Zellweger syndrome knockout mouse models challenge putative peroxisomal beta-oxidation involvement in docosahexaenoic acid (22:6n-3) biosynthesis

The putative involvement of peroxisomal beta-oxidation in the biosynthetic pathway of docosahexaenoic acid (22:6n-3, DHA) synthesis is critically reviewed in light of experiments with two recently developed knockout mouse models for Zellweger syndrome, a peroxisomal disorder affecting brain development. These mice were generated by targeted disruption of the PEX2 and PEX5 peroxisomal assembly genes encoding targeting signal receptor peroxins for the recognition and transport of a set of peroxisomal enzymes, including those of peroxisomal beta-oxidation, to the peroxisomal matrix. Analysis of esterified 22:6n-3 concentrations in PEX2-/- and PEX5-/- mice do not support the hypothesized requirement of peroxisomal beta-oxidation in 22:6n-3 synthesis, as only brain, but not liver or plasma, 22:6n-3 levels were decreased. Supplementation of PEX5+/- dams with 22:6n-3, although restoring the levels of brain 22:6n-3 in total lipids to that of controls, did not normalize the phenotype. These decreased brain 22:6n-3 concentrations appear to be secondary to impaired plasmalogen (sn-1-alkyl-, alkenyl-2-acyl glycerophospholipids) synthesis, probably at the level of the dihydroxyacetonephosphate acyltransferase (DHAP-AT), a peroxisomal enzyme catalyzing the first step in the synthesis of 22:6n-3-rich plasmalogens. To diminish the confounding effects of impaired plasmalogen synthesis in the brains of these Zellweger syndrome mouse models, kinetic experiments with labeled precursors, such as 18:3n-3 or 20:5n-3, in liver or isolated hepatocytes, which have negligible amounts of plasmalogens, are suggested to establish the rates of 22:6n-3 biosynthesis and precursor-product relationships. Similar experiments using brain of the acyl-CoA oxidase knockout mouse model are proposed to confirm the lack of peroxisomal beta-oxidation involvement in 22:6n-3 synthesis, since this mutation would not impair plasmalogen synthesis.     

Nonsense and temperature-sensitive mutations in PEX13 are the cause of complementation group H of peroxisome biogenesis disorders.

Peroxisome biogenesis disorders, including Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease, are lethal hereditary diseases caused by abnormalities in peroxisomal assembly. To date, 12 genotypes have been identified. We now have evidence that the complete human cDNA encoding Pex13p, an SH3 protein of a docking factor for the peroxisome targeting signal 1 receptor (Pex5p), rescues peroxisomal matrix protein import and its assembly in fibroblasts from PBD patients of complementation group H. In addition, we detected mutations on the human PEX13 cDNA in two patients of group H. A severe phenotype of a ZS patient (H-02) was homozygous for a nonsense mutation, W234ter, which results in the loss of not only the SH3 domain but also the putative transmembrane domain of Pex13p. A more mildly affected NALD patient (H-01), whose fibroblasts showed the temperature-sensitive (TS) phenotype, was homozygous for a missense mutation in the SH3 domain of Pex13p, I326T. This mutant PEX13 cDNA expression in a PEX13-defective CHO mutant showed I326T to be a TS mutation and thus suggested that Pex13p with the I326T mutation in the SH3 domain is stable at 30 degrees C but is somewhat unstable at 37 degrees C.     

A very long-chain acyl-CoA synthetase-deficient mouse and its relevance to X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative and endocrine disorder resulting from mutations in ABCD1 which encodes a peroxisomal membrane protein in the ATP binding cassette superfamily. The biochemical signature of X-ALD is increased levels of saturated very long-chain fatty acids (VLCFA; carbon chains of 22 or more) in tissues and plasma that has been associated with decreased peroxisomal very long-chain acyl-CoA synthetase (VLCS) activity and decreased peroxisomal VLCFA beta-oxidation. It has been hypothesized that ABCD1, which has no demonstrable VLCS activity itself, has an indirect effect on peroxisomal VLCS activity and VLCFA beta-oxidation by transporting fatty acid substrates, VLCS protein or some required co-factor into peroxisomes. Here we report the characterization of a Vlcs knockout mouse that exhibits decreased peroxisomal VLCS activity and VLCFA beta-oxidation but does not accumulate VLCFA. The XALD/Vlcs double knockout mouse has the biochemical abnormalities observed in the individual knockout mice but does not display a more severe X-ALD phenotype. These data lead us to conclude that (1) VLCFA levels are independent of peroxisomal fatty acid beta-oxidation, (2) there is no ABCD1/VLCS interaction and (3) the common severe forms of X-ALD cannot be modeled by decreasing peroxisomal VLCS activity in the XALD mouse.     

Straight-chain acyl-CoA oxidase knockout mouse accumulates extremely long chain fatty acids from alpha-linolenic acid: evidence for runaway carousel-type enzyme kinetics in peroxisomal beta-oxidation diseases

Extremely long chain polyunsaturated fatty acids (ELCPs) with >24 carbons and four or more double bonds are normally found in excitatory tissues but have no known function, and are greatly increased in brain and other tissues of humans with peroxisomal disorders. Straight-chain acyl-CoA oxidase (AOX) catalyzes the first, rate-limiting step of peroxisomal beta-oxidation of very-long-chain saturated and unsaturated fatty acids. We have studied the polyunsaturated fatty acid metabolism of AOX knockout mice (AOX-/- as a model of human AOX deficiency (pseudo-neonatal adrenoleukodystrophy), and as a genetic tool to test the putative peroxisomal beta-oxidation involvement in polyunsaturated fatty acid synthesis. Liver lipids of 26-day-old weanling AOX-/- mice livers accumulate n-3 and n-6 ELCPs from C24 to C30 with 5 and 6 double bonds, have 356 +/- 66 microg/g docosahexaenoic acid (22:6n-3), similar to congenic (AOX -/* = AOX+/+ and AOX+/-) controls (401 +/- 96 microg/g), but increased 22:5n-6 (22.4 +/- 3.7 vs 6.4 +/- 1.5 microg/g). AOX+/* mice injected intraperitoneally at 23 days with [U-(13)C]-18:3n-3 show strong labeling of 22:6n-3 after 72 h, whereas AOX -/- mice display less labeling of 22:6n-3 but strong tracer incorporation into 24:6n-3, 26:6n-3, and 28:6n-3, after the same period. These data suggest that ELCPs are natural runaway elongation by-products of 22:6n-3 and 22:5n-6 synthesis, which are normally disposed of by peroxisomal beta-oxidation. Under conditions with impaired peroxisomal beta-oxidation, such as Zellweger syndrome and adrenoleukodystrophies, ELCPs accumulate due to increased synthesis and impaired disposal. Two mechanisms for the formation of these runaway elongation by-products and the involvement of secondary carnitine deficiency in this process are proposed: n-3 ELCPs are synthesized by a carnitine-dependent multifunctional mitochondrial docosahexaenoic acid synthase (mtDHAS) which normally synthesizes primarily 22:6n-3, while n-6 ELCPs are synthesized by independent elongation enzymes in the endoplasmic reticulum.     

Gene disruption of the calcium channel Orai1 results in inhibition of osteoclast and osteoblast differentiation and impairs skeletal development

Calcium signaling plays a central role in the regulation of bone cells, although uncertainty remains with regard to the channels involved. In previous studies, we determined that the calcium channel Orai1 was required for the formation of multinucleated osteoclasts in vitro. To define the skeletal functions of calcium release-activated calcium currents, we compared the mice with targeted deletion of the calcium channel Orai1 to wild-type littermate controls, and examined differentiation and function of osteoblast and osteoclast precursors in vitro with and without Orai1 inhibition. Consistent with in vitro findings, Orai1(-/-) mice lacked multinucleated osteoclasts. Yet, they did not develop osteopetrosis. Mononuclear cells expressing osteoclast products were found in Orai1(-/-) mice, and in vitro studies showed significantly reduced, but not absent, mineral resorption by the mononuclear osteoclast-like cells that form in culture from peripheral blood monocytic cells when Orai1 is inhibited. More prominent in Orai1(-/-) mice was a decrease in bone with retention of fetal cartilage. Micro-computed tomography showed reduced cortical ossification and thinned trabeculae in Orai1(-/-) animals compared with controls; bone deposition was markedly decreased in the knockout mice. This suggested a previously unrecognized role for Orai1 within osteoblasts. Analysis of osteoblasts and precursors in Orai1(-/-) and control mice showed a significant decrease in alkaline phosphatase-expressing osteoblasts. In vitro studies confirmed that inhibiting Orai1 activity impaired differentiation and function of human osteoblasts, supporting a critical function for Orai1 in osteoblasts, in addition to its role as a regulator of osteoclast formation.     

The bcl-2 knockout mouse exhibits marked changes in osteoblast phenotype and collagen deposition in bone as well as a mild growth plate phenotype

Histological examination of long bones from 1-day-old bcl-2 knockout and age-matched control mice revealed no obvious differences in length of bone, growth plate architecture or stage of endochondral ossification. In 35-day-old bcl-2 knockout mice that are growth retarded or 'dwarfed'. the proliferative zone of the growth plate appeared slightly thinner and the secondary centres of ossification less well developed than their age-matched wild-type controls. The most marked histological effects of bcl-2 ablation were on osteoblasts and bone. 35-day-old knockout mouse bones exhibited far greater numbers of osteoblasts than controls and the osteoblasts had a cuboidal phenotype in comparison with the normal flattened cell appearance. In addition, the collagen deposited by the osteoblasts in the bcl-2 knockout mouse bone was disorganized in comparison with control tissue and had a pseudo-woven appearance. The results suggest an important role for Bcl-2 in controlling osteoblast phenotype and bone deposition in vivo.     

Mutation analysis of PEX7 in 60 probands with rhizomelic chondrodysplasia punctata and functional correlations of genotype with phenotype

PEX7 encodes the cytosolic receptor for the set of peroxisomal matrix enzymes targeted to the organelle by the peroxisome targeting signal 2 (PTS2). Mutations in PEX7 cause rhizomelic chondrodysplasia punctata (RCDP), a distinct peroxisome biogenesis disorder. In previous work we described three novel PEX7 mutant alleles, including one, L292X, with a high frequency due to a founder effect. We have now extended our analysis to 60 RCDP probands and identified a total of 24 PEX7 alleles, accounting for 95% of the mutant PEX7 genes in our sample. Of these, 50% are L292X, 13% are IVS9+1G>C, and the remainder are mostly private. IVS9+1G>C occurs on at least three different haplotypes and thus appears to result from recurrent mutation. The phenotypic spectrum of RCDP is broader than commonly recognized and includes minimally affected individuals at the mild end of the spectrum. To relate PEX7 genotype and phenotype, we evaluated the consequence of the disease mutation on PEX7 RNA by Northern analysis and RT/PCR. We evaluated the function of the encoded Pex7 protein (Pex7p) by expressing selected alleles in fibroblasts from RCDP patients and assaying their ability to restore import of a PTS2 marker protein. We find that residual activity of mutant Pex7p and reduced amounts of normal Pex7p are associated with milder and variant phenotypes.     

Identification of PEX3 as the gene mutated in a Zellweger syndrome patient lacking peroxisomal remnant structures

Peroxisome biogenesis disorders, of which 13 complementation groups have been identified, are subdivided with regard to two major dysfunctions: peroxisomal matrix protein import and peroxisomal membrane synthesis. Detectable remnant membrane structures are evident only in the former. Molecular defects have been defined in 10 PEX genes, including eight related to protein import and two to membrane synthesis. We now have evidence that the human complete cDNA encoding Pex3p, a peroxisomal membrane protein (PMP) factor for the proper localization of PMPs, rescues the import of both PMP and the matrix protein in fibroblasts from a Zellweger syndrome patient of complementation group G. This patient was homozygous for a 1 base insertion in the codon for V182, which resulted in a change of codon (182-183) and introduced a termination codon (184), which inactivated PMP and matrix protein import by Pex3p. A PEX3-defective CHO mutant clone, ZPG208, was of the same complementation group as group G.     

Endothelial nitric oxide synthase gene-deficient mice demonstrate marked retardation in postnatal bone formation, reduced bone volume, and defects in osteoblast maturation and activity

Nitric oxide (NO) has been implicated in the local regulation of bone metabolism. However, the contribution made by specific NO synthase (NOS) enzymes is unclear. Here we show that endothelial NOS gene knockout mice (eNOS-/-) have marked abnormalities in bone formation. Histomorphometric analysis of eNOS-/- femurs showed bone volume and bone formation rate was reduced by up to 45% (P: < 0.01) and 52% (P: < 0.01), respectively. These abnormalities were prevalent in young (6 to 9 weeks old) adults but by 12 to 18 weeks bone phenotype was restored toward wild-type. Dual energy X-ray absorptiometry analysis confirmed the age-related bone abnormalities revealing significant reductions in femoral (P: < 0.05) and spinal bone mineral densities (P: < 0.01) at 8 weeks that were normalized at 12 weeks. Reduction in bone formation and volume was not related to increased osteoclast numbers or activity but rather to dysfunctional osteoblasts. Osteoblast numbers and mineralizing activity were reduced in eNOS-/- mice. In vitro, osteoblasts from calvarial explants showed retarded proliferation and differentiation (alkaline phosphatase activity and mineral deposition) that could be restored by exogenous administration of a NO donor. These cells were also unresponsive to 17ss-estradiol and had an attenuated chemotactic response to transforming growth factor-beta. In conclusion, eNOS is involved in the postnatal regulation of bone mass and lack of eNOS gene results in reduced bone formation and volume and this is related to impaired osteoblast function.     

Selenium deficiency and fulvic acid supplementation induces fibrosis of cartilage and disturbs subchondral ossification in knee joints of mice: An animal model study of Kashin-Beck disease

Kashin-Beck disease is an acquired, chronic and degenerative osteoarticular disorder. Selenium deficiency and fulvic acid in drinking water have been implicated in the cause of this disease. Pathologically, chondronecrosis of the growth plate and articular cartilage and subconsequent disturbance of ossification were observed in the joints. In this animal model study, mice were fed with a selenium deficient diet and fulvic acid supplemented drinking water for two generations. In undecalcified histological preparations of bone we carried out histological staining to detect mineralized and unmineralized bone and cartilage. The results revealed that selenium deficiency and fulvic acid supplementation induced degeneration of the articular cartilage in the knee joints of mice. Dynamic fluorescent labelling of ossification, enzyme histochemical detection of alkaline phosphatase activity in osteoblasts and a typical immunohistochemical localization of collagens type I and II indicated the development of fibrocartilage at the articular surface of knee joints, resembling the early stages of osteoarthrosis. This became obvious by disturbed development of the articular space and meniscus, markedly impaired formation of subchondral bone and early differentiation failure during enchondral ossification. This animal model provides an approach to study the molecular pathogenesis of Kashin-Beck disease.     

Suppression of peroxisomal membrane protein defects by peroxisomal ATP binding cassette (ABC) proteins

X-Linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder characterized by reduced peroxisomal very long chain fatty acid (VLCFA) beta-oxidation. The X - ALD gene product (ALDP) is a peroxisomal transmembrane protein with an ATP binding cassette (ABC). ALDP and three other ABC proteins (PMP70, ALDR, P70R) localize to the peroxisomal membrane. The function of this family of peroxisomal membrane proteins is unknown. We used complementation studies to begin analysis of their role in VLCFA beta-oxidation and on the peroxisomal membrane. Expression of either ALDP or PMP70 restores VLCFA beta- oxidation in X-ALD fibroblasts, indicating overlapping functions. Their expression also restores peroxisome biogenesis in cells that are deficient in the peroxisomal membrane protein Pex2p. Thus it is likely that complex protein interactions are involved in the function and biogenesis of peroxisomal membranes that may contribute to disease heterogeneity.      

The host Pex19p plays a role in peroxisomal localization of tombusvirus replication proteins

Replication of Tomato bushy stunt virus (TBSV) RNA takes place on the cytosolic membrane surface of peroxisomes in plants and in yeast, a model host. To identify the host proteins involved in assisting the peroxisomal localization of the tombusvirus p33 replication protein, we tested if p33 could bind directly to yeast proteins involved in peroxisomal transport in vitro. This work has led to the demonstration of Pex19p-p33 interaction via pull-down and co-purification experiments. Pex19p was also detected in the tombusvirus replicase after protein cross-linking, suggesting that Pex19p transiently binds to the replicase as could be expected from a transporter. To validate the importance of Pex19p-p33 interaction in TBSV replication in yeast, we re-targeted Pex19p to the mitochondria, which resulted in the re-distribution of a large fraction of p33 to the mitochondria. The expression of the mitochondrial-targeted Pex19p inhibited TBSV RNA accumulation by 2-4-fold in vivo and reduced the in vitro activity of the tombusvirus replicase by 80%. These data support the model that Pex19p is a cellular transporter for localization of p33 replication protein to the host peroxisomal membranes.     

Delayed tooth eruption in membrane type-1 matrix metalloproteinase deficient mice

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is expressed highly in mineralizing tissues including bones and teeth. Mice deficient in MT1-MMP (-/-) display severe defects in skeletal development including dwarfism, osteopenia, and craniofacial abnormalities. Death occurs in these mice by about 3 weeks of age. Since MT1-MMP is expressed by the ameloblasts of the enamel organ and by the odontoblasts of the dental papilla, we asked if the developing teeth were adversely affected in the knockout animals. Molars from MT1-MMP -/- mice and controls were examined by histological, X-ray, and SEM analysis at 4, 18-20, and 25 days of postnatal development. At 4 days of development the molars from the -/- mice appeared histologically normal. At 18-20 days of development, the first molars of the -/- mice had apparently normal tooth crowns with normal dentin and enamel; however, the roots were truncated and the teeth had not yet erupted. In contrast to the -/- mice, the first molars of the 18-20-day control animals had erupted. SEM analysis of a -/- first molar and incisor revealed a normal enamel prism pattern. However, X-ray analysis demonstrated that tooth eruption was delayed by approximately 5 days and that the tooth roots were abnormally short in the knockout animals. Since MT1-MMP-deficient mice have been demonstrated to display a generalized increase in bone resorption, these data suggest that inefficient growth of bone surrounding the tooth root complex causes a delay in tooth eruption.     

The Pex1-G844D mouse: A model for mild human Zellweger spectrum disorder

Zellweger spectrum disorder (ZSD) is a disease continuum that results from inherited defects in PEX genes essential for normal peroxisome assembly. These autosomal recessive disorders impact brain development and also cause postnatal liver, adrenal, and kidney dysfunction, as well as loss of vision and hearing. The hypomorphic PEX1-G843D missense allele, observed in approximately 30% of ZSD patients, is associated with milder clinical and biochemical phenotypes, with some homozygous individuals surviving into early adulthood. Nonetheless, affected children with the PEX1-G843D allele have intellectual disability, failure to thrive, and significant sensory deficits. To enhance our ability to test candidate therapies that improve human PEX1-G843D function, we created the novel Pex1-G844D knock-in mouse model that represents the murine equivalent of the common human mutation. We show that Pex1-G844D homozygous mice recapitulate many classic features of mild ZSD cases, including growth retardation and fatty livers with cholestasis. In addition, electrophysiology, histology, and gene expression studies provide evidence that these animals develop a retinopathy similar to that observed in human patients, with evidence of cone photoreceptor cell death. Similar to skin fibroblasts obtained from ZSD patients with a PEX1-G843D allele, we demonstrate that murine cells homozygous for the Pex1-G844D allele respond to chaperone-like compounds, which normalizes peroxisomal β-oxidation. Thus, the Pex1-G844D mouse provides a powerful model system for testing candidate therapies that address the most common genetic cause of ZSD. In addition, this murine model will enhance studies focused on mechanisms of pathogenesis.     

Impaired inhibitory control of cortical synchronization in fragile X syndrome

Fragile X syndrome (FXS) is a neurodevelopmental disorder characterized by severe cognitive impairments, sensory hypersensitivity, and comorbidities with autism and epilepsy. Fmr1 knockout (KO) mouse models of FXS exhibit alterations in excitatory and inhibitory neurotransmission, but it is largely unknown how aberrant function of specific neuronal subtypes contributes to these deficits. In this study we show specific inhibitory circuit dysfunction in layer II/III of somatosensory cortex of Fmr1 KO mice. We demonstrate reduced activation of somatostatin-expressing low-threshold-spiking (LTS) interneurons in response to the group I metabotropic glutamate receptor (mGluR) agonist 3,5-dihydroxyphenylglycine (DHPG) in Fmr1 KO mice, resulting in impaired synaptic inhibition. Paired recordings from pyramidal neurons revealed reductions in synchronized synaptic inhibition and coordinated spike synchrony in response to DHPG, indicating a weakened LTS interneuron network in Fmr1 KO mice. Together, these findings reveal a functional defect in a single subtype of cortical interneuron in Fmr1 KO mice. This defect is linked to altered activity of the cortical network in line with the FXS phenotype.     

A mouse model for alpha-methylacyl-CoA racemase deficiency: adjustment of bile acid synthesis and intolerance to dietary methyl-branched lipids

alpha-Methylacyl-CoA racemase (Amacr) deficiency in humans leads to sensory motor neuronal and liver abnormalities. The disorder is recessively inherited and caused by mutations in the AMACR gene, which encodes Amacr, an enzyme presumed to be essential for bile acid synthesis and to participate in the degradation of methyl-branched fatty acids. To generate a model to study the pathophysiology in Amacr deficiency we inactivated the mouse Amacr gene. As per human Amacr deficiency, the Amacr(-/-) mice showed accumulation (44-fold) of C27 bile acid precursors and decreased (over 50%) primary (C24) bile acids in bile, serum and liver, however the Amacr(-/-) mice were clinically symptomless. Real-time quantitative PCR analysis showed that, among other responses, the level of mRNA for peroxisomal multifunctional enzyme type 1 (pMFE-1) was increased 3-fold in Amacr(-/-) mice. This enzyme can be placed, together with CYP3A11 and CYP46A1, to make an Amacr-independent pathway for the generation of C24 bile acids. Exposure of Amacr(-/-) mice to a diet supplemented with phytol, a source for branched-chain fatty acids, triggered the development of a disease state with liver manifestations, redefining the physiological significance of Amacr. Amacr is indispensable for the detoxification of dietary methyl-branched lipids and, although it contributes normally to bile acid synthesis from cholesterol, the putative pMFE-1-mediated cholesterol degradation can provide for generation of bile acids, allowing survival without Amacr. Based upon our mouse model, we propose elimination of phytol from the diet of patients suffering from Amacr deficiency.     

Retrovirally transduced bone marrow has a therapeutic effect on brain in the mouse model of mucopolysaccharidosis IIIB

Mucopolysaccharidosis IIIB (MPS IIIB) is a lysosomal storage disorder caused by mutations in NAGLU, the gene encoding alpha-N-acetylglucosaminidase. The disease is characterized by profound mental retardation and eventual neurodegeneration, but relatively mild somatic manifestations. There is no available therapy. We have used a mouse knockout model of the disease to test therapy by genetically modified bone marrow. Bone marrow from Naglu -/- male mice was transduced with human NAGLU cDNA in an MND-MFG vector, and transplanted into 6- to 8-week-old lethally irradiated female -/- mice. Sham-treated mice received bone marrow transduced with eGFP cDNA in an MND vector. alpha-N-Acetylglucosaminidase activity in plasma and leukocytes, measured 3 and 6 months after transplantation, varied from marginal to nearly 30 times wild-type. A low level of alpha-N-acetylglucosaminidase activity, as little as provided by transplantation of unmodified Naglu +/+ bone marrow, could normalize biochemical defects (glycosaminoglycan storage and beta-hexosaminidase elevation) in liver and spleen, but a very high level was required for an effect on kidney. Effects on the brain were best seen by examination of cellular morphology using light and electron microcopy. Mice that expressed very high levels of alpha-N-acetylglucosaminidase in blood had an increased number of normal-appearing neurons in the cortex and other parts of the brain, while microglia with engorged lysosomes had almost completely disappeared. Immunohistochemistry showed a marked decrease of staining for subunit c of mitochondrial ATP synthase and for Lamp1, markers of neuronal and microglial pathology, respectively, as well as a decrease in staining for glial fibrillary acid protein, a marker of activated astrocytes. These results show that genetically modified cells of hematopoietic origin can reduce the pathologic manifestations of MPS IIIB in the Naglu -/- mouse brain.     

Interferon-gamma-induced nitric oxide causes intrinsic intestinal denervation in Trypanosoma cruzi-infected mice

In this study, the role of nitric oxide (NO) in neuronal destruction during acute-phase Trypanosoma cruzi infection was evaluated in male C57BL/6 (WT, wild-type) mice and knockout mice [inducible nitric oxide synthase (iNOS)(-/-) and interferon (IFN)(-/-)]. Selected animals were infected by intraperitoneal injection of 100 trypomastigote forms of the Y strain of T. cruzi. Others were injected intraperitoneally with an equal volume of saline solution and served as controls. Our findings support those of previous studies regarding myenteric denervation in acute-phase T. cruzi infection. In addition, we clearly demonstrate that, despite the fact that parasite nests and similar inflammatory infiltrate in the intestinal wall were more pronounced in infected iNOS(-/-) mice than in infected WT mice, the former presented no reduction in myenteric plexus neuron numbers. Neuronal nerve profile expression, as revealed by the general nerve marker PGP 9.5, was preserved in all knockout animals. Infected IFN(-/-) mice suffered no significant neuronal loss and there was no inflammatory infiltrate in the intestinal wall. On days 5 and 10 after infection, iNOS activity was greater in infected WT mice than in controls, whereas iNOS activity in infected knockout mice remained unchanged. These findings clearly demonstrate that neuronal damage does not occur in NO-impaired infected knockout mice, regardless of whether inflammatory infiltrate is present (iNOS(-/-)) or absent (IFN(-/-)). In conclusion, our observations strongly indicate that myenteric denervation in acute-phase T. cruzi infection is because of IFN-gamma-elicited NO production resulting from iNOS activation in the inflammatory foci along the intestinal wall.