Hirohide Sawada

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.

Identification and characterization of novel mouse and human ADAM33s with potential metalloprotease activity.

The ADAM family of membrane-anchored proteins has a unique domain structure, with each containing a disintegrin and metalloprotease (ADAM) domain. We have isolated mouse and human cDNAs encoding a novel member of the ADAM family. The mouse and human predicted proteins consisted of 797 and 813 amino acids, respectively, and they shared 70% homology of the entire amino acid sequence. The mouse ADAM gene exists at a single gene locus. The human gene was ubiquitously expressed in tissues other than liver, was mapped to human chromosome 20p13, and was found to consist of 22 exons. Both proteins have domain organization identical to that of previously reported members of the ADAM family, and contain the typical zinc-binding consensus sequence (HEXGHXXGXXHD) in their metalloprotease domain and a pattern of cysteine localization (C(x)(3)C(x)(5)C(x)(5)CxC(x)(8)C) in their EGF-like domain that is typical of an EGF-like motif. The human protein shows homology with Xenopus ADAM13 (44%), human ADAM19 (40%), and human ADAM12 (39%). From the results of phylogenic analysis based on primary amino acid sequence and distribution of the mRNA, these novel ADAM genes were thus named ADAM33.

Age-dependent and tissue-specific CAG repeat instability occurs in mouse knock-in for a mutant Huntington's disease gene

Huntingtons disease (HD) is a neurodegenerative disorder characterized by the expansion of CAG repeats in exon 1 of the HD gene. To clarify the instability of expanded CAG repeats in HD patients, an HD model mouse has been generated by gene replacement with human exon 1 of the HD gene with expansion to 77 CAG repeats. Chimeric proteins composed of human mutated exon 1 and mouse huntingtin are expressed ubiquitously in brain and peripheral tissues. One or two CAG repeat expansion was found in litters from paternal transmission, whereas contraction of CAG repeat in litters was observed through maternal transmission. Elderly mice show greater CAG repeat instability than younger mice, and a unique case was observed of an expanded 97 CAG repeat mouse. Somatic CAG repeat instability is particularly pronounced in the liver, kidney, stomach, and brain but not in the cerebellum of 100‐week‐old mice. The same results of expanded CAG repeat instability as observed in this HD model mouse were confirmed in the human brain of HD patients. Glial fibrillary acidic protein (GFAP)‐positive cells have been found to be increased in the substantia nigra (SN), globus pallidus (GP), and striatum (St) in the brains of 40‐week‐old affected mice, although without neuronal cell death. The CAG repeat instability and increase in GFAP‐positive cells in this mouse model appear to mirror the abnormalities in HD patients. The HD model mouse may therefore have advantages for investigations of molecular mechanisms underlying instability of CAG repeats. J. Neurosci. Res. 65:289–297, 2001.

Mutant huntingtin impairs Ku70-mediated DNA repair

Mutant huntingtin prevents interaction of the DNA damage repair complex component Ku70 with damaged DNA, blocking repair of double-strand breaks.

Effects of Aging on Neuroprotective and Neurotoxic Properties of Microglia in Neurodegenerative Diseases

The inflammatory process in the brain, which is accompanied by changes in the levels of proinflammatory cytokines and neurotrophins, along with the presence of activated microglia, has recently gained much attention in neurodegenerative diseases. Activated microglia produce either neuroprotective or neurotoxic factors. Many reports indicate that activated microglia promote degeneration of dopamine (DA) neurons in Parkinson’s disease (PD). On the other hand, there are several lines of evidence that microglia also have a neuroprotective function. Microglia activated with lipopolysaccharide in the nigrostriatum of neonatal mice protect DA neurons against the PD-producing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), whereas activated microglia in aged mice promote DA cell death by MPTP. These results suggest that the function of activated microglia may change in vivo from neuroprotective to neurotoxic during aging as neurodegeneration progresses in the PD brain.

Water-dispersed single-wall carbon nanohorns as drug carriers for local cancer chemotherapy

AIM Functional analyses of water-dispersed carbon nanohorns with antitumor activity were performed to explore their potential as a drug carrier for local cancer chemotherapy. MATERIALS & METHODS Water-dispersed carbon nanohorns were prepared through adsorption of polyethylene glycol-doxorubicin conjugate (PEG-DXR) onto oxidized single-wall carbon nanohorns (oxSWNHs). PEG-DXR-bound oxSWNHs were administered intratumorally to human nonsmall cell lung cancer-cell NCI-H460-bearing mice. RESULTS & DISCUSSION When injected intratumorally, PEG-DXR-bound oxSWNHs caused significant retardation of tumor growth associated with prolonged DXR retention in the tumor. In accordance with this DXR retention, a large number of oxSWNH agglomerates was found in the periphery of the tumor. Histological analyses showed migration of oxSWNHs to the axillary lymph node, which is a major site of breast cancer metastasis near the tumor, possibly by means of interstitial lymphatic-fluid transport. CONCLUSIONS These results suggest that water-dispersed oxSWNHs may thus be useful as a drug carrier for local chemotherapy.

Motor and learning dysfunction during postnatal development in mice defective in dopamine neuronal transmission

Mice lacking expression of tyrosine hydroxylase (TH), the first and rate‐limiting enzyme of the catecholamine biosynthetic pathway, in dopaminergic neuronal cell types were generated by a transgenic rescue approach to clarify the role of dopamine signaling during postnatal development. Introduction of the TH transgene directed by the dopamine β‐hydroxylase gene promoter into TH knockout mice restored noradrenaline and adrenaline synthesis, preventing perinatal lethality and cardiac dysfunction in the knockout mice. Lack of TH expression in the cells that normally express the dopaminergic phenotype resulted in a marked reduction of dopamine accumulation in the tissues, which led to multiple behavioral abnormalities at the juvenile stage. These abnormalities were characterized by a reduction in spontaneous locomotor activity, blockade of methamphetamine‐induced hyperactivity, cataleptic behavior, and defects in active avoidance learning. In contrast, development of the pituitary gland as well as production and secretion of the pituitary peptide hormones dependent on hypothalamic dopaminergic control were normally maintained, despite defective dopamine synthesis. These results demonstrate that dopamine neurotransmission is essential for controlling spontaneous and voluntary movement and associative learning during postnatal development through the nigrostriatal and mesocorticolimbic pathways. J. Neurosci. Res. 54: 450–464, 1998.

Activated microglia affect the nigro-striatal dopamine neurons differently in neonatal and aged mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

Microglia play an important role in the inflammatory process that occurs in Parkinsons disease (PD). Activated microglia produce cytokines and neurotrophins and may have neurotoxic or neurotrophic effects. Because microglia are most proliferative and easily activated during the neonatal period, we examined the effects of neonatal microglia activated with lipopolysaccharide (LPS) on the nigro‐striatal dopamine neurons in mice treated with 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP), in comparison with activated microglia from the aged mice. By MPTP administration to neonatal mice, the number of dopamine neurons in the substantia nigra (SN) was decreased significantly, whereas that in the mice treated with LPS and MPTP was recovered to normal, along with significant microglial activation. Tyrosine hydroxylase (TH) activity, the levels of dopamine and 3,4‐dihydroxyphenylacetic acid (DOPAC), and the levels of pro‐inflammatory cytokines IL‐1β and IL‐6 in the midbrain were elevated in the neonates treated with LPS and MPTP. On the contrary, although the number of dopamine neurons in the 60‐week‐old mice treated with MPTP was also decreased significantly, the microglial activation by LPS treatment caused a further decrease in their number. These results suggest that the activated microglia in neonatal mice are different from those in aged mice, with the former having neurotrophic potential toward the dopamine neurons in the SN, in contrast to the neurotoxic effect of the latter.

Analysis of the Alternative Promoters that Regulate Tissue‐Specific Expression of Human Aromatic l‐Amino Acid Decarboxylase

Abstract: Previously we identified two alternative first exons (exon N1 and exon L1) coding for 5′ untranslated regions of human aromatic l‐amino acid decarboxylase (AADC) and found that their alternative usage produced two types of mRNAs in a tissue‐specific manner. To determine the cis‐acting element regulating the tissue‐specific expression of human AADC, we produced three kinds of transgenic mice harboring 5′ flanking regions of the human AADC gene fused to the bacterial chloramphenicol acetyltransferase (CAT) gene. The transgene termed ACA contained −7.0 kb to −30 bp in exon N1, including the entire exon L1; ACN contained −3.6 kb to −30 bp in exon N1; and ACL contained −2.8 kb to −42 bp in exon L1. The ACA transgenic mice expressed CAT at extremely high levels in peripheral nonneuronal tissues, such as pancreas, liver, kidney, small intestine, and colon, that contained endogenous high AADC activity, whereas CAT immunoreactivity was not detected in either catecholaminergic or serotonergic neurons in the CNS. Thus, it was suggested that the ACA transgene contained the major part of cis‐regulatory elements for the expression of AADC in peripheral nonneuronal tissues. On the other hand, the ACN transgenic mice moderately expressed CAT in various tissues except for the lung and liver, and the ACL transgenic mice showed moderate CAT expression only in the kidney.

Alteration of Catecholamine Phenotype in Transgenic Mice Influences Expression of Adrenergic Receptor Subtypes

Abstract: Agonist‐induced regulation of adrenergic receptors (ARs) has an important role in controlling physiological functions in response to changes in catecholamine stimulation. We previously generated transgenic mice expressing phenylethanolamine N‐methyltransferase (PNMT) under the control of a human dopamine β‐hydroxylase gene promoter to switch catecholamine specificity from the norepinephrine phenotype to the epinephrine phenotype. In the present study, we first examined changes in catecholamine metabolism in peripheral tissues innervated by sympathetic neurons of the transgenic mice. In the transgenic target tissues, a high‐level expression of PNMT led to a dramatic increase in the epinephrine levels, whereas the norepinephrine levels were decreased to 48.6–87.9% of the nontransgenic control levels. Analysis of plasma catecholamines in adrenalectomized mice showed large amounts of epinephrine derived from sympathetic neurons in the transgenic mice. Subsequently, we performed radioligand binding assays with (−)‐[125I]iodocyanopindolol to determine changes in binding sites of β‐AR subtypes. In transgenic mice, the number of β2‐AR binding sites was 56.4–74.9% of their nontransgenic values in the lung, spleen, submaxillary gland, and kidney, whereas the β1‐AR binding sites were regulated in a different fashion among these tissues. Moreover, northern blot analysis of total RNA from the lung tissues showed that down‐regulation of β2 binding sites was accompanied by a significant decrease in steady‐state levels of the receptor mRNA. These results strongly suggest that alteration of catecholamine specificity in the transgenic sympathetic neurons leads to regulated expression of the β‐AR subtypes in their target tissues.