Ikuko Nagatsu

Use of high concentrations of glutaraldehyde for immunocytochemistry of transmitter-synthesizing enzymes in the central nervous system

Aldehyde fixatives containing high concentrations of glutaraldehyde, usually used for conventional electron microscope studies, were successfully used for immunocytochemistry of transmitter synthesizing enzymes, glutamate decarboxylase and tyrosine hydroxylase, in the rat central nervous system. Although a high concentration of glutaraldehyde could cause tremendous non-specific staining, this was almost completely absent after treating sections with 1% sodium borohydride for 30 min. Furthermore, it was shown that a high concentration of glutaraldehyde might cause no appreciable reduction of the antigenicities of glutamate decarboxylase and tyrosine hydroxylase when compared with fixatives containing a low concentration of glutaraldehyde. It is suggested that fixatives containing high concentrations of glutaraldehyde are very useful, not only for conventional electron microscope studies, but also for light and electron microscope immunocytochemistry of some antigens, including glutamate decarboxylase and tyrosine hydroxylase.

Targeted Disruption of the Tyrosine Hydroxylase Locus Results in Severe Catecholamine Depletion and Perinatal Lethality in Mice

Tyrosine 3-hydroxylase (TH, EC 1.14.16.2) catalyzes the first and rate-limiting step of the catecholamine biosynthetic pathway in the nervous and endocrine systems. The TH locus was disrupted in mouse embryonic stem cells by homologous recombination. Mice heterozygous for the TH mutation were apparently normal. In these mice, TH activity in the embryos and adult tissues was less than 50% of the wild-type values, but the catecholamine level was decreased only moderately in the developing animals and was maintained normally at adulthood, suggesting the presence of a regulatory mechanism for ensuring the proper catecholamine level during animal development. In contrast, the homozygous mutant mice died at a late stage of embryonic development or shortly after birth. Both TH mRNA and enzyme activity were lacking in the homozygous mutants, which thus explained the severe depletion of catecholamines. These changes, however, did not affect gross morphological development of the cells that normally express high catecholamine levels. Analysis of electrocardiograms of surviving newborn mutants showed bradycardia, suggesting an alteration of cardiac functions in the homozygous mice that may lead to the lethality of this mutation. In addition, transfer of a human TH transgene into the homozygous mice corrected the mutant phenotype, showing recovery of TH activity by expression of the human enzyme. These results indicate that TH is essential for survival of the animals during the late gestational development and after birth.

Triple Transduction with Adeno-Associated Virus Vectors Expressing Tyrosine Hydroxylase, Aromatic-L-Amino-Acid Decarboxylase, and GTP Cyclohydrolase I for Gene Therapy of Parkinson's Disease

Parkinsons disease (PD), a neurological disease suited to gene therapy, is biochemically characterized by a severe decrease in the dopamine content of the striatum. One current strategy for gene therapy of PD involves local production of dopamine in the striatum achieved by inducing the expression of enzymes involved in the biosynthetic pathway for dopamine. We previously showed that the coexpression of tyrosine hydroxylase (TH) and aromatic-L-amino-acid decarboxylase (AADC), using two separate adeno-associated virus (AAV) vectors, resulted in more effective dopamine production and more remarkable behavioral recovery in 6-hydroxydopamine-lesioned parkinsonian rats, compared with the expression of TH alone. Not only levels of TH and AADC but also levels of tetrahydrobiopterin (BH4), a cofactor of TH, and GTP cyclohydrolase I (GCH), a rate-limiting enzymes for BH4 biosynthesis, are reduced in parkinsonian striatum. In the present study, we investigated whether transduction with separate AAV vectors expressing TH, AADC, and GCH was effective for gene therapy of PD. In vitro experiments showed that triple transduction with AAV-TH, AAV-AADC, and AAV-GCH resulted in greater dopamine production than double transduction with AAV-TH and AAV-AADC in 293 cells. Furthermore, triple transduction enhanced BH4 and dopamine production in denervated striatum of parkinsonian rats and improved the rotational behavior of the rats more efficiently than did double transduction. Behavioral recovery persisted for at least 12 months after stereotaxic intrastriatal injection. These results suggest that GCH, in addition to TH and AADC, is important for effective gene therapy of PD.

Coexistence of immunoreactivities for glutamate decar☐ylase and tyrosine hydroxylase in some neurons in the periglomerular region of the rat main olfactory bulb: possible coexistence of gamma-aminobutyric acid (GABA) and dopamine

The coexistence of immunoreactivities for glutamate decarboxylase (GAD) and tyrosine hydroxylase (TH) was revealed in some neurons in the periglomerular region and in the superficial part of the external plexiform layer of the rat main olfactory bulb. In neurons showing the immunoreactivity for either GAD or TH, about 10-55% showed both immunoreactivities.

L-DOPA is converted to dopamine in serotonergic fibers of the striatum of the rat: a double-labeling immunofluorescence study.

The aim of the present study is to examine whether serotonergic terminals of the rat brain are induced to contain dopamine by L-DOPA administration. In rats that received intraperitoneally L-DOPA plus a peripheral decarboxylase inhibitor, we showed by use of a double-labeling immunofluorescence method that dopamine was localized in serotonergic fibers of the striatum and cerebral cortex as well as in serotonergic cell bodies of the midbrain raphe nuclei. In normal rats, no dopamine was detected in the serotonergic fibers or cell bodies. The finding suggests that a part of the administered L-DOPA may enter the serotonergic terminals and be converted to dopamine in the rat brain.

Immunohistochemical evidence that central serotonin neurons produce dopamine from exogenousl-DOPA in the rat, with reference to the involvement of aromaticl-amino acid decar☐ylase

The aim of the present study is to examine whether aromatic L-amino acid decarboxylase (AADC) catalyzes the conversion of exogenous L-3,4-dihydroxyphenylalanine (L-DOPA) to dopamine in serotonin neurons of the rat dorsal raphe nucleus. First, in order to confirm the localization of AADC in central serotonin neurons, we used an immunoperoxidase method for AADC and demonstrated that the distribution of AADC-containing neurons in the dorsal raphe nucleus corresponds very closely to the previous description on the distribution of serotonin-immunoreactive neurons. Second, in the rat that received intraperitoneally L-DOPA plus a peripheral AADC inhibitor, we used a double-labeling immunofluorescence method and showed that serotonin-stained neurons of the dorsal raphe nucleus were also immunoreactive to dopamine. The present result suggests that AADC decarboxylating L-5-hydroxytryptophan to serotonin in physiological conditions is also able to catalyze the in vivo decarboxylation of exogenous L-DOPA.

Reduced mastication stimulates impairment of spatial memory and degeneration of hippocampal neurons in aged SAMP8 mice

The involvement of reduced mastication in senile dementia was evaluated by examining the effect of cutting off the upper molars (molarless) on spatial memory and numbers of hippocampal neurons in aged SAMP8 mice. Molarless mice showed a decrease in both learning ability in a water maze and neuron density in the hippocampal CA1 region compared with control mice. These changes increased the longer the molarless condition persisted. The data suggest a possible link between reduced mastication and hippocampal neuron loss that may be one risk factor for senile impairment of spatial memory.

Tyrosine hydroxylase-like immunoreactive neurons in the striatum of the rat.

Striatal neurons exhibiting tyrosine hydroxylase-like immunoreactivity (TH-LI) were found in the adult rat. Most of these neurons had a cell body with a 10-20 microns diameter and several spiny dendrites. The number of striatal neurons with TH-LI was increased on the side ipsilateral to an electrothermic or 6-hydroxydopamine-induced lesion which had been placed in the regions around the substantia nigra.

Successive cleavage of N-terminal Arg1--Pro2 and Lys3-Pro4 from substance P but no release of Arg1-Pro2 from bradykinin, by X-Pro dipeptidyl-aminopeptidase.

X-Pro dipeptidyl-aminopeptidase (EC 3.4.14.1) purified homogeneously from the human submaxillary gland was proved to hydrolyze N-terminal dipeptide Arg1-Pro2 and subsequent dipeptide Lys3-Pro4 from substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-gly-Leu-Met-NH2). Km and V values of hydrolysis of substance P were 2.0 mM and 3.6 mumol/min per mg protein, respectively. In contrast, the N-terminal Arg-Pro of bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) was not cleaved by the enzyme.

Biopterin in human brain and urine from controls and parkinsonian patients: Application of a new radioimmunoassay

Total biopterin concentrations in the post-mortem human brain (caudate nucleus) and in the urine of controls and parkinsonian patients were measured by a newly developed radioimmunoassay. There was good correlation between the total biopterin level and tyrosine hydroxylase activity in the human brain. Biopterin concentrations in the caudate nucleus were greatly reduced in parkinsonian patients. In contrast, the reduction of urinary biopterin in parkinsonian patients was slight and not statistically significant, as compared with normal controls.