Fedias L. Christofi

SMN deficiency disrupts gastrointestinal and enteric nervous system function in mice

The 2007 Consensus Statement for Standard of Care in Spinal Muscular Atrophy (SMA) notes that patients suffer from gastroesophageal reflux, constipation and delayed gastric emptying. We used two mouse models of SMA to determine whether functional GI complications are a direct consequence of or are secondary to survival motor neuron (Smn) deficiency. Our results show that despite normal activity levels and food and water intake, Smn deficiency caused constipation, delayed gastric emptying, slow intestinal transit and reduced colonic motility without gross anatomical or histopathological abnormalities. These changes indicate alterations to the intrinsic neural control of gut functions mediated by the enteric nervous system (ENS). Indeed, Smn deficiency led to disrupted ENS signaling to the smooth muscle of the colon but did not cause enteric neuron loss. High-frequency electrical field stimulation (EFS) of distal colon segments produced up to a 10-fold greater contractile response in Smn deficient tissues. EFS responses were not corrected by the addition of a neuronal nitric oxide synthase inhibitor indicating that the increased contractility was due to hyperexcitability and not disinhibition of the circuitry. The GI symptoms observed in mice are similar to those reported in SMA patients. Together these data suggest that ENS cells are susceptible to Smn deficiency and may underlie the patient GI symptoms.

Intravenous AAV9 efficiently transduces myenteric neurons in neonate and juvenile mice

Gene therapies for neurological diseases with autonomic or gastrointestinal involvement may require global gene expression. Gastrointestinal complications are often associated with Parkinsons disease and autism. Lewy bodies, a pathological hallmark of Parkinsons brains, are routinely identified in the neurons of the enteric nervous system (ENS) following colon biopsies from patients. The ENS is the intrinsic nervous system of the gut, and is responsible for coordinating the secretory and motor functions of the gastrointestinal tract. ENS dysfunction can cause severe patient discomfort, malnourishment, or even death as in intestinal pseudo-obstruction (Ogilvie syndrome). Importantly, ENS transduction following systemic vector administration has not been thoroughly evaluated. Here we show that systemic injection of AAV9 into neonate or juvenile mice results in transduction of 25–57% of ENS myenteric neurons. Transgene expression was prominent in choline acetyltransferase positive cells, but not within vasoactive intestinal peptide or neuronal nitric oxide synthase cells, suggesting a bias for cells involved in excitatory signaling. AAV9 transduction in enteric glia is very low compared to CNS astrocytes. Enteric glial transduction was enhanced by using a glial specific promoter. Furthermore, we show that AAV8 results in comparable transduction in neonatal mice to AAV9 though AAV1, 5, and 6 are less efficient. These data demonstrate that systemic AAV9 has high affinity for peripheral neural tissue and is useful for future therapeutic development and basic studies of the ENS.

Enteric glial cells are major contributors to formation of cyclic AMP in myenteric plexus cultures from adult guinea-pig small intestine

Cultures derived from ganglia isolated from the small intestine of adult guinea-pigs were used to determine relative contribution of neurons and glial cells to stimulation of cAMP formation by forskolin in myenteric ganglia. In untreated cultures (8-12 days), the ratio of glial cells to neurons was 5-fold higher than the ratio in intact myenteric plexus preparations. Treatment with cytosine arabinoside virtually eliminated the glia by the 12th day. Microelectrode recording of excitatory responses to forskolin in AH/Type 2 neurons confirmed the viability of cultured neurons in cytosine arabinoside. Forskolin elevated the cAMP content of cultures and cytosine arabinoside reduced this effect by 80-90%. This suggests that enteric glial cells are the major contributors to cAMP formation in the cultures and that glial cells contribute significantly to elevation of cAMP levels seen in intact myenteric ganglia.

Corrigendum to SMN deficiency disrupts gastrointestinal and enteric nervous system function in mice [Human Molecular Genetics, 24, 13, (2015) 3847-3860, doi: 10.1093/hmg/ddv127]

The 2007 Consensus Statement for Standard of Care in Spinal Muscular Atrophy (SMA) notes that patients suffer from gastroesophageal reflux, constipation and delayed gastric emptying. We used twomousemodels of SMA to determinewhether functional GI complications are a direct consequence of or are secondary to survivalmotor neuron (Smn) deficiency. Our results show that despite normal activity levels and food and water intake, Smn deficiency caused constipation, delayed gastric emptying, slow intestinal transit and reduced colonic motility without gross anatomical or histopathological abnormalities. These changes indicate alterations to the intrinsic neural control of gut functionsmediated by the enteric nervous system (ENS). Indeed, Smn deficiency led to disrupted ENS signaling to the smoothmuscle of the colon but did not cause enteric neuron loss. High-frequencyelectricalfield stimulation (EFS) of distal colon segmentsproducedup to a 10-fold greater contractile response in Smn deficient tissues. EFS responses were not corrected by the addition of a neuronal nitric oxide synthase inhibitor indicating that the increased contractility was due to hyperexcitability and not disinhibition of the circuitry. The GI symptoms observed in mice are similar to those reported in SMA patients. Together these data suggest that ENS cells are susceptible to Smn deficiency and may underlie the patient GI symptoms.

Characterization of CD38 in the major cell types of the heart: endothelial cells highly express CD38 with activation by hypoxia-reoxygenation triggering NAD(P)H depletion

The NAD(P)+-hydrolyzing enzyme CD38 is activated in the heart during the process of ischemia and reperfusion, triggering NAD(P)(H) depletion. However, the presence and role of CD38 in the major cell types of the heart are unknown. Therefore, we characterize the presence and function of CD38 in cardiac myocytes, endothelial cells, and fibroblasts. To comprehensively evaluate CD38 in these cells, we measured gene transcription via mRNA, as well as protein expression and enzymatic activity. Endothelial cells strongly expressed CD38, while only low expression was present in cardiac myocytes with intermediate levels in fibroblasts. In view of this high level expression in endothelial cells and the proposed role of CD38 in the pathogenesis of endothelial dysfunction, endothelial cells were subjected to hypoxia-reoxygenation to characterize the effect of this stress on CD38 expression and activity. An activity-based CD38 imaging method and CD38 activity assays were used to characterize CD38 activity in normoxic and hypoxic-reoxygenated endothelial cells, with marked CD38 activation seen following hypoxia-reoxygenation. To test the impact of hypoxia-reoxygenation-induced CD38 activation on endothelial cells, NAD(P)(H) levels and endothelial nitric oxide synthase (eNOS)-derived NO production were measured. Marked NADP(H) depletion with loss of NO and increase in superoxide production occurred following hypoxia-reoxygenation that was prevented by CD38 inhibition or knockdown. Thus, endothelial cells have high expression of CD38 which is activated by hypoxia-reoxygenation triggering CD38-mediated NADP(H) depletion with loss of eNOS-mediated NO generation and increased eNOS uncoupling. This demonstrates the importance of CD38 in the endothelium and explains the basis by which CD38 triggers post-ischemic endothelial dysfunction.

Chronic ileal inflammation leads to transmural changes in the iNOS - NO signaling pathway

Alterations in mucosal production of NO and expression of iNOS are known to occur in lBD . Changes in the iNOS-NO pathway during lBD were further studied using a rabbit model of chronic ileitis. Chronic inflammation in pathogen free rabbits was produced by intragastric inoculation with Eimeria magna oocytes . Experiments were done on ileal specimens from 14 rabbits day 14 post-inoculation; means are average values from 3-4 rabbits. NO concentration was measured by chemiluminescence. The production of NO in the mucosa of inflamed animals increased from 22.23±.04 ~M to 84.76=: .51~M (p=O.OOOI). The activity of mucosal NOS increased from 2.75=:.29 to 7.34:!:.45 nmoles/mg DNA/hr (p=0.0222). Inflammation increased blood NO from 12.39:!:.40 to 19.36:!:.34 p.M (p=0.0153. No clear change in NO production occured in smooth muscle-myenteric plexus tissues (l2.95:!:.22p.M vs 15.35:!:.35p.M, p> .05). Labeling studies with a monoclonal anti-iNOS IgGI antibody were used to analyze the cellular distribution of iNOS immunoreactivity (IR) . Laser confocal imaging showed that inflammation did not increase the number of iNOS-positive myenteric or submucous neurons (inflamed,6.39:!:.95 myenteric neurons / ganglion, n=2,738 iNOS cells counted vs normal,6.77 :!: 0.92 myenteric neurons / ganglion , n= 2,734 iNOS cells counted, p> .05). Inflammation caused a 21% increase in iNOS intensity/expression in myenteric neurons (intensity increased from 163.81:!:.70 to 197.87:!:.11 units. n=514 neurons , p< .OOOOI). Inflammation caused a tiny 7:!:.02% increase in iNOS expression in submucous neurons (n=327 cells, p= .OO8). Inducible NOS was expressed in neurons with diverse morphologies. Intense focal expression of iNOS was observed in inflamed villus and crypt epithelia near abscesses, but was marginally detectable in non-inflamed villus epithelium. Diffuse staining occurred in the cytoplasm of epithelia. MCID Imaging Analysis revealed a 4-5 fold increase in iNOS IR / mm? proportional area. Intense iNOS staining occured at the apical and basolateral borders of the inflamed villus epithe lia, apical borders of some inflamed crypts , as well as in mononuclear leukocytes of the inflamed lamina propria. The results are consistent with mucosal alterations in the iNOS-NO pathway known to occur in IBD, suggesting that the rabbit enteritis model is a useful model to further study the pathway. Chronic enteritis increases the expression of iNOS-NO production in epithelia, mononuclear leukocytes and myenteric neurons (seed funds & NIH NCRR ISlO RRI1434).