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In intestinal cells, arginine (Arg) is 1 of the 2 most potent amino acid activators of p70(s6k), a key regulator of 5'- terminal oligopyrimidine mRNA translation, a necessary condition for increased cell migration. To investigate the mechanism of response to Arg, we used the rat crypt cell line cdx2-transformed IEC-6 cells (cdx2-IEC) and measured cell migration, immunocytochemical analysis of p70(s6k) activation in response to Arg, and production of nitric oxide (NO). When treated with Arg, cdx2-IEC increased in phosphorylation on Thr-389 of p70(s6k) (pp70(s6k)) compared with control (P < 0.01). Phospho-Thr-421/Ser-424-p70(s6k) was located in the nucleus shortly after Arg treatment. Arg enhanced pp70(s6k), cell migration (55% wound coverage), and NO production. In comparison, the branched-chain amino acid leucine (Leu) activated pp70(s6k), was a weaker stimulator of migration (23% coverage), and did not increase NO. A total of 25 micromol/L DETA-NONOate (DETA/NO) did not significantly enhance phosphorylation of p70(s6k) but enhanced the rate of cell migration by approximately 25%. Wound coverage with Leu plus DETA/NO (25 micromol/L) was greater than coverage with DETA/NO alone (P < 0.01). These and our previous studies lead to a model in which Arg must stimulate both pp70(s6k) (in the nucleus) and NO release to enhance intestinal epithelial cell migration, which may be relevant to diseases that involve intestinal villous injury.

This study descibes gene expression in the fetus hearts obtained from mouse model for Phenylketonuria. These hearts have cardiovascular disease (CVD). Therefore genes involved in CVD were examined. Several genes associated with heart development and inflammation were found to be altered. In order to investigate whether the abnormal gene expression alters transcription and translation, the levels of troponin mRNA and protein were determined. One step real time RT-PCR showed a reduction in cardiac troponin I, troponin T2 and ryanodine receptor 2. Determination of troponin I and T protein levels showed reduced levels of these proteins. Our results suggest that altered gene expression affects protein production. These changes are likely involved in the cardiovascular defects seen in the mouse.

Phenylketonuria patients harboring a subset of phenylalanine hydroxylase (PAH) mutations have recently shown normalization of blood phenylalanine levels upon oral administration of the PAH cofactor tetrahydrobiopterin [(6 R )- l - erythro -5,6,7,8-tetrahydrobiopterin (BH 4 )]. Several hypotheses have been put forward to explain BH 4 responsiveness, but the molecular basis for the corrective effect(s) of BH 4 has not been understood. We have investigated the biochemical, kinetic, and structural changes associated with BH 4 -responsive mutations (F39L, I65T, R68S, H170D, E178G, V190A, R261Q, A300S, L308F, A313T, A373T, V388M, E390G, P407S, and Y414C). The biochemical and kinetic characterization of the 15 mutants studied points toward a multifactorial basis for the BH 4 responsiveness; the mutants show residual activity (>30% of WT) and display various kinetic defects, including increased K m (BH 4 ) and reduced cooperativity of substrate binding, but no decoupling of cofactor (BH 4 ) oxidation. For some, BH 4 seems to function through stabilization and protection of the enzyme from inactivation and proteolytic degradation. In the crystal structures of a phenylketonuria mutant, A313T, minor changes were seen when compared with the WT PAH structures, consistent with the mild effects the mutant has upon activity of the enzyme both in vitro and in vivo . Truncations made in the A313T mutant PAH form revealed that the N and C termini of the enzyme influence active site binding. Of fundamental importance is the observation that BH 4 appears to increase Phe catabolism if at least one of the two heterozygous mutations has any residual activity remaining.

Purpose: Phenylketonuria (PKU) is an autosomal recessive disorder caused by mutations in the phenylalanine hydroxylase (PAH) gene. There have been more than 400 mutations identified in the PAH gene leading to variable degrees of deficiency in PAH activity, and consequently a wide spectrum of clinical severity. A pilot study was undertaken to examine the response to 6-R-l-erythro-5,6,7,8-tetrahydrobiopterin (BH4) in patients with atypical and classical PKU. Methods: PAH gene mutation analysis was performed using denaturing gradient gel electrophoresis and gene sequencing. Patients with classical, atypical, or mild PKU were orally given BH4 10 mg/kg. Blood phenylalanine and tyrosine levels were determined using tandem MS/MS at 0 hours, 4 hours, 8 hours, and 24 hours intervals. Results: Thirty-six patients were given a single oral dose of 10 mg/kg of BH4. Twenty one patients (58.33%) responded with a decrease in blood phenylalanine level. Of the patients that responded, 12 were classical, 7 atypical, and 2 mild. The mean decline in blood phenylalanine at 24 hours was > 30% of baseline. There were 15 patients who did not respond to the BH4 challenge, 14 of those had classical and one had atypical PKU. Mapping the mutations that responded to BH4 on the PAH enzyme showed that mutations were in the catalytic, regulatory, oligomerization, and BH4 binding domains. Five patients responding to BH4 had mutations not previously identified. Conclusion: The data presented suggest higher than anticipated number of PKU mutations respond to BH4, and such mutations are on all the domains of PAH.

Aspartoacylase/aminoacylase II (ASPA/ACY II) is mainly synthesized in oligodendrocytes to contribute in myelin synthesis. Although axonal damage is seen in the brain with human immunodeficiency virus encephalitis (HIVE), ASPA contribution in the pathology is not known. Immunostaining study showed that ASPA protein is reduced in the white matter of patients with HIVE compared to the control. Western blot study further confirmed ASPA deficiency in the HIVE brain compared to the control. This paper suggests that HIVE condition affects ASPA to contribute in myelin loss/axonal damage seen in the disease.

Astrocytes play an important role in astrocyte–neuron homeostasis. In HIV-1-infected brain, interleukin 1 beta (IL-1β) activation of astrocytes contributes to neurodegeneration. However, the molecular mechanisms underlying IL-1β-activated-astrocytes-induced neurodegeneration in HIV-1-infected brain are largely unknown. We hypothesize that secretory factors from the activated astrocytes affect N -methyl- d -aspartate (NMDA) receptor, a major pathway implicated in HIV-1-associated neurodegeneration. To test this hypothesis, we studied effects of IL-1β-stimulated astrocyte conditioned medium (ACM+) for its ability to activate NR1a/NR2B receptors expressed on Xenopus oocytes. Astrocytes treated with IL-1β 20 ng/ml for 24 h induced CXCL8, CCL2, MMP1 and MMP7. Pressure ejection of the ACM(+) produced an inward current in NR1a/NR2B-expressing oocytes. The inward current produced by ACM(+) was blocked by NMDA receptor antagonist, APV but not by non-NMDA receptor antagonist, CNQX. These results suggest that IL-1β stimulated astrocytes activate NR1a/NR2B receptors which may have implications in HIV-1-associated neurodegeneration.

In intestinal cells, arginine (Arg) is 1 of the 2 most potent amino acid activators of p70(s6k), a key regulator of 5'- terminal oligopyrimidine mRNA translation, a necessary condition for increased cell migration. To investigate the mechanism of response to Arg, we used the rat crypt cell line cdx2-transformed IEC-6 cells (cdx2-IEC) and measured cell migration, immunocytochemical analysis of p70(s6k) activation in response to Arg, and production of nitric oxide (NO). When treated with Arg, cdx2-IEC increased in phosphorylation on Thr-389 of p70(s6k) (pp70(s6k)) compared with control (P0.01). Phospho-Thr-421/Ser-424-p70(s6k) was located in the nucleus shortly after Arg treatment. Arg enhanced pp70(s6k), cell migration (55% wound coverage), and NO production. In comparison, the branched-chain amino acid leucine (Leu) activated pp70(s6k), was a weaker stimulator of migration (23% coverage), and did not increase NO. A total of 25 micromol/L DETA-NONOate (DETA/NO) did not significantly enhance phosphorylation of p70(s6k) but enhanced the rate of cell migration by approximately 25%. Wound coverage with Leu plus DETA/NO (25 micromol/L) was greater than coverage with DETA/NO alone (P0.01). These and our previous studies lead to a model in which Arg must stimulate both pp70(s6k) (in the nucleus) and NO release to enhance intestinal epithelial cell migration, which may be relevant to diseases that involve intestinal villous injury.

The Spontaneously Epileptic Rat (SER), a double-mutant for tremor and zitter mutations, shows spontaneous occurrences of absence-like and tonic seizures. Several lines of evidence suggest that the combined effect of Aspa and Atrn mutations is the most likely cause of the epileptic phenotype of the SER. To address this issue, we produced a new double-mutant mouse line carrying both homozygous Aspa-knockout and Atrn mg-3J mutant alleles. The Aspa/Atrn double-mutant mice exhibited absence-like and tonic seizures that were characterized by the appearance of 5-7 Hz spike-wave-like complexes and low voltage fast waves on EEGs. These results demonstrate directly that the simultaneous loss of the Aspa and Atrn gene functions causes epileptic seizures in the mouse and suggest that both Aspa and Atrn deficiencies might be responsible for epileptic seizures in the SER.

N-acetylaspartate (NAA) attains one of the highest concentrations of any molecule in the human CNS (1), yet the functions it serves remain controversial. NAA is synthesized from l-aspartate and acetyl CoA in neuronal mitochondria by the enzyme aspartate N-acetyltransferase (Asp-NAT) (EC 2.3.1.17) (2, 3). NAA is found predominantly in neurons, (4) but the catabolic enzyme aspartoacylase (ASPA) is present primarily in oligodendrocytes in the CNS (5). The high concentration of NAA in the CNS and its characteristic peak in water-suppressed proton magnetic resonance spectroscopy (MRS) permits noninvasive determinations of NAA concentrations in the human brain. MRS determinations of NAA levels are commonly used for evaluating the integrity of neurons in a number of neurological disorders, and this method has emerged as a preferred technique for following the clinical course of several CNS pathologies (6–8). MRS studies operate on the assumptions that NAA is synthesized by and accumulated in neurons and that the steady-state NAA levels in the brain can be interpreted as indicating overall neuronal health or integrity (9, 10). Canavan's disease (CD) is a fatal, hereditary leukodystrophy that compromises normal CNS development and is caused by mutations in the gene for the enzyme ASPA (11, 12). ASPA currently is thought to function exclusively to hydrolyze NAA, a neuron-specific amino acid derivative, into l-aspartate and free acetate. However, ASPA is strongly expressed in other tissues, such as kidney, even though the only known substrate, NAA, is present predominantly in the nervous system (13). Despite the established connection between mutations in the gene for ASPA in CD and the lost capacity to deacetylate NAA, the specific connection between ASPA deficiency and the failure of proper CNS development is unclear (14). Further, the precise roles that NAA plays in CNS development and function remain a matter of inquiry. Several hypotheses have been proposed for the roles served by NAA in the nervous system. Because of its high concentration and lack of known actions on neurons or glia, NAA has been proposed to act as an organic osmolyte that removes excess water from neurons by acting as a molecular water pump (15). In this regard, it has been proposed that excess NAA leads to osmotic dysregulation or has other cytotoxic effects that are responsible for the pathology observed in CD patients (16). NAA has also been proposed as a required substrate for the enzymatic synthesis of the neuron-specific dipeptide N-acetylaspartylglutamate (17). There is more compelling evidence that NAA is essential for lipid synthesis and myelination in the CNS, in addition to the well established connection between ASPA mutations and leukodystrophy. In particular, the levels of NAA, ASPA, and Asp-NAT rise with a temporal course similar to those of myelin proteins (18, 19). Further, it has been shown that NAA contributes acetyl groups for the synthesis of lipids, which in turn are incorporated into myelin (20, 21). Finally, radiolabeled NAA is transported down optic nerve axons, and the acetate moiety is incorporated into their ensheathing myelin lipids (22). These and other observations led to our hypothesis that mutations in ASPA result in a deficiency in the supply of NAA-derived acetate, which in turn results in decreased synthesis of myelin-related fatty acids and lipids. Under our hypothesis, it is this lipogenic deficiency that compromises CNS myelination, impairs CNS development, and ultimately results in the white matter degeneration observed in CD (23, 24). In most cell types, such as hepatocytes, the citrate–citrate lyase system provides the acetyl groups for fatty acid synthesis (25). It remains to be demonstrated that the NAA–ASPA system contributes significantly to lipid synthesis in the CNS. We addressed this issue by examining the rate of myelin lipid synthesis during the period of maximal postnatal myelination in ASPA knockout (ASPA–/–) mice, which were developed as a model for CD (26). These mice exhibit pathology similar to that of human CD patients. We also examined acetate levels in the brains of ASPA–/– mice and normal mice, as well as the lipid content in postmortem white matter samples from a 4.75-yearold CD patient compared with the lipid levels in normal human white matter.

Canavan disease (CD) is a neurodegenerative disorder characterized by the spongy degeneration of the white matter of the brain. Aspartoacylase (ASPA) gene mutation resulting enzyme deficiency is the basic cause of CD. Whether the ASPA defect in CD affects the spinal cord has been investigated using the ASPA gene knockout mouse. Luxol fast blue-hematoxylin and eosin staining in the spinal cord of the knockout mouse showed vacuolation in both white matter and gray matter areas of cervical, thoracic, lumbar, and sacral segments of the spinal cord. However, more vacuoles were seen in the gray matter than the white matter of the spinal cord. ASPA activity in the cervical, thoracic, lumbar, and sacrococcygeal regions of the spinal cord was significantly lower in the knockout mouse compared to the wild type. The enzyme defect in the knockout mouse was also confirmed using the Western blot method. These observations suggest that the ASPA gene defect in the mouse leads to spinal cord pathology, and that these changes may be partly involved in the cause of the physiological/behavioral abnormalities seen in the knockout mouse, if documented also in patients with CD.

Phenylketonuria (PKU) is an inborn error of amino acid metabolism caused by phenylalanine hydroxylase (PAH) deficiency. Dietary treatment has been the cornerstone for controlling systemic phenylalanine (Phe) levels in PKU for the past 4 decades. Over the years, it has become clear that blood Phe concentration needs to be controlled for the life of the patient, a difficult task taking into consideration that the diet becomes very difficult to maintain. Therefore alternative models of therapy are being pursued. This review describes the progress made in enzyme replacement therapy for PKU. Two modalities are discussed, PAH and phenylalanine ammonia-lyase PAH. Developing stable and functional forms of both enzymes has proven difficult, but recent success in producing polyethylene glycol-modified forms of active and stable PAH shows promise.

Canavan disease (CD) is an autosomal recessive leukodystrophy caused by deficiency of aspartoacylase (ASPA). Deficiency of ASPA leads to elevation of N-acetyl-L-aspartic acid (NAA) in the brain and urine. To explore the feasibility of gene transfer to replace ASPA in CD, we generated a knockout mouse and constructed an AAV vector that encodes human ASPA cDNA (hASPA) followed by green fluorescent protein (GFP) after an intraribosomal entry site. We injected CD mice with rAAV-hASPA-GFP in the striatum and thalamus or injected rAAV-GFP identically into control animals. Three to five months after the injection, we determined the presence of ASPA in the CD mouse brain by ASPA activity assay, GFP expression, and Western blot analysis. While rAAV-GFP-injected animals displayed undetectable levels of ASPA, all detection methods revealed significant ASPA levels in rAAV-hASPA-GFP-injected CD mice. We evaluated the functional effects of rAAV-hASPA-GFP-mediated ASPA expression by standard histological methods, magnetic resonance spectroscopy (MRS) for in vivo NAA levels, and magnetic resonance imaging of CD mice. rAAV-hASPA-injected animals displayed a remarkable lack of spongiform degeneration in the thalamus. However, pathology in sites unrelated to the injected areas showed no improvement in histopathology. The improvement in thalamic neuropathology was also detectable via in vivo MRI. MRS revealed that in vivo NAA levels were also reduced. These data indicate that rAAV-mediated ASPA delivery may be an interesting avenue for the treatment of CD.