxicity, and enteric neurodegeneration in PD may be occurring through independent somatic and neuritic mechanisms. Thus, therapies targeting both axonal and somal protection can be important in finding interventions for enteric symptoms in PD.

Role of oxygen consumption in hypoxia protection by translation factor depletion.

The reduction of protein synthesis has been associated with resistance to hypoxic cell death. Which components of the translation machinery control hypoxic sensitivity and the precise mechanism has not been systematically investigated, although a reduction in oxygen consumption has been widely assumed to be the mechanism. Using genetic reagents in Caenorhabditis elegans, we examined the effect on organismal survival after hypoxia of knockdown of 10 factors functioning at the three principal steps in translation. Reduction-of-function of all 10 translation factors significantly increased hypoxic survival to varying degrees, not fully accounted for by the level of translational suppression. Measurement of oxygen consumption showed that strong hypoxia resistance was possible without a significant decrease in oxygen consumption. Hypoxic sensitivity had no correlation with lifespan or reactive oxygen species sensitivity, two phenotypes associated with reduced translation. Resistance to tunicamycin, which produces misfolded protein toxicity, was the only phenotype that significantly correlated with hypoxic sensitivity. Translation factor knockdown was also hypoxia protective for mouse primary neurons. These data show that translation factor knockdown is hypoxia protective in both C. elegans and mouse neurons and that oxygen consumption does not necessarily determine survival; rather, mitigation of misfolded protein toxicity is more strongly associated with hypoxic protection.

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.

Tributyltin Alters Calcium Levels, Mitochondrial Dynamics, and Activates Calpains Within Dorsal Root Ganglion Neurons.

Tributyltin (TBT) remains a global health concern. The primary route of human exposure to TBT is either through ingestion or skin absorption, but TBT's effects on the peripheral nervous system have still not been investigated. Therefore, we exposed in vitro sensory dorsal root ganglion (DRG) neurons to TBT at a concentration of 50-200 nM, which is similar to the observed concentrations of TBT in human blood samples. We observed that TBT causes extensive axon degeneration and neuronal death in the DRG neurons. Furthermore, we discovered that TBT causes an increase in both cytosolic and mitochondrial calcium levels, disrupts mitochondrial dynamics, decreases neuronal ATP levels, and leads to the activation of calpains. Additional experiments demonstrated that inhibition of calpain activation prevented TBT-induced fragmentation of neuronal cytoskeletal proteins and neuronal cell death. Thus, we conclude that calpain activation is the key executioner of TBT-induced peripheral neurodegeneration.

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 (Wld(s)) protein can delay axonal degeneration initiated via axotomy, chemotherapeutic agents, or genetic mutations. The Wld(s) 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 Wld(s)-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 wld(s) mutant mice have demonstrated that delaying axonal degeneration slows disease course and prolongs survival in neurodegenerative disease models. The Wld(s) 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 Wld(s)-mediated axonal protection, several important questions remain including whether the Ube4b component of Wld(s) 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 Wld(s) 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 Wld(s) 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.

Calpain activation impairs neuromuscular transmission in a mouse model of the slow-channel myasthenic syndrome.

The slow-channel myasthenic syndrome (SCS) is a hereditary disorder of the acetylcholine receptor (AChR) of the neuromuscular junction (NMJ) that leads to prolonged AChR channel opening, Ca(2+) overload, and degeneration of the NMJ. We used an SCS transgenic mouse model to investigate the role of the calcium-activated protease calpain in the pathogenesis of synaptic dysfunction in SCS. Cleavage of a fluorogenic calpain substrate was increased at the NMJ of dissociated muscle fibers. Inhibition of calpain using a calpastatin (CS) transgene improved strength and neuromuscular transmission. CS caused a 2-fold increase in the frequency of miniature endplate currents (MEPCs) and an increase in NMJ size, but MEPC amplitudes remained reduced. Persistent degeneration of the NMJ was associated with localized activation of the non-calpain protease caspase-3. This study suggests that calpain may act presynaptically to impair NMJ function in SCS but further reveals a role for other cysteine proteases whose inhibition may be of additional therapeutic benefit in SCS and other excitotoxic disorders.

Lead exposure reduces survival, neuronal determination, and differentiation of P19 stem cells.

Lead (Pb) is a teratogen that poses health risks after acute and chronic exposure. Lead is deposited in the bones of adults and is continuously leached into the blood for decades. While this chronic lead exposure can have detrimental effects on adults such as high blood pressure and kidney damage, developing fetuses and young children are particularly vulnerable. During pregnancy, bone-deposited lead is released into the blood at increased rates and can cross the placental barrier, exposing the embryo to the toxin. Embryos exposed to lead display serious developmental and cognitive defects throughout life. Although studies have investigated lead's effect on late-stage embryos, few studies have examined how lead affects stem cell determination and differentiation. For example, it is unknown whether lead is more detrimental to neuronal determination or differentiation of stem cells. We sought to determine the effect of lead on the determination and differentiation of pluripotent embryonic testicular carcinoma (P19) cells into neurons. Our data indicate that lead exposure significantly inhibits the determination of P19 cells to the neuronal lineage by alteration of N-cadherin and Sox2 expression. We also observed that lead significantly alters subsequent neuronal and glial differentiation. Consequently, this research emphasizes the need to reduce public exposure to lead.

Protein kinase Czeta and glycogen synthase kinase-3beta 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 Czeta (PKCzeta), and glycogen synthase kinase 3beta (GSK3beta) are expressed in developing enteric neurons with a polarized distribution. Blocking PKCzeta or GSK3beta 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 PKCzeta or GSK3beta might alter Hirschsprung disease penetrance or expressivity by affecting ENS precursor migration.

Nicotinamide Mononucleotide Adenylyltransferase 2 maintains neuronal structural integrity through the maintenance of golgi structure.

Golgi fragmentation and loss of Nicotinamide Mononucleotide Adenylyltransferase 2 (NMNAT2) are the early key features of many neurodegenerative disorders. We investigated the link between NMNAT2 loss, Golgi fragmentation and axon degeneration. Golgi fragmentation in the cultured dorsal root ganglion (DRG) neurons resulted in caspase dependent axon degeneration and neuronal cell death. NMNAT2 depletion in the DRG neurons caused Golgi fragmentation and caspase dependent axon degeneration. NMNAT2 depletion did not cause ATP loss in the axons. These results indicate that NMNAT2 is required for maintenance of Golgi structure. Loss of Golgi structure or Nmnat2 depletion causes caspase dependent neurodegeneration. cytNmnat1 overexpression inhibited the axon degeneration induced by Golgi fragmentation or NMNAT2 depletion. These results also suggest that these degeneration signals converge on a common cytNmnat1 mediated axon protective program and are distinct from the SARM1 mediated caspase independent axon degeneration.

Mitochondrial Dynamics Decrease Prior to Axon Degeneration Induced by Vincristine and are Partially Rescued by Overexpressed cytNmnat1.

Axon degeneration is a prominent feature of various neurodegenerative diseases, such as Parkinson's and Alzheimer's, and is often characterized by aberrant mitochondrial dynamics. Mitochondrial fission, fusion, and motility have been shown to be particularly important in progressive neurodegeneration. Thus we investigated these imperative dynamics, as well as mitochondrial fragmentation in vincristine induced axon degradation in cultured dorsal root ganglia (DRG) neurons. CytNmnat1 inhibits axon degeneration in various paradigms including vincristine toxicity. The mechanism of its protection is not yet fully understood; therefore, we also investigated the effect of cytNmnat1 on mitochondrial dynamics in vincristine treated neurons. We observed that vincristine treatment decreases the rate of mitochondrial fission, fusion and motility and induces mitochondrial fragmentation. These mitochondrial events precede visible axon degeneration. Overexpression of cytNmnat1 inhibits axon degeneration and preserves the normal mitochondrial dynamics and motility in vincristine treated neurons. We suggest the alterations in mitochondrial structure and dynamics are early events which lead to axon degeneration and cytNmnat1 blocks axon degeneration by halting the vincristine induced changes to mitochondrial structure and dynamics.

Withania somnifera (Ashwagandha) attenuates antioxidant defense in aged spinal cord and inhibits copper induced lipid peroxidation and protein oxidative modifications.

Withania somnifera is classified in Ayurveda, the ancient Indian system of medicine, as a rasayana, a group of plant-derived drugs which promote physical and mental health, augment resistance of the body against disease and diverse adverse environmental factors, revitalize the body in debilitated conditions and increase longevity. We investigated the effects of Withania somnifera on copper-induced lipid peroxidation and antioxidant enzymes in aging spinal cord of Wistar rats. The activity of glutathione peroxidase (GPx) decreased significantly in the spinal cord from adult to aged mice. Treatment with Withania somnifera successfully attenuated GPx activity and inhibited lipid peroxidation in a dose dependent manner. Withania somnifera inhibited both the lipid peroxidation and protein oxidative modification induced by copper. These effects were similar to those of superoxide dismutase and mannitol. The results indicate the therapeutic potential of Withania somnifera in aging and copper-induced pathophysiological conditions.

Axon degeneration is key component of neuronal death in amyloid-ß toxicity.

Depending upon the stimulus, neuronal cell death can either be triggered from the cell body (soma) or the axon. We investigated the origin of the degeneration signal in amyloid ß (Aß) induced neuronal cell death in cultured in vitro hippocampal neurons. We discovered that Aß1-42 toxicity-induced axon degeneration precedes cell death in hippocampal neurons. Overexpression of Bcl-xl inhibited both axonal and cell body degeneration in the Aß-42 treated neurons. Nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1) blocks axon degeneration in a variety of paradigms, but it cannot block neuronal cell body death. Therefore, if the neuronal death signals in Aß1-42 toxicity originate from degenerating axons, we should be able to block neuronal death by inhibiting axon degeneration. To explore this possibility we over-expressed Nmnat1 in hippocampal neurons. We found that inhibition of axon degeneration in Aß1-42 treated neurons prevented neuronal cell death. Thus, we conclude that axon degeneration is the key component of Aß1-42 induced neuronal degeneration, and therapies targeting axonal protection can be important in finding a treatment for Alzheimer's disease.

Differential regional and subtype-specific vulnerability of enteric neurons to mitochondrial dysfunction.

Mitochondrial dysfunction is a central mediator of disease progression in diverse neurodegenerative diseases that often present with prominent gastrointestinal abnormalities. Gastrointestinal dysfunction in these disorders is related, at least in part, to defects in the enteric nervous system (ENS). The role of mitochondrial deficits in ENS neurodegeneration and their relative contribution to gastrointestinal dysfunction, however, are unclear. To better understand how mitochondrial abnormalities in the ENS influence enteric neurodegeneration and affect intestinal function, we generated mice (Tfam-ENSKOs) with impaired mitochondrial metabolism in enteric neurons and glia through the targeted deletion of the mitochondrial transcription factor A gene (Tfam). Tfam-ENSKO mice were initially viable but, at an early age, they developed severe gastrointestinal motility problems characterized by intestinal pseudo-obstruction resulting in premature death. This gastrointestinal dysfunction was caused by extensive, progressive neurodegeneration of the ENS involving both neurons and glia. Interestingly, mitochondrial defects differentially affected specific subpopulations of enteric neurons and regions of the gastrointestinal tract. Mitochondrial deficiency-related neuronal and glial loss was most prominent in the proximal small intestine, but the first affected neurons, nitrergic inhibitory neurons, had the greatest losses in the distal small intestine. This regional and subtype-specific variability in susceptibility to mitochondrial defects resulted in an imbalance of inhibitory and excitatory neurons that likely accounts for the observed phenotype in Tfam-ENSKO mice. Mitochondrial dysfunction, therefore, is likely to be an important driving force of neurodegeneration in the ENS and contribute to gastrointestinal symptoms in people with neurodegenerative disorders.

Reduced endothelin converting enzyme-1 and endothelin-3 mRNA in the developing bowel of male mice may increase expressivity and penetrance of Hirschsprung disease-like distal intestinal aganglionosis.

Hirschsprung disease (distal intestinal aganglionosis, HSCR) is a multigenic disorder with incomplete penetrance, variable expressivity, and a strong male gender bias. Recent studies demonstrated that these genetic patterns arise because gene interactions determine whether enteric nervous system (ENS) precursors successfully proliferate and migrate into the distal bowel. We now demonstrate that male gender bias in the extent of distal intestinal aganglionosis occurs in mice with Ret dominant-negative mutations (RetDN) that mimic human HSCR. We hypothesized that male gender bias could result from reduced expression of a gene already known to be essential for ENS development. Using quantitative real-time polymerase chain reaction (PCR) we demonstrated reduced levels of endothelin converting enzyme-1 and endothelin-3 mRNA in the male mouse bowel at the time that ENS precursors migrate into the colon. Other HSCR-associated genes are expressed at comparable levels in male and female mice. Testosterone and Mullerian inhibiting substance had no deleterious effect on ENS precursor development, but adding EDN3 peptide to E11.5 male RetDN heterozygous mouse gut explants in organ culture significantly increased the rate of ENS precursor migration through the bowel.

Activation of apoptotic pathways at muscle fiber synapses is circumscribed and reversible in a slow-channel syndrome model.

In the slow-channel syndrome (SCS) mutant acetylcholine receptors elicit calcium overload and myonuclear degeneration at the neuromuscular junction (NMJ), without muscle fiber death. Activated caspases are present at SCS motor endplates. We hypothesized that SCS represents a limited form of apoptosis. We found condensed chromatin and occasional single-strand DNA nicks in degenerating synaptic nuclei. Cleaved forms of caspases-3 and -9 were present in mouse SCS muscle homogenates and were specifically localized to NMJs. Finally, interruption of cholinergic activity by axotomy markedly reduced NMJ caspase activity and improved the morphological features of apoptosis at NMJs. These results demonstrate that in SCS processes leading to apoptosis may remain compartmentalized and reversible. Use of cysteine protease inhibitors may aid in treatment of this and other dystrophic muscle and excitotoxic disorders. Identification of extrasynaptic factors that prevent the spread of apoptosis in SCS muscle fibers may aid in developing treatments for neurological disorders characterized by excitotoxicity or apoptosis.

BMP signaling regulates murine enteric nervous system precursor migration, neurite fasciculation, and patterning via altered Ncam1 polysialic acid addition.

The enteric nervous system (ENS) forms from migrating neural crest-derived precursors that differentiate into neurons and glia, aggregate into ganglion cell clusters, and extend neuronal processes to form a complex interacting network that controls many aspects of intestinal function. Bone morphogenetic proteins (BMPs) have diverse roles in development and influence the differentiation, proliferation, and survival of ENS precursors. We hypothesized that BMP signaling might also be important for the ENS precursor migration, ganglion cell aggregation, and neurite fasciculation necessary to form the enteric nervous system. We now demonstrate that BMP signaling restricts murine ENS precursors to the outer bowel wall during migration. In addition, blocking BMP signaling causes faster colonization of the murine colon, reduces ganglion cell aggregation, and reduces neurite fasciculation. BMP signaling also influences patterns of neurite extension within the developing bowel wall. These effects on ENS precursor migration and neurite fasciculation appear to be mediated at least in part by increased polysialic acid addition to neural cell adhesion molecule (Ncam1) in response to BMP. Removing PSA enzymatically reverses the BMP effects on ENS precursor migration and neurite fasciculation. These studies demonstrate several novel roles for BMP signaling and highlight new functions for sialyltransferases in the developing ENS.

Differential gene expression and functional analysis implicate novel mechanisms in enteric nervous system precursor migration and neuritogenesis.

Enteric nervous system (ENS) development requires complex interactions between migrating neural-crest-derived cells and the intestinal microenvironment. Although some molecules influencing ENS development are known, many aspects remain poorly understood. To identify additional molecules critical for ENS development, we used DNA microarray, quantitative real-time PCR and in situ hybridization to compare gene expression in E14 and P0 aganglionic or wild type mouse intestine. Eighty-three genes were identified with at least two-fold higher expression in wild type than aganglionic bowel. ENS expression was verified for 39 of 42 selected genes by in situ hybridization. Additionally, nine identified genes had higher levels in aganglionic bowel than in WT animals suggesting that intestinal innervation may influence gene expression in adjacent cells. Strikingly, many synaptic function genes were expressed at E14, a time when the ENS is not needed for survival. To test for developmental roles for these genes, we used pharmacologic inhibitors of Snap25 or vesicle-associated membrane protein (VAMP)/synaptobrevin and found reduced neural-crest-derived cell migration and decreased neurite extension from ENS precursors. These results provide an extensive set of ENS biomarkers, demonstrate a role for SNARE proteins in ENS development and highlight additional candidate genes that could modify Hirschsprung's disease penetrance.