Glyceryl triacetate for Canavan disease: a low-dose trial in infants and evaluation of a higher dose for toxicity in the tremor rat model.

Canavan disease (CD) is a fatal dysmyelinating genetic disorder associated with aspartoacylase deficiency, resulting in decreased brain acetate levels and reduced myelin lipid synthesis in the developing brain. Here we tested tolerability of a potent acetate precursor, glyceryl triacetate (GTA), at low doses in two infants diagnosed with CD, aged 8 and 13 months. Much higher doses of GTA were evaluated for toxicity in the tremor rat model of CD. GTA was given orally to the infants for up to 4.5 and 6 months, starting at 25 mg/kg twice daily, doubling the dose weekly until a maximum of 250 mg/kg reached. Wild-type and tremor rat pups were given GTA orally twice daily, initially at a dose of 4.2 g/kg from postnatal days 7 through 14, and at 5.8 g/kg from day 15 through 23, and thereafter in food (7.5%) and water (5%). At the end of the trial (approximately 90 to 120 days) sera and tissues from rats were analysed for changes in blood chemistry and histopathology. GTA treatment caused no detectable toxicity and the patients showed no deterioration in clinical status. In the high-dose animal studies, no significant differences in the mean blood chemistry values occurred between treated and untreated groups, and no lesions indicating toxicity were detectable in any of the tissues examined. Lack of GTA toxicity in two CD patients in low-dose trials, as well as in high-dose animal studies, suggests that higher, effective dose studies in human CD patients are warranted.

Temporal and spatial changes of quinolinic acid immunoreactivity in the immune system of lipopolysaccharide-stimulated mice.

Quinolinic acid (Quin), a metabolite of tryptophan, is a neurotoxin that has been implicated in a variety of neuropathologic disorders that have immune components. The goal of this study was to characterize the changes in the cellular localization of Quin immunoreactivity in a paradigm of immune stimulation with lipopolysaccharide (LPS) in vivo to provide a basis for further studies on the physiological role of Quin in the immune system. Intraperitoneal LPS injection significantly increased Quin immunoreactivity (IR) in lymphoid tissues within 24 h. Spatial changes in splenic Quin-IR demonstrated a shift from the periarterial lymphoid sheaths to the follicles before returning to control levels by 72 h post-LPS. The strongly Quin-IR cells were tentatively identified as interdigitating dendritic cells and macrophages. Only minimal Quin-IR was detected in liver and lung, even under conditions of LPS stimulation combined with tryptophan loading. These data emphasize the temporally and spatially specific nature of Quin-IR changes in lymphoid tissues under conditions of immune stimulation and raise the possibility that Quin may have an immunomodulatory function.

Localization of quinolinic acid in the murine AIDS model of retrovirus-induced immunodeficiency: implications for neurotoxicity and dendritic cell immunopathogenesis.

OBJECTIVE AND DESIGN: Using murine AIDS (MAIDS) as a model of retrovirus-induced immunodeficiency, the aims of this study were (1) to determine the cellular source(s) of quinolinic acid (Quin) with regard to its significance as a potential neuroexcitotoxin in AIDS dementia complex, and (2) to characterize the relationship between dendritic cell Quin immunoreactivity and the histopathological changes associated with the progression of disease. METHODS: Mice with MAIDS were sacrificed from 1 to 16 weeks post-infection. Temporal and spatial changes in the in vivo distribution of Quin at the cellular level were determined by carbodiimide-based immunohistochemical methods. RESULTS: Cellular Quin immunoreactivity was chronically elevated in lymphoid tissues of mice with MAIDS. In contrast, no cellular Quin immunoreactivity was visible in the brain parenchyma at any timepoint studied. CONCLUSION: These findings are consistent with the view that select immune cells in the peripheral lymphoid tissues may be the primary source of Quin, which may contribute to neurotoxic complications in retrovirus-induced immunodeficiency syndromes. The predominant Quin immunoreactive cell types changed with the progression of disease. A significant finding was the marked increase in the number of Quin immunoreactive dendritic cells in the early phase of MAIDS, suggesting a relationship between dendritic cells and Quin in retroviral infection.

Two forms of the GABAA receptor distinguished by anion-exchange chromatography.

The GABAA receptor complex was solubilized from rat brain membranes in Triton X-100, enriched by 1012-S affinity chromatography, and subjected to DEAE anion-exchange chromatography. Two forms were distinguished by their differential elution during this HPLC with a KCl gradient. They displayed similar [3H]muscimol- and [3H]flunitrazepam-binding characteristics, as well as [3H]flunitrazepam-binding inhibition by CL 218872. Rechromatography of these distinct ionic forms indicated that they were not in dynamic equilibrium during chromatography. Resolution of these two pharmacologically similar populations of GABAA receptor by anion-exchange HPLC suggests that they differ in charge densities, a condition which may reflect differing glycosylation or phosphorylation states of the complex.

Comparative distribution of N-acetylaspartylglutamate and GAD67 in the cerebellum and precerebellar nuclei of the rat utilizing enhanced carbodiimide fixation and immunohistochemistry.

The most prevalent peptide in the nervous system, N-acetylaspartylglutamate (NAAG), specifically activates N-methyl D-aspartate (NMDA) receptors and a subclass of metabotropic glutamate receptors. One action of this peptide may be to modulate the release of other neurotransmitters, including gamma-aminobutyric acid (GABA). The present study describes the cellular distribution of NAAG, relative to GABA, in the cerebellum and precerebellar nuclei as a foundation for further physiological investigations. Numerous cells of origin for mossy fibers, including many of the larger neurons of the pontine nuclei, lateral reticular nuclei, vestibular nuclei, reticulotegmental nuclei, and spinal grey, were moderately to strongly stained for NAAG. Many NAAG-labeled fibers were clearly visible in the cerebellar peduncles and central white matter. Mossy fibers and mossy endings were among the most prominent NAAG-immunoreactive elements in the cerebellar cortex. Most neurons in the inferior olive were not stained for NAAG, and only sparse, lightly immunoreactive, climbing fiber-like endings could be identified in restricted regions of the cortical molecular layer. Purkinje neurons ranged from nonreactive to moderately positive, with the great majority being unstained. Cerebellar granule cells did not exhibit any NAAG immunoreactivity. A population of neurons in the deep cerebellar nuclei was highly immunoreactive for NAAG. Additionally, many neurons of the red nucleus were intensely stained for NAAG. Comparisons with staining for the 67 kD form of glutamic acid decarboxylase in serial sections revealed complementary distributions, with NAAG in excitatory pathways and cell groups, and glutamic acid decarboxylase in inhibitory systems. These findings suggest a significant functional involvement of NAAG in the excitatory afferent and efferent projection systems and provide an anatomical basis for investigations into the interactions of NAAG and GABA in the cerebellum.

Quinolinic acid immunoreactive cells in the choroid plexus, leptomeninges and brain vasculature of the immune-stimulated gerbil.

Antibodies to quinolinic acid were utilized to study the cellular localization of this endogenous neurotoxin in the gerbil brain subsequent to systemic immune stimulation with pokeweed mitogen. Immunohistochemistry of carbodiimide fixed spleen revealed a dramatic increase in the number of quinolinic acid-positive cells in the red pulp in the immune-stimulated animals. Quinolinic acid immunoreactivity in the brain was observed in cells within the choroid plexus, vasculature and leptomeninges of the stimulated group only. No immunoreactivity was observed in brain parenchyma. These results are supportive of an immune system origin for the increases in quinolinic acid in CSF and brain during immune stimulation in a rodent model system.

Enhanced carbodiimide fixation for immunohistochemistry: application to the comparative distributions of N-acetylaspartylglutamate and N-acetylaspartate immunoreactivities in rat brain.

To improve carbodiimide-based immunohistochemistry, carbodiimide-mediated coupling of radiolabeled N-acetylaspartylglutamate (NAAG) to bovine serum albumin was assayed in vitro. Various perfusion protocols, based on assay results, were tested for their ability to improve the immunohistochemical localization of two nervous system-specific molecules, NAAG and N-acetylaspartate (NAA) in the spinal cord, medulla, hippocampus, and cerebral cortex of the rat. Coupling of [3H]-NAAG to BSA in vitro was optimal with 100 mM carbodiimide and 1 mM N-hydroxysuccinimide in water at 37 degrees C. Optimal fixation of tissue was defined as permitting the identification of the NAAG and NAA in neuronal somata, dendritic arborizations, fine axons, and synaptic terminals with minimal diffuse background immunoreactivity. These conditions were obtained at 37 degrees C with 6% carbodiimide, 1 mM N-hydroxysuccinimide, and 5% dimethylsulfoxide perfused transcardially. Strong NAAG and NAA immunoreactivities were co-distributed in the majority of neurons in the spinal cord. Large-diameter spinal sensory afferents were stained for NAAG in the dorsal horn. The dorsal column nuclei were immunoreactive for NAAG and NAA, but only NAA staining was observed in the nucleus of the solitary tract. In cerebral cortex and hippocampus, NAAG and NAA immunoreactivities appeared to be exclusive, with NAAG staining observed in interneurons throughout all cortical layers, and NAA immunoreactivity present in most pyramidal neurons.

Cloning and characterization of the epsilon and zeta isoforms of the 14-3-3 proteins.

Two prominent proteins (30 and 33 kD) in a purified preparation of the sheep pineal gland were studied. Amino acid analysis of tryptic peptides indicated that the 33-kD protein was the epsilon isoform of the 14-3-3 family of proteins, and that the 30-kD protein was the zeta isoform. The sheep pineal gland was found to have six other 14-3-3 isoforms in addition to the epsilon and zeta, suggesting that copurification of the epsilon and zeta forms may reflect the existence of homo- or heterodimers comprised of these isoforms. To characterize 14-3-3 proteins further in the pineal gland, the full sequence of the epsilon isoform and a partial sequence of the zeta isoform were cloned from a rat pineal cDNA library and are reported here. Tissue distribution studies using Western blot analysis revealed that rat pineal and retina have levels of 14-3-3 protein similar to those found in brain, and that relatively low levels occur in other tissues. This investigation also revealed the epsilon isoform was present at high levels in the rat pineal gland early in development and decreased steadily thereafter and that 30-kD isoforms exhibited the inverse developmental pattern.

Immunohistochemical localization of N-acetylaspartate in rat brain.

N-acetylaspartate (NAA) is one of the most prevalent compounds in the mammalian nervous system. As such, NAA largely contributes to the major peak on water-suppressed proton magnetic resonance spectra. Highly specific antibodies to NAA demonstrate that this compound is discretely localized in a substantial number of neurons throughout the extent of the rat CNS. N-acetylaspartylglutamate (NAAG) is a structurally related neuronal dipeptide which is less widely distributed than NAA. NAAG and NAA immunoreactivities were extensively colocalized in many brainstem areas, where NAAG containing neurons were more numerous than in forebrain structures.

Selective distribution of N-acetylaspartylglutamate immunoreactivity in the extrapyramidal system of the rat.

N-Acetylaspartylglutamate (NAAG), a neuron-specific dipeptide, was found by immunocytochemistry to be localized in specific compartments of the extrapyramidal system of the rat. Cellular and neuropil NAAG-like immunoreactivity (NAAG-IR) was evident throughout the pallidum, entopeduncular nucleus, subthalamic nucleus, and all subdivisions of the substantia nigra. By contrast, only a minority of cells of the caudate-putamen and those of the accumbens nucleus stained positively for NAAG. In general, neuronal NAAG-IR was widespread in all ventroposterior output zones of the extrapyramidal system, while cellular immunostaining was greatly reduced in the corticostriate receptive zones. These data suggest that NAAG may serve some neuronal communication function in the extrapyramidal processing circuits and output projections to the thalamus and midbrain. Additionally, neuronal NAAG immunolabeling differentiates corticostriate receptive zones from pallidal and nigral subdivisions of the extrapyramidal system in the rat.

Effect of eye removal on N-acetylaspartylglutamate immunoreactivity in retinal targets of the cat.

The endogenous brain dipeptide N-acetylaspartylglutamate (NAAG) has previously been demonstrated in the somata of retinal ganglion cells and the neuropil of retinal targets. In this paper we report that the NAAG immunoreactivity of the neuropil in the retinal targets is dependent on an intact optic pathway. Removal of one eye produced a marked decrease in the staining of the neuropil in layer A of the contralateral geniculate nucleus (LGN) and layer A1 of the ipsilateral LGN. There was also decreased staining in the superficial layers of the superior colliculus contralateral to the removal. These results suggest that NAAG is present in the terminals of retinal ganglion cells and is consistent with a role for NAAG in visual synaptic transmission.

Effect of optic nerve transection on N-acetylaspartylglutamate immunoreactivity in the primary and accessory optic projection systems in the rat.

Evidence has been presented in recent years that support the hypothesis that N-acetylaspartylglutamate (NAAG) may be involved in synaptic transmission in the optic tract of mammals. Using a modified fixation protocol, we have determined the detailed distribution of NAAG immunoreactivity (NAAG-IR) in retinal ganglion cells and optic projections of the rat. Following optic nerve transection, dramatic losses of NAAG-IR were observed in the neuropil of all retinal target zones including the lateral geniculate nucleus, superior colliculus, nucleus of the optic tract, the dorsal and medial terminal nuclei and suprachiasmatic nucleus. Brain regions were microdissected and NAAG levels measured by a radioimmunoassay (RIA) (IC50: NAAG = 2.5 nM, NAA = 100 microM; smallest detectable amount = 1-2 pg/assay). Large decreases (50-60%) in NAAG levels were detected in the lateral geniculate, superior colliculus and suprachiasmatic nucleus. Moderate losses (25-45%) were noted in the pretectal nucleus and the nucleus of the optic tract. Smaller changes (15-20%) were detected in the paraventricular nucleus and the pretectal area. These results are consistent with a synaptic communication role for NAAG in the visual system.

Antibodies to quinolinic acid reveal localization in select immune cells rather than neurons or astroglia.

Polyclonal antibodies were produced against quinolinic acid. No immunoreactivity was observed in any cell type in carbodiimide-fixed brain tissue from control rats. When the antibodies were applied to carbodiimide-fixed spleen tissue, strong quinolinic acid immunoreactivity was observed in some cells with the appearance of macrophages and dendritic cells. These findings indicate an immune system origin for quinolinic acid, and implicate immune cells in excitotoxic CNS pathologies. These findings also raise the possibility that quinolinic acid is a unique cytokine in immune system signal transmission.

N-acetylaspartylglutamate identified in the rat retinal ganglion cells and their projections in the brain.

N-Acetylaspartylglutamate like immunoreactivity (NAAG-L) was identified in retinal ganglion cell bodies and their axons. The presence of the dipeptide in ganglion cell projection areas, the lateral geniculate nucleus (LGN) and superior colliculus (SC), was confirmed following NAAG purification from these tissues by a high-performance liquid chromatographic method. NAAG-L was identified in the optic tract as well as within fibers and puncta in the LGN and SC. The hypothesis that NAAG is present within ganglion cell axons in the brain was tested by unilateral enucleation which resulted in loss of NAAG and NAAG-L within the contralateral LGN and SC.

Effect of the proteasome inhibitor MLN519 on the expression of inflammatory molecules following middle cerebral artery occlusion and reperfusion in the rat.

Anti-inflammatory treatment with the proteasome inhibitor MLN519 has been previously reported to be neuroprotective against ischemic brain injury in rats. These effects have been related to inhibition of the transcription factor NF-kappaB, which is activated through ubiquitin-proteasomal degradation. The aim of this study was to evaluate the effects of MLN519 to alter the expression of several inflammatory genes under the control of NF-kappaB. Male Sprague-Dawley rats underwent middle cerebral artery occlusion (MCAo) followed by vehicle or MLN519 (1.0 g/kg, i.v.) treatment immediately after reperfusion of blood to the brain at 2h. Gene expression was evaluated 3-72 h post-MCAo. The most striking effects of intravenous treatment with MLN519 were associated with reductions in ICAM-1 expression at 3 h followed by reductions in E-selectin (12-72 h). Less dramatic reductions were observed in IL-1Beta (3-24 h) and TNF-Alpha (24 h) with no apparent effects on IL-6 and VCAM-1 mRNA levels. Immunohistochemical analysis revealed that the genes most dramatically affected by MLN519 had highest expression in endothelial cells and leukocytes (E-selectin, ICAM-1),indicating that these cell types may be the primary targets of intravenously delivered MLN519 treatment.

Regulation of melatonin synthesis in the ovine pineal gland.

The results presented here show clearly that the rapid nocturnal increase in circulating melatonin in sheep is associated with an equally rapid increase in melatonin production in the pineal gland. Pineal NAT activity and NAS concentration also increased under this condition, indicating that NAT activity is an important factor in this process. According to the current model for the regulation of pineal melatonin production in the rat, the large nocturnal increase in NAT activity is the major factor responsible for the daily rhythm in melatonin, as well as for the opposite rhythm in serotonin and its oxidation products in the pineal gland. Increased NAT activity in the pineal gland at night channels serotonin toward melatonin production at an enhanced rate, thereby causing pineal serotonin levels to drop. This, in turn, leads to decreased production of oxidative metabolites due to reduced substrate availability for MAO. Thus, the large increase in NAT activity acts as the key factor, directly or indirectly, for the generation of all rhythms in serotonin metabolites in the pineal gland, and possibly in the circulation. Such an exclusive role for NAT is doubtful in the sheep pineal gland for the following reasons: First, the nocturnal increase in NAT activity is relatively small (3-5-fold) compared to the rat (50-100-fold). Second, in at least two instances--a 30 min light pulse and prazosin treatment--there was a clear dissociation between melatonin production and NAT activity. This lack of correlation between NAT activity and melatonin production does not seem to be due to serotonin availability since serotonin levels did not change under the above conditions. Further, serotonin levels were found to decrease rather than increase when melatonin levels increased at night. Finally, our observation that melatonin production can be increased during the day by increasing serotonin levels in the pineal gland may reflect only the incompletely saturated nature of pineal NAT and may have little relevance for the physiological regulation of pineal melatonin production. Even though NAT activity may not be the only factor responsible for the rhythmic production of serotonin metabolites in sheep pineal gland, activation of the serotonin----melatonin pathway seems to be the primary metabolic response involved in this regulation, since the rhythms in the serotonin metabolites are similar in both rat and sheep. In the rat, the activation of the serotonin----melatonin pathway is brought about exclusively by the increase in NAT activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential distribution of N-acetylaspartylglutamate and N-acetylaspartate immunoreactivities in rat forebrain.

Contradictory immunohistochemical data have been reported on the localization of N-acetylaspartylglutamate in the rat forebrain, using different carbodiimide fixation protocols and antibody purification methods. In one case, N-acetylaspartylglutamate immunoreactivity was observed in apparent interneurons throughout all allocortical and isocortical regions, suggesting possible colocalization with GABA. In another case, strong immunoreactivity was observed in numerous pyramidal cells in neocortex and hippocampus, suggesting colocalization with glutamate or aspartate. Reconciling these disparate findings is crucial to understanding the role of N-acetylaspartylglutamate in nervous system function. Antibodies to N-acetylaspartylglutamate and a structurally related molecule, N-acetylaspartate, were purified in stages, and their cross-reactivities with protein conjugates of N-acetylaspartylglutamate and N-acetylaspartate were monitored at each stage by solid-phase immunoassay. Reduction of the cross-reactivity of the anti-N-acetylaspartylglutamate antibodies of N-acetylaspartate-protein conjugates to about 1% eliminated significant staining of most pyramidal neurons in the rat forebrain. Utilizing highly purified antibodies, the distributions of N-acetylaspartylglutamate and N-acetylaspartate were examined in several major telencephalic and diencephalic regions of the rat, and were found to be distinct. N-acetylaspartylglutamate-immunoreactivity was observed in specific neuronal populations, including many groups thought to use GABA as a neurotransmitter. Among these were the globus pallidus, ventral pallidum, entopeducular nucleus, thalamic reticular nucleus, and scattered non-pyramidal neurons in all layers of isocortex and allocortex. N-acetylaspartate-immunoreactivity was more broadly distributed than N-acetylaspartylglutamate-immunoreactivity in the rat forebrain, appearing strongest in many pyramidal neurons. Although N-acetylaspartate-immunoreactivity was found in most neurons, it exhibited a great range of intensities between different neuronal types.

Antibodies to quinolinic acid and the determination of its cellular distribution within the rat immune system.

Antibodies to quinolinic acid were produced in rabbits with protein-conjugated and gold particle-adsorbed quinolinic acid. Quinolinic acid immunoreactivity was below detection limits in carbodiimide-fixed rat brain. In contrast, strong quinolinic acid immunoreactivity was observed in spleen cells with variable, complex morphology located predominantly in the periarterial lymphocyte sheaths. In the thymus, quinolinic acid immunoreactivity was observed in cells with variable morphology, located almost exclusively in the medulla. Lymph nodes and gut-associated lymphoid tissue contained many, strongly stained cells of similar complex morphology in perifollicular areas. Immunoreactivity in liver and lung was restricted to widely scattered, perivascular cells and alveolar cells respectively. Additional stained cells with complex morphology were observed in bronchus-associated lymphoid tissue, in skin, and in the lamina propria of intestinal villi. Follicles in all secondary lymphoid organs were diffusely stained, ranging from mildly to moderately immunoreactive in spleen, to intensely immunoreactive in gut-associated lymphoid tissue. These results suggest that quinolinic acid is an immune system-specific molecule. Two hypothetical schemes are proposed to account for high levels of quinolinic acid in specific cells of the immune system.

Differential effects of kynurenine and tryptophan treatment on quinolinate immunoreactivity in rat lymphoid and non-lymphoid organs.

Quinolinate is a tryptophan metabolite and an intermediary in nicotinamide adenine dinucleotide (NAD+) synthesis in hepatocytes. Kynurenine is an upstream metabolite in the same biochemical pathway. Under normal physiological conditions, kynurenine is thought to be produced primarily in the liver as an NAD+ precursor. However, during immune stimulation or inflammation, numerous extrahepatic tissues convert systemic tryptophan to kynurenine, and its concentration subsequently rises dramatically in blood. The fate and role of extrahepatic kynurenine are uncertain. In order to begin addressing this question, the present study was performed to determine which cell types can produce quinolinate from either systemic tryptophan or kynurenine. By using highly specific antibodies to protein-coupled quinolinate, we found that intraperitoneal injections of tryptophan led to increased quinolinate immunoreactivity primarily in hepatocytes, with moderate increases in tissue macrophages and splenic follicles. In contrast, intraperitoneal injections of kynurenine did not result in any significant increase in hepatocyte quinolinate immunoreactivity, but rather led to dramatic increases in immunoreactivity in tissue macrophages, splenic white pulp, and thymic medulla. These findings suggest that hepatocytes do not make significant use of extracellular kynurenine for quinolinate or NAD+ synthesis, and that, instead, extrahepatic kynurenine is preferentially metabolized by immune cells throughout the body. The possible significance of the preferential metabolism of kynurenine by immune cells during an immune response is discussed.

N-acetylaspartylglutamate: a transmitter candidate for the retinohypothalamic tract.

The retinohypothalamic tract is the neural pathway mediating the photic entrainment of circadian rhythms in mammals. Important targets for these retinal fibers are the suprachiasmatic nuclei (SCN) of the hypothalamus, which are thought to be primary sites for the biological clock. The neurotransmitters that operate in this projection system have not yet been determined. Immunohistochemistry and radioimmunoassay performed with affinity-purified antibodies to N-acetylaspartylglutamate (NAAG) demonstrate that this neuron-specific dipeptide, which may act as an excitatory neurotransmitter, is localized extensively in the retinohypothalamic tract and its target zones, including the SCN. Optic nerve transections resulted in significant reductions in NAAG immunoreactivity in the optic chiasm and SCN. Analysis of NAAG concentrations in micropunches of SCN, by means of radioimmunoassay, showed approximately 50% reductions in NAAG levels. These results suggest that this peptide may act as one of the neurotransmitters involved in retinohypothalamic communication and circadian rhythm entrainment.