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The author identified the genes and proteins of human enzymes involved in the biosynthesis of catecholamines (dopamine, norepinephrine, epinephrine) and tetrahydrobiopterin (BH4): tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC), dopamine β-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), and GTP cyclohydrolase I (GCH1). In Parkinson's disease (PD), the activities and levels of mRNA and protein of all catecholamine-synthesizing enzymes are decreased, especially in dopamine neurons in the substantia nigra. Hereditary GCH1 deficiency results in reductions in the levels of BH4 and the activities of TH, causing decreases in dopamine levels. Severe deficiencies in GCH1 or TH cause severe decreases in dopamine levels leading to severe neurological symptoms, whereas mild decreases in TH activity in mild GCH1 deficiency or in mild TH deficiency result in only modest reductions in dopamine levels and symptoms of DOPA-responsive dystonia (DRD, Segawa disease) or juvenile Parkinsonism. DRD is a treatable disease and small doses of L-DOPA can halt progression. The death of dopamine neurons in PD in the substantia nigra may be related to (i) inflammatory effect of extra neuronal neuromelanin, (ii) inflammatory cytokines which are produced by activated microglia, (iii) decreased levels of BDNF, and/or (iv) increased levels of apoptosis-related factors. This review also discusses progress in gene therapies for the treatment of PD, and of GCH1, TH and AADC deficiencies, by transfection of TH, AADC, and GCH1 via adeno-associated virus (AAV) vectors., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Since the description of some peculiar symptoms by James Parkinson in 1817, attempts have been made to define its cause or at least to enlighten the pathology of "Parkinson's disease (PD)." The vast majority of PD subtypes and most cases of sporadic PD share Lewy bodies (LBs) as a characteristic pathological hallmark. However, the processes underlying LBs generation and its causal triggers are still unknown. ɑ-Synuclein (ɑ-syn, encoded by the SNCA gene) is a major component of LBs, and SNCA missense mutations or duplications/triplications are causal for rare hereditary forms of PD. Thus, it is imperative to study ɑ-syn protein and its pathology, including oligomerization, fibril formation, aggregation, and spreading mechanisms. Furthermore, there are synergistic effects in the underlying pathogenic mechanisms of PD, and multiple factors-contributing with different ratios-appear to be causal pathological triggers and progression factors. For example, oxidative stress, reduced antioxidative capacity, mitochondrial dysfunction, and proteasomal disturbances have each been suggested to be causal for ɑ-syn fibril formation and aggregation and to contribute to neuroinflammation and neural cell death. Aging is also a major risk factor for PD. Iron, as well as neuromelanin (NM), show age-dependent increases, and iron is significantly increased in the Parkinsonian substantia nigra (SN). Iron-induced pathological mechanisms include changes of the molecular structure of ɑ-syn. However, more recent PD research demonstrates that (i) LBs are detected not only in dopaminergic neurons and glia but in various neurotransmitter systems, (ii) sympathetic nerve fibres degenerate first, and (iii) at least in "brain-first" cases dopaminergic deficiency is evident before pathology induced by iron and NM. These recent findings support that the ɑ-syn/LBs pathology as well as iron- and NM-induced pathology in "brain-first" cases are important facts of PD pathology and via their interaction potentiate the disease process in the SN. As such, multifactorial toxic processes posted on a personal genetic risk are assumed to be causal for the neurodegenerative processes underlying PD. Differences in ratios of multiple factors and their spatiotemporal development, and the fact that common triggers of PD are hard to identify, imply the existence of several phenotypical subtypes, which is supported by arguments from both the "bottom-up/dual-hit" and "brain-first" models. Therapeutic strategies are necessary to avoid single initiation triggers leading to PD., (© 2023. The Author(s).)

The dark pigment neuromelanin (NM) is abundant in cell bodies of dopamine (DA) neurons in the substantia nigra (SN) and norepinephrine (NE) neurons in the locus coeruleus (LC) in the human brain. During the progression of Parkinson's disease (PD), together with the degeneration of the respective catecholamine (CA) neurons, the NM levels in the SN and LC markedly decrease. However, questions remain among others on how NM is associated with PD and how it is synthesized. The biosynthesis pathway of NM in the human brain has been controversial because the presence of tyrosinase in CA neurons in the SN and LC has been elusive. We propose the following NM synthesis pathway in these CA neurons: (1) Tyrosine is converted by tyrosine hydroxylase (TH) to L-3,4-dihydroxyphenylalanine (L-DOPA), which is converted by aromatic L-amino acid decarboxylase to DA, which in LC neurons is converted by dopamine β-hydroxylase to NE; (2) DA or NE is autoxidized to dopamine quinone (DAQ) or norepinephrine quinone (NEQ); and (3) DAQ or NEQ is converted to eumelanic NM (euNM) and pheomelanic NM (pheoNM) in the absence and presence of cysteine, respectively. This process involves proteins as cysteine source and iron. We also discuss whether the NM amounts per neuromelanin-positive (NM + ) CA neuron are higher in PD brain, whether NM quantitatively correlates with neurodegeneration, and whether an active lifestyle may reduce NM formation., (© 2023. The Author(s).)

Psychotherapies aim to relieve patients from mental distress by guiding them toward healthier attitudes and behaviors. Psychotherapies can differ substantially in concepts and approaches. In this review article, we compare the methods and science of three established psychotherapies: Morita Therapy (MT), which is a 100-year-old method established in Japan; Cognitive Behavioral Therapy (CBT), which-worldwide-has become the major psychotherapy; and Acceptance and Commitment Therapy (ACT), which is a relatively young psychotherapy that shares some characteristics with MT. The neuroscience of psychotherapy as a system is only beginning to be understood, but relatively solid scientific information is available about some of its important aspects such as learning, physical health, and social interactions. On average, psychotherapies work best if combined with pharmacotherapies. This synergy may rely on the drugs helping to "kickstart" the use of neural pathways (behaviors) to which a patient otherwise has poor access. Improved behavior, guided by psychotherapy, can then consolidate these pathways by their continued usage throughout a patient's life., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

5'-Nucleotidase domain-containing protein 2 (NT5DC2) has been revealed by genome-wide association studies (GWAS) as a gene implicated in neuropsychiatric disorders related to the abnormality of dopamine (DA) activity in the brain. Based on its amino acid sequence, NT5DC2 is assumed to be a member of the family of haloacid dehalogenase-type phosphatases; although there is no information about its function and structural conformation. We recently reported that NT5DC2 binds to tyrosine hydroxylase (TH) and that the down-regulation of NT5DC2 tended to increase DA synthesis. In this study, we investigated whether NT5DC2 could regulate the catalytic activity of TH, which converts tyrosine to DOPA, because the phosphorylation level of TH, controlled by protein kinases and phosphatases, is well known to regulate its catalytic activity. The down-regulation of NT5DC2 by siRNA increased mainly DOPA synthesis by TH in PC12D cells, although this down-regulation tended to increase the conversion of DOPA to DA by aromatic L-amino acid decarboxylase. The increased DOPA synthesis should be attributed to the catalytic activity of TH controlled by its phosphorylation, because Western blot analysis revealed that the down-regulation of NT5DC2 tended to increase the level of TH phosphorylated at its Ser residues, but not that of the TH protein. Moreover, the induction of kinase activity by forskolin markedly potentiated the phosphorylation of TH at its Ser40 in PC12D cells having down-regulated NT5DC2. Immunocytochemical analysis of PC12D cells demonstrated that NT5DC2, TH protein, and TH phosphorylated at its Ser40 were predominantly localized in the cytoplasm and that the localization of NT5DC2 and TH proteins partially overlapped. Collectively, our results indicate that NT5DC2 could work to inhibit the DOPA synthesis by decreasing the phosphorylation of TH at its Ser40. We propose that NT5DC2 might decrease this phosphorylation of TH by promoting dephosphorylation or by inhibiting kinase activity.

Cognitive behavioral therapy (CBT) for depression and anxiety, established since the 1960s in the USA, and now in Europe, and all over the world has been found to be effective for treating depression in Parkinson's disease (PD). CBT is further developed to acceptance and commitment therapy (ACT) in Europe and the USA. The neural mechanism of CBT or ACT is still under investigation. In Japan, Morita therapy, a psychotherapy founded in 1919 by Masatake (Shoma) Morita, has been used for common mental problems such as anxiety and depression, but rarely for the psychological symptoms in PD. Morita Therapy is in sharp contrast to western CBT in teaching that undesired mental symptoms such as anxiety and depression are natural features of human emotion in health and disease rather than something to control or eliminate, but it is speculated to be similar to ACT in the approach to acceptance but not elimination of mental symptoms. I speculate that the neural basis might be similar in CBT, ACT, and Morita Therapy. In this commentary, a hypothesis is proposed that CBT, ACT, as well as Morita Therapy might be effective for the treatment of the psychological symptoms such as anxiety and depression in PD and in other mental and physical diseases, probably by similar neural mechanisms in the brain.

Parkinson's disease (PD) is an aging-related movement disorder mainly caused by a deficiency of neurotransmitter dopamine (DA) in the striatum of the brain and is considered to be due to progressive degeneration of nigro-striatal DA neurons. Most PD is sporadic without family history (sPD), and there are only a few percent of cases of young-onset familial PD (fPD, PARKs) with the chromosomal locations and the genes identified. Tyrosine hydroxylase (TH), tetrahydrobiopterin (BH4)-dependent and iron-containing monooxygenase, catalyzes the conversion of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA), which is the initial and rate-limiting step in the biosynthesis of catecholamines (DA, noradrenaline, and adrenaline). PD affects specifically TH-containing catecholamine neurons. The most marked neurodegeneration in patients with DA deficiency is observed in the nigro-striatal DA neurons, which contain abundant TH. Accordingly, TH has been speculated to play some important roles in the pathophysiology in PD. However, this decrease in TH is thought to be secondary due to neurodegeneration of DA neurons caused by some as yet unidentified genetic and environmental factors, and thus, TH deficiency may not play a direct role in PD. This manuscript provides an overview of the role of human TH in the pathophysiology of PD, covering the following aspects: (1) structures of the gene and protein of human TH in relation to PD; (2) similarity and dissimilarity between the phenotypes of aging-related sPD and those of young-onset fPD or DOPA-responsive dystonia due to DA deficiency in the striatum with decreased TH activity caused by mutations in either the TH gene or GTP cyclohydrolase I (GCH1) gene; and (3) genetic variants of the TH gene (polymorphisms, rare variants, and mutations) in PD, as discovered recently by advanced genome analysis.

Prolyl oligopeptidase (also named prolyl endopeptidase; PREP) hydrolyzes the Pro-Xaa bonds of biologically active oligopeptides on their carboxyl side. In 1987, we detected PREP activity in human cerebrospinal fluid (CSF) using highly sensitive liquid chromatography-fluorometry with succinyl-Gly-Pro-4-methyl-coumarin amide as a new synthetic substrate, and found a marked decrease in its activity in the cerebrospinal fluid (CSF) from patients with Parkinson's disease (PD) as compared with its level in control patients without neurological diseases. In 2013, Hannula et al. found co-localization of PREP with α-synuclein in the postmortem PD brain. Several recent studies also suggest that the level of PREP in the brain of PD patients may be related to dopamine (DA) cell death via promotion of α-synuclein oligomerization and that inhibitors of PREP may play a neuroprotective role in PD. Although the relationship between another family of prolyl oligopeptidase enzymes, dipeptidyl peptidase II (DPP II) and dipeptidyl peptidase IV (DPP IV), and α-synuclein in the PD brain is not yet clear, we found that the DPP II activity/DPP IV activity ratio in the CSF was significantly increased in PD patients. This review discusses the possibility of PREP as well as the DPP II/DPP IV ratio in the CSF as potential biomarkers of PD.

Tyrosine hydroxylase (TH), which was discovered at the National Institutes of Health (NIH) in 1964, is a tetrahydrobiopterin (BH4)-requiring monooxygenase that catalyzes the first and rate-limiting step in the biosynthesis of catecholamines (CAs), such as dopamine, noradrenaline, and adrenaline. Since deficiencies of dopamine and noradrenaline in the brain stem, caused by neurodegeneration of dopamine and noradrenaline neurons, are mainly related to non-motor and motor symptoms of Parkinson's disease (PD), we have studied human CA-synthesizing enzymes [TH; BH4-related enzymes, especially GTP-cyclohydrolase I (GCH1); aromatic L-amino acid decarboxylase (AADC); dopamine β-hydroxylase (DBH); and phenylethanolamine N-methyltransferase (PNMT)] and their genes in relation to PD in postmortem brains from PD patients, patients with CA-related genetic diseases, mice with genetically engineered CA neurons, and animal models of PD. We purified all human CA-synthesizing enzymes, produced their antibodies for immunohistochemistry and immunoassay, and cloned all human genes, especially the human TH gene and the human gene for GCH1, which synthesizes BH4 as a cofactor of TH. This review discusses the historical overview of TH, BH4-, and other CA-related enzymes and their genes in relation to the pathophysiology of PD, the development of drugs, such as L-DOPA, and future prospects for drug and gene therapy for PD, especially the potential of induced pluripotent stem (iPS) cells.

We previously showed that aripiprazole increases intracellular NADPH and glucose-6-phosphate dehydrogenase mRNA in PC12 cells. Aripiprazole presumably activates a system that concurrently detoxifies reactive oxygen species and replenishes NADPH. Nrf2, a master transcriptional regulator of redox homeostasis genes, also activates the pentose phosphate pathway, including NADPH production. Therefore, our aim was to determine whether aripiprazole activates Nrf2 in PC12 cells. Aripiprazole increased mRNA expression of Nrf2-dependent genes (NAD(P)H-quinone oxidoreductase-1, Nqo1; heme oxygenase-1, HO1; and glutamate-cysteine ligase catalytic subunit) and protein expression of Nqo1 and HO1 in these cells (p < 0.05). To maintain increased Nrf2 activity, it is necessary to inhibit Nrf2 degradation; this is done by causing Nrf2 to dissociate from Keap1 or β-TrCP. However, in aripiprazole-treated cells, the relative amount of Nrf2 anchored to Keap1 or β-TrCP was unaffected and Nrf2 in the nuclear fraction decreased (p < 0.05). Aripiprazole did not affect phosphorylation of Nrf2 at Ser40 and decreased the relative amount of acetylated Nrf2 (p < 0.05). The increase in Nqo1 and HO1 in aripiprazole-treated cells cannot be explained by the canonical Nrf2-degrading pathways. Further experiments are needed to determine the biochemical mechanisms underlying the aripiprazole-induced increase in these enzymes.

We previously reported that an optimal dose of lipopolysaccharide (LPS) markedly extends the lifespan of murine primary-cultured microglia by suppressing cell death pathways. In this study, we investigated the effects of LPS pretreatment on UV light-induced apoptosis of cells from the microglial cell line BV-2. More than half of BV-2 cells were apoptotic, and procaspase-3 was cleaved into its active form at 3 h of UV irradiation. In contrast, in BV-2 cells treated with LPS for 24 h, UV irradiation caused neither apoptosis nor procaspase-3 cleavage. LPS treatment arrested the cell cycle in G1 phase and upregulated cyclin-dependent kinase inhibitor p21(Waf1/Cip1) and growth arrest and DNA damage-inducible (GADD) 45α in BV-2 cells. When p21(Waf1/Cip1) and GADD45α were knocked down by small interfering RNA, procaspase-3 was cleaved into its active form to induce apoptosis. Our findings suggest that LPS inhibits UV-induced apoptosis in BV-2 cells through arrest of the cell cycle in G1 phase by upregulation of p21(Waf1/Cip1) and GADD45α. Excessive activation of microglia may play a critical role in the exacerbation of neurodegeneration, therefore, normalizing the precise regulation of apoptosis may be a new strategy to prevent the deterioration caused by neurodegenerative disorders.

In aripiprazole-treated PC12 cells, we previously showed that the mitochondrial membrane potential (Δψm) was rather increased in spite of lowered cytochrome c oxidase activity. To address these inconsistent results, we focused the NADPH generation by glucose-6-phosphate dehydrogenase (G6PD), a rate-limiting enzyme of the pentose phosphate pathway (PPP), to titrate reactive oxygen species (ROS) that results in the Δψm maintenance. G6PD may be also involved in another inconsistent result of lowered intracellular lactate level in aripiprazole-treated PC12 cells, because PPP competes glucose-6-phosphate with the glycolytic pathway, resulting in the downregulation of glycolysis. Therefore, we assayed intracellular amounts of NADPH, ROS, and the activities of the enzymes generating or consuming NADPH (G6PD, NADP(+)-dependent isocitrate dehydrogenase, NADP(+)-dependent malic enzyme, glutathione reductase, and NADPH oxidase [NOX]) and estimated glycolysis in 50 μM aripiprazole-, clozapine-, and haloperidol-treated PC12 cells. NADPH levels were enhanced only in aripiprazole-treated ones. Only haloperidol increased ROS. However, the enzyme activities did not show significant changes toward enhancing NADPH level except for the aripiprazole-induced decrease in NOX activity. Thus, the lowered NOX activity could have contributed to the aripiprazole-induced increase in the NADPH level by lowering ROS generation, resulting in maintained Δψm. Although the aforementioned assumption was invalid, the ratio of fructose-1,6-bisphosphate to fructose-6-phosphate was decreased by all antipsychotics examined. Pyruvate kinase activity was enhanced only by aripiprazole. In summary, these observations indicate that aripiprazole possibly possesses the pharmacological superiority to clozapine and haloperidol in the ROS generation and the adjustment of glycolytic pathway.

Postmortem brain biochemistry has revealed that the main symptom of movement disorder in Parkinson's disease (PD) is caused by a deficiency in dopamine (DA) at the nerve terminals of degenerating nigro-striatal DA neurons in the striatum. Since tyrosine hydroxylase (TH) is the rate-limiting enzyme for the biosynthesis of DA, TH may play an important role in the disease process of PD. DA regulated by TH activity is thought to interact with α-synuclein protein, which results in intracellular aggregates called Lewy bodies and causes apoptotic cell death during the aging process. Human TH has several isoforms produced by alternative mRNA splicing, which may affect activation by phosphorylation of serine residues in the N-terminus of TH. The activity and protein level of TH are decreased to cause DA deficiency in the striatum in PD. However, the homo-specific activity (activity/enzyme protein) of TH is increased. This increase in TH homo-specific activity suggests activation by increased phosphorylation at the N-terminus of the TH protein for a compensatory increase in DA synthesis. We recently found that phosphorylation of the N-terminal portion of TH triggers proteasomal degradation of the enzyme to increase TH turnover. We propose a hypothesis that this compensatory activation of TH by phosphorylation in the remaining DA neurons may contribute to a further decrease in TH protein and activity in DA neurons in PD, causing a vicious circle of decreasing TH activity, protein level and DA contents. Furthermore, increased TH homo-specific activity leading to an increase in DA may cause toxic reactive oxygen species in the neurons to promote neurodegeneration.

Aripiprazole is the only atypical antipsychotic drug known to cause the phosphorylation of AMP-activated protein kinase (AMPK) in PC12 cells. However, the molecular mechanisms underlying this phosphorylation in aripiprazole-treated PC12 cells have not yet been clarified. Here, using PC12 cells, we show that these cells incubated for 24 h with aripiprazole at 50 μM and 25 mM glucose underwent a decrease in their NAD⁺/NADH ratio. Aripiprazole suppressed cytochrome c oxidase (COX) activity but enhanced the activities of pyruvate dehydrogenase (PDH), citrate synthase and Complex I. The changes in enzyme activities coincided well with those in NADH, NAD⁺, and NAD⁺/NADH ratio. However, the bioenergetic peril judged by the lowered COX activity might not be accompanied by excessive occurrence of apoptotic cell death in aripiprazole-treated cells, because the mitochondrial membrane potential was not decreased, but rather increased. On the other hand, when PC12 cells were incubated for 24 h with clozapine at 50 μM and 25 mM glucose, the NAD⁺/NADH ratio did not change. Also, the COX activity was decreased; and the PDH activity was enhanced. These results suggest that aripiprazole-treated PC12 cells responded to the bioenergetic peril more effectively than the clozapine-treated ones to return the ATP biosynthesis back toward its ordinary level. This finding might be related to the fact that aripiprazole alone causes phosphorylation of AMPK in PC12 cells.

By converting changes in intracellular energy status to changes in cell membrane polarization, ATP-sensitive K(+) (K(ATP)) channels in hypothalamic appetite-regulating neurons play a critical role in linking neuronal electrochemical function, metabolic and energy status, and feeding behavior. Most atypical antipsychotics (AAPs) increase the appetite of patients with schizophrenia and thus cause obesity. This study aimed to explain the mechanism underlying AAP-induced appetite stimulation, based on the fact that the efficiency of fatty acid uptake into mitochondria generating ATP through β-oxidation is determined by the rate of fatty acid synthesis. Using PC12 cells exposed to clozapine, olanzapine, risperidone, quetiapine, ziprasidone, aripiprazole, and haloperidol, we measured intracellular ATP and mRNA and protein expression of enzymes and related substances involved in fatty acid synthesis and K(ATP) channel function. Forty-eight-hour treatment of cells with 50 μM aripiprazole in 5.6 mM glucose decreased intracellular ATP. Only 50 μM aripiprazole phosphorylated AMP-activated protein kinase (AMPK); none of the other antipsychotics did so to a detectable level. Expression of carnitine palmitoyltransferase 1a, uncoupling protein 2, and sulfonylurea receptor 1 was unaffected by the antipsychotics, although expression of their mRNA was affected by AAPs. Pyrilamine (H(1) receptor antagonist), ketanserin (5HT(2) receptor antagonist), and raclopride (D(2) receptor antagonist) alone or in combination had no effect on expression of the aforementioned proteins. Therefore, although this study did not differentiate orexigenic and non-orexigenic AAPs, it suggests that aripiprazole is unique in its ability to activate AMPK.

Tyrosine hydroxylase (TH) catalyzes the conversion of L: -tyrosine to L: -dopa, which is the initial and rate-limiting step in the biosynthesis of catecholamines [CA; dopamine (DA), noradrenaline, and adrenaline], and plays a central role in the neurotransmission and hormonal actions of CA. Thus, TH is related to various neuro-psychiatric diseases such as TH deficiency, Parkinson's disease (PD), and schizophrenia. Four isoforms of human TH (hTH1-hTH4) are produced from a single gene by alternative mRNA splicing in the N-terminal region, whereas two isoforms exist in monkeys and only a single protein exist in all non-primate mammals. A catalytic domain is located within the C-terminal two-thirds of molecule, whereas the part of the enzyme controlling enzyme activity is assigned to the N-terminal end as the regulatory domain. The catalytic activity of TH is end product inhibited by CA, and the phosphorylation of Ser residues (Ser(19), Ser(31), and especially Ser(40) of hTH1) in the N-terminus relieves the CA-mediated inhibition. Ota and Nakashima et al. have investigated the role of the N-terminus of TH enzyme in the regulation of both the catalytic activity and the intracellular stability by producing various mutants of the N-terminus of hTH1. The expression of the following three enzymes, TH, GTP cyclohydrolase I, which synthesizes the tetrahydrobiopterin cofactor of TH, and aromatic-L: -amino acid decarboxylase, which produces DA from L: -dopa, were induced in the monkey striatum using harmless adeno-associated virus vectors, resulting in a remarkable improvement in the symptoms affecting PD model monkeys Muramatsu (Hum Gene Ther 13:345-354, 2002). Increased knowledge concerning the amino acid sequences of the N-terminus of TH that control enzyme activity and stability will extend the spectrum of the gene-therapy approach for PD.

We previously reported that nicotine withdrawal up-regulates transcription of some immediately early genes (IEGs), c-fos (Ichino et al., 1999) and egr1, nur77 (Ichino et al., 2002) in cultures of pheochromocytoma PC12 cells, which are of neuronal lineage. In the present study we aimed at further elucidating the effects of nicotine withdrawal on the expression of the genes downstream of IEGs. We examined the changes in the protein levels of 2 GTP-binding proteins, Rab2 (Ras-related protein) and Rac1. PC12 cells were cultured in the presence of nicotine for 24 hours, and then the nicotine was removed from the medium. The protein level of Rab2 was low in the presence of nicotine, but was rapidly increased after nicotine withdrawal. In contrast, that of Rac1 did not change after the withdrawal. Considering the neuroprotective effect of nicotine, we also examined the level of Bag-1 protein, which is a binding protein for Bcl-2, an anti-apoptotic factor, and found a slight increase in the gene expression of Bag-1 following nicotine withdrawal. Among 56-kDa, 50-kDa, and 36-kDa protein components of the Bag-1 protein complex, the levels of 56-kDa and 50-kDa proteins were not changed by the addition or withdrawal of nicotine; but the level of the 36-kDa protein, which had been increased in the presence of nicotine, was markedly decreased after nicotine withdrawal. The present results suggest that such changes may also occur in individuals during abstaining from smoking and be related to the withdrawal symptoms experienced after smokers stop smoking.

The influence of nicotine on the expression of egr-1 and nur77 genes by nicotine treatment and withdrawal was assessed using PC12 cells. Nicotine treatment significantly increased the amount of mRNA for egr-1 and nur77 genes at 0.5 h post-nicotine treatment in the PC12 cells. In addition, nicotine withdrawal also elevated transcriptional levels of egr-1 and nur77 genes in Northern blot analyses. Nicotine treatment (200 microM) was also found to significantly increase expressional levels of Egr-1 and Nur77 proteins at 0.5 h post-nicotine treatment. In contrast, Egr-1 and Nur77 protein levels were dramatically decreased by nicotine withdrawal. These results suggest that expressional levels of Egr-1 and Nur77 proteins in neural cells may affect the transcriptional activity of late-response genes after nicotine withdrawal.

Specific degeneration of the nigrostriatal dopamine (DA) neurons of the substantia nigra pars compacta and the resulting loss of nerve terminals accompanied by DA deficiency in the striatum are responsible for most of the movement disturbances called parkinsonism, i.e., muscle rigidity, akinesia, and resting tremor, observed in Parkinson's disease (PD). We and other workers have found changes in the levels of cytokines, neurotrophins, and other apoptosis-related factors in the nigro-striatal region of postmortem brain and/or in the cerebrospinal fluid (CSF) from PD patients, or from animal models of PD such as 1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine (MPTP)-induced PD in mice or 6-hydroxydopamine (6-OHDA)-induced PD in rats. The most remarkable changes observed specifically in the nigrostriatal region were decreased levels of neurotrophins supporting DA neurons. These results indicate that the process of cell death in the nigrostriatal DA neurons in PD may be the so-called programmed cell death, i.e., apoptosis. Thus gene therapy for PD should aim both at supplementing the decreased striatal DA level by introducing the genes of DA-synthesizing enzymes into non-DA cells in the striatum and at supporting or restoring DA neurons by preventing apoptosis by introducing genes that block the process of apoptosis.

The activities of caspase-1 and caspase-3 were measured by use of fluoropeptides as substrates for the first time in the brain (substantia nigra, caudate nucleus, putamen, cerebellum, and frontal cortex) from control and parkinsonian patients. The activities of caspases in the brain were significantly higher in the substantia nigra from parkinsonian patients than those in the brain from control patients (p < 0.01). However, the activities of caspases in the caudate nucleus, putamen, cerebellum, and frontal cortex showed no significant difference between parkinsonian and control patients. The tumor necrosis factor (TNF) receptor R1 (TNF-R1, p55) level was also elevated in the substantia nigra of the parkinsonian brain in comparison with that of controls (p < 0.05). Since both caspases and TNF-R1 may play important roles in apoptotic cell death through TNF-alpha-induced signaling pathway, our present data suggest the presence of a proapoptotic environment in the substantia nigra of parkinsonian brain, probably inducing vulnerability of neurons and glias towards a variety of noxious factors.

Wild-type and N-terminal 35-, 38-, and 44-amino acid-deleted mutants of human tyrosine hydroxylase type 1 (hTH1) fused to maltose-binding protein via the target sequence for a restriction protease were expressed in Escherichia coli and purified. The fused protein was treated with the restriction protease factor Xa or enterokinase to isolate hTH1 from the fused form. The treatment of fused wild-type and 35-amino acid-deleted mutant with factor Xa and enterokinase caused non-specific cleavages in the vicinity of the phosphorylation sites, Ser19 and Ser40, due to the flexible conformation of the N-terminus of hTH1.

Guanosine triphosphate (GTP) cyclohydrolase I (GCH) is the first and rate-limiting enzyme for biosynthesis of tetrahydrobiopterin, the cofactor of tyrosine hydroxylase (TH). Our previous study reported the presence of GCH in several neuronal groups in animal brains using a newly raised anti-GCH antibody. The present study aims at elucidating whether GCH and TH coexist in the same neurons of the human brain with the aid of immunohistochemical dual labeling. GCH-immunoreactivity was observed in the cell bodies and fibers of monoaminergic neurons of the human brain. Neurons which contain both enzymes are seen in the human substantia nigra, ventral tegmental area, locus coeruleus, dorsal raphe, and zona incerta. In these regions, almost all the cells also show immunoreactivity for aromatic L-amino acid decarboxylase (AADC), the second step enzyme for catecholamine synthesis, indicating that these neurons are catecholaminergic. However, some neurons in the dorsal and dorsomedial hypothalamic nuclei are stained only for GCH or TH. They appear to constitute an independent cell group in the human brain. The present observation suggests that L-dopa is not produced in the cells immunoreactive for TH but not for GCH, and that TH in these cells which lack GCH may have an unidentified role other than dopa synthesis.

The catecholaminergic neurons of senescence-accelerated mice (SAM-P8) were analyzed by immunohistochemical microphotometry in terms of immunoreactivities to aromatic L-amino acid decarboxylase (AADC), dopamine (DA), or noradrenaline (NA). Accelerated senescence-resistant mice (SAM-R1) were used as control mice. The immunoreactivities to AADC, DA, and NA of the catecholaminergic neurons of the SAM-P8 mice were weaker than those of the SAM-R1 mice in all the brain regions. Immunoelectron microscopy revealed progressive degeneration of dopaminergic neurons and their terminal fibers in the substantia nigra as well as in noradrenergic neurons and their proximal dendrites in the locus coeruleus of the SAM-P8 mice. In contrast, there was no difference between the SAM-P8 and SAM-R1 mice in the distribution of AADC-only positive neurons (designated as D neurons in the rat brain by Jaeger et al.) nor in their immunoreactivities. These results may indicate that DA neurons in the substantia nigra and NA neurons in the locus coeruleus degenarate more rapidly during aging in SAM-P8 mice than in control SAM-R1 mice and that D neurons may function as a part of a compensatory system for the decreases in catecholaminergic neurons during aging.

The human aromatic L-amino acid decarboxylase (AADC) gene is transcribed in a tissue-specific manner by an alternative promoter. In this study using human cultured cell lines, we analyzed the alternative promoter that regulates tissue-specific expression of AADC. Neither neuronal nor nonneuronal-type mRNA of AADC was detected in HeLa cells, nonneuronal-type mRNA of AADC was expressed in HepG2 cells, and the neuronal-type was expressed in the SK-N-SH cell line. We examined the promoter activities located in 5'- and 3'-flanking regions of exon N1 and exon L1 by transfection experiments. Plasmids containing 5'-flanking regions of exon L1, the shortest of which was 0.3 kb, could promote specifically high expression of the reporter gene HepG2 cells. On the other hand, plasmids containing 5'-flanking regions of exon N1 (3.6 kb to 0.5 kb) could promote the reporter gene expression not only in SK-N-SH cells but also in HeLa and HepG2. More enhanced expression were observed by transfection of plasmids containing parts of the first intron in these cell lines. Thus, these results suggest that the basal liver-specific promoter activity is located in the 5'-flanking region of exon L1 and that the first intron may also be needed for enhanced expression rather than determination of cell-specificity.

The N-terminal 52-, 70-, and 157-amino acids-deleted mutants and wild-type tyrosine hydroxylases were expressed in Escherichia coli and utilized to investigate the roles of the N-terminus in the catecholamine inhibition on enzyme activity. Their lysate's supernatants were used as enzyme samples. Three catecholamines, namely dopamine, norepinephrine, and epinephrine, affected both wild-type and mutant enzymes after preincubation in the mode of mixed inhibition, and the most marked alteration among the kinetic parameters produced by the deletion was the increase in the inhibition constants. The deletions also abolished the catecholamine-induced shift of the pH profile of the enzyme activity toward a more acidic pH optimum. All three mutants responded to catecholamines almost in the same way. These results suggest that the three catecholamine end products exert their inhibition on tyrosine hydroxylase to the same extent and that the N-terminal 52 amino acid residues contain the key sequence in mediating the inhibitory action.

The contents of interleukin (IL)-2 and basic fibroblast growth factor (bFGF) were measured in the brain (caudate nucleus, putamen, and cerebral cortex) from control and parkinsonian patients by highly sensitive enzyme-linked immunosorbent assay (ELISA). The concentrations of IL-2 in the brain were in the order of pg/mg protein, and the values were significantly higher in the caudate and putamen from parkinsonian patients than those from control patients. However, the levels of IL-2 in the cerebral cortex showed no significant difference between parkinsonian and control patients. In contrast to IL-2, the bFGF levels in the brain were high and in the order of ng/mg protein, and there was no significant difference in the caudate and putamen between parkinsonian and control patients. Although both IL-2 and bFGF may play important roles in dopaminergic neurons as neurotrophic factors, IL-2 but not bFGF may relate to the compensatory response in the nigrostriatal dopaminergic regions in parkinsonian brain during progress of neurodegeneration.