Catecholamines and Parkinson's disease: tyrosine hydroxylase (TH) over tetrahydrobiopterin (BH4) and GTP cyclohydrolase I (GCH1) to cytokines, neuromelanin, and gene therapy: a historical overview.

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 ß-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.

Lewy bodies, iron, inflammation and neuromelanin: pathological aspects underlying Parkinson's disease.

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.

The role of tyrosine hydroxylase as a key player in neuromelanin synthesis and the association of neuromelanin with Parkinson's disease.

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 ß-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.

Remembering Keisuke Fujita, M.D., Ph.D., President and Founder of Fujita Health University, and his contributions to medical science and education.

Keisuke Fujita, M.D., Ph.D. (1925-1995), founded and was President of Fujita Gakuen (academy) in 1964, Fujita Health University in 1968, and Fujita Health University (FHU) School of Medicine in 1972. He also established the Institute for Comprehensive Medical Science (ICMS) at FHU in 1972, at the same time as the founding of FHU School of Medicine, to promote the institutional development of FHU School of Medicine by providing a strong foundation for science. I collaborated with Dr. Fujita from 1965. Returning from the study abroad at the NIH in the USA in 1965, I joined Professor Fujita's Department of Biochemistry at the Aichi Gakuin University School of Dentistry, as an Associate Professor. Dr. Fujita's major research interest was in the biochemistry of diseases, namely, cancer, neuropsychiatric diseases, and various intractable diseases, which he investigated by applying analytical chemistry and molecular/cellular biochemistry. He was also interested in the pharmacognosy of aloe plants and established "Syoyaku Kenkyu Juku (Center for Pharmacognosy)," and where he studied by himself and trained many FHU graduates. I herein present an overview of the research carried out by Dr. Fujita to share his legacy and praise his memoir and contributions to medical science and education for all faculty members, staffs and students of FHU. It is assumed that individuals at FHU have already made significant contributions to medical science. I hope that his vision of FHU producing a Nobel Prize laureate will be realized someday.

Identification by nano-LC-MS/MS of NT5DC2 as a protein binding to tyrosine hydroxylase: Down-regulation of NT5DC2 by siRNA increases catecholamine synthesis in PC12D cells.

Tyrosine hydroxylase (TH), which catalyzes the conversion of l-tyrosine to l-DOPA, is the rate-limiting enzyme in the biosynthesis of catecholamines. It is well known that both α-synuclein and 14-3-3 protein family members bind to the TH molecule and regulate phosphorylation of its N-terminus by kinases to control the catalytic activity. In this present study we investigated whether other proteins aside from these 2 proteins might also bind to TH molecules. Nano-LC-MS/MS analysis revealed that 5'-nucleotidase domain-containing protein 2 (NT5DC2), belonging to a family of haloacid dehalogenase-type (HAD) phosphatases, was detected in the immunoprecipitate of PC12D cell lysates that had been reacted with Dynabeads protein G-anti-TH antibody conjugate. Surprisingly, NT5DC2 had already been revealed by Genome-Wide Association Studies (GWAS) as a gene implicated in neuropsychiatric disorders such as schizophrenia, bipolar disorder, which are diseases related to the abnormality of dopamine activity in the brain, although the role that NT5DC2 plays in these diseases remains unknown. Therefore, we investigated the effect of NT5DC2 on the TH molecule. The down-regulation of NT5DC2 by siRNA increased the synthesis of catecholamines (dopamine, noradrenaline, and adrenaline) in PC12D cells. These increases might be attributed to the catalytic activity of TH and not to the intracellular stability of TH, because the intracellular content of TH assessed by Western blotting was not changed by the down-regulation of NT5DC2. Collectively, our results indicate that NT5DC2 inhibited the synthesis of dopamine by decreasing the enzymatic activity of TH.

Prolyl oligopeptidase and dipeptidyl peptidase II/dipeptidyl peptidase IV ratio in the cerebrospinal fluid in Parkinson's disease: historical overview and future prospects.

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.

Inhibition of deubiquitinating activity of USP14 decreases tyrosine hydroxylase phosphorylated at Ser19 in PC12D cells.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis, and its stability is a fundamental factor to maintain the level of the catecholamines in cells. However, the intracellular stability determined by the degradation pathway remains unknown. In this study, we investigated the mechanism by which phosphorylation of TH affected the proteasome pathway. The inhibition of proteasomes by MG-132 increased the percentage of TH molecules phosphorylated at their Ser19, Ser31 and/or Ser40 among the total TH proteins to about 70% in PC12D cells over a 24-hr period; although the percentage of phosphorylated TH molecules was about 20% under basal conditions. Moreover, the inhibition of proteasomes by epoxomicin with high specificity increased primarily the quantity of TH molecules phosphorylated at their Ser19. The phosphorylation of Ser19 potentiated Ser40 phosphorylation in cells by a process known as hierarchical phosphorylation. Therefore, the proteasome inhibition might result in an increase in the levels of all 3 phosphorylated TH forms, thus complicating interpretation of data. Conversely, activation of proteasome degradation by IU-1, which is an inhibitor for the deubiquitinating activity of USP14, decreased only the quantity of TH molecules phosphorylated at their Ser19, although it did not decrease that of TH phosphorylated at its Ser31 and Ser40 or that of TH molecules. These results suggest that the phosphorylation of Ser19 in the N-terminal portion of TH is critical as a trigger for the degradation of this enzyme by the ubiquitin-proteasome pathway.

Aripiprazole increases NAD(P)H-quinone oxidoreductase-1 and heme oxygenase-1 in PC12 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 ß-TrCP. However, in aripiprazole-treated cells, the relative amount of Nrf2 anchored to Keap1 or ß-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.

Aripiprazole increases NADPH level in PC12 cells: the role of NADPH oxidase.

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.

Effects of aripiprazole and clozapine on the treatment of glycolytic carbon in PC12 cells.

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.

Phosphorylation of the N-terminal portion of tyrosine hydroxylase triggers proteasomal digestion of the enzyme.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis, and its N-terminus plays a critical role in the intracellular stability of the enzyme. In the present study, we investigated the mechanism by which the N-terminal region of TH affects this stability. TH molecules phosphorylated at their Ser31 and Ser40 were localized predominantly in the cytoplasm of PC12D cells. However, those molecules phosphorylated at Ser19 were found mainly in the nucleus, whereas they seemed to be negligible in the cytoplasm. The inhibition of proteasomes increased the quantity of TH molecules phosphorylated at their Ser19 and Ser40, although it did not increase that of TH molecules or that of TH phosphorylated at its Ser31. The inhibition of autophagy did not affect the amount of the TH molecule or that of its three phosphorylated forms. Deletion mutants of human TH type-1 lacking the N-terminal region containing the three phosphorylation sites possessed high stability of the enzyme in PC12D cells. These results suggest that the phosphorylation of the N-terminal portion of TH regulates the degradation of this enzyme by the ubiquitin-proteasome pathway.

Effects of atypical antipsychotics and haloperidol on PC12 cells: only aripiprazole phosphorylates AMP-activated protein kinase.

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 ß-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.

Peripherally injected lipopolysaccharide induces apoptosis in the subventricular zone of young adult mice.

Because the subventricular zone (SVZ) constantly supplies newly generated neurons to the olfactory bulb (OB) along the rostral migratory stream (RMS) in adult brain, SVZ-RMS-OB axis has been thought to work as a unit. We previously reported that peripherally injected lipopolysaccharide (LPS) induces apoptosis in the OB in young adult mice. Therefore, this study was undertaken to examine whether peripherally injected LPS induces apoptotic cell death also in the SVZ. Two mouse strains were used: C3H/HeN and Toll-like receptor 4-mutated C3H/HeJ, and wild-type C57BL/6 and TNFR1(-/-)-2(-/-), in which the genes tumor necrosis factor receptor (TNFR)1 and TNFR2 are knocked out. Immunohistochemical study and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay done on the SVZ-RMS pathway of young adult male mice showed that peripherally injected LPS switches on the apoptotic signal by cleaving pro-caspase-3, thus possibly increasing the number of cells dying from apoptosis in these areas in adult mice. Activation of the tumor necrosis factor (TNF)-alpha-TNFR system played a critical role in fully inducing apoptosis in this pathway. We suggest that TNF-alpha was probably released not from microglia but from astrocytes in the SVZ and RMS.

Lipopolysaccharide extends the lifespan of mouse primary-cultured microglia.

Microglial activation has been implicated in the recognition and phagocytic removal of degenerating neurons; however, this process must be tightly regulated in the central nervous system, because prolonged activation could damage normal neurons. We report that mouse primary-cultured microglia, which are destined to die within a few days under ordinary culture conditions, can live for more than 1 month when kept activated by lipopolysaccharide (LPS) treatment. Primary-cultured microglia treated with sublethal doses of LPS remained viable, without any measurable increase in apoptotic or necrotic cell death. LPS-treated microglia had an arborescent shape, with enlarged somata and thickened cell bodies. Although the amount of intracellular ATP in these microglia was reduced by 2 h after the start of LPS treatment, this had no effect on the viability of the cells. LPS treatment of microglia increased the antiapoptotic factor Bcl-xL protein level at day 1, although the level of the proapoptotic Bcl-associated X-protein was unaffected. Furthermore, the level of microtubule-associated light chain 3, a marker protein for autophagy, decreased at 3 h after exposure to LPS. These data show that the optimal dose of LPS suppresses the induction of both apoptosis and autophagy in primary-cultured microglia, allowing the cells to stay alive for more than 1 month. Because long-lived microglia may play critical roles in the exacerbation of neurodegeneration, our findings suggest that inducing a resting stage in active microglia could be a new and promising strategy to inhibit the deterioration of neurodegenerative disease.

Role of N-terminus of tyrosine hydroxylase in the biosynthesis of catecholamines.

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.

Peripheral lipopolysaccharide administration affects the olfactory dopamine system in mice.

Peripheral administration of lipopolysaccharide (LPS) in an amount that produces acute stress has been found to affect the catecholamine systems in the brain. Acute peripheral LPS administration activated norepinephrine (NE) metabolism in the locus ceruleus (LC). Approximately 40% of murine LC neurons project to the olfactory bulb (OB) and the anterior olfactory nucleus (AON). Thus, we investigated the effects of a single intra-peritoneal (i.p.) LPS injection on catecholamine biosynthesis in the OB and AON in 8-week-old C3H/HeN male mice. In the AON, the content of dopamine (DA), but not that of NE, was highly increased 6 h after LPS injection. In the OB, the contents of DA and NE did not change; but within 2 h after a single i.p. LPS injection, the mRNA levels of IkappaB, TNF-alpha, and TNF-alpha receptor type 1 were significantly enhanced. Almost all TNF-alpha-immunoreactive cells in the OB of the LPS-injected mice were located in the granule cell layer, and unexpectedly, they were not microglia but astroglia. The number of TUNEL-positive cells identified exclusively in the granule cell layer was significantly increased at 24 h after LPS injection. Therefore, our data suggest that astroglia activated by peripherally injected LPS may release TNF-alpha, which may trigger apoptosis in the granule cell layer in the OB. The increase in DA content in the AON and the production of TNF-alpha and apoptotic cells in the OB by acute peripheral LPS administration are not likely to be related.

RNAi of 14-3-3eta protein increases intracellular stability of tyrosine hydroxylase.

Tyrosine hydroxylase is the rate-limiting enzyme in catecholamine biosynthesis, and its N-terminus plays a critical role in the intracellular stability of the enzyme. In the present study, we investigated the mechanism by which the N-terminus of human tyrosine hydroxylase type 1 (hTH1) affects the stability. The results obtained by using N-terminus-deleted hTH1 mutants identified the sequence up to Ala(23) as mediating the stability. The down-regulation of 14-3-3eta proteins in PC12D cells exogenously expressing hTH1, enhanced the stability of the wild-type enzyme and that of the mutant lacking the N-terminus up to Ala(23). However, the stability of the mutant was reduced compared to the wild-type enzyme. The stability of the mutant with the N-terminus deleted up to Glu(43) was not affected by the down-regulation of 14-3-3eta. These results suggest that the 14-3-3eta protein regulates hTH1 stability by acting on the N-terminus.

p53 protein, interferon-gamma, and NF-kappaB levels are elevated in the parkinsonian brain.

We and other workers found markedly increased levels of proinflammatory cytokines and apoptosis-related proteins in parkinsonian brain. Although the pathogenesis of Parkinson's disease (PD) remains enigmatic, apoptosis might be involved in the degeneration of dopaminergic neurons in PD. To investigate the possible presence of other inflammatory cytokines and/or apoptosis-related protein, the levels of p53 protein, interferon-gamma, and NF-kappaB were measured for the first time in the brain (substantia nigra, caudate nucleus, putamen, cerebellum, and frontal cortex) from control and parkinsonian patients by a highly sensitive sandwich enzyme-linked immunosorbent assay. The p53 protein level in the caudate nucleus was significantly higher in parkinsonian patients than in controls (P<0.05), whereas this protein in the substantia nigra, putamen, and cerebral cortex showed no significant difference between parkinsonian and control subjects. The interferon-gamma level was significantly higher in the nigrostriatal dopaminergic regions (substantia nigra, caudate nucleus, and putamen) in parkinsonian patients than in the controls (P<0.05), but was not significantly different in the cerebellum or frontal cortex between the two groups. In accordance with previous immunohistochemical analysis, the NF-kappaB level in the nigrostriatal dopaminergic regions was significantly higher in parkinsonian patients than in the controls (P<0.05). These data suggest that the significant increase in the levels of p53 protein, interferon-gamma, and NF-kappaB reflect apoptosis and the inflammatory state in the parkinsonian brain and that their elevation is involved in the degeneration of the nigrostriatal dopaminergic neurons.

Cytosolic catechols inhibit alpha-synuclein aggregation and facilitate the formation of intracellular soluble oligomeric intermediates.

Aberrant aggregation of alpha-synuclein (alpha-syn) to form fibrils and insoluble aggregates has been implicated in the pathogenic processes of many neurodegenerative diseases. Despite the dramatic effects of dopamine in inhibiting the formation of alpha-syn fibrils by stabilization of oligomeric intermediates in cell-free systems, no studies have examined the effects of intracellular dopamine on alpha-syn aggregation. To study this process and its association with neurodegeneration, intracellular catechol levels were increased to various levels by expressing different forms of tyrosine hydroxylase, in cells induced to form alpha-syn aggregates. The increase in the steady-state dopamine levels inhibited the formation of alpha-syn aggregates and induced the formation of innocuous oligomeric intermediates. Analysis of transgenic mice expressing the disease-associated A53T mutant alpha-syn revealed the presence of oligomeric alpha-syn in nondegenerating dopaminergic neurons that do contain insoluble alpha-syn. These data indicate that intraneuronal dopamine levels can be a major modulator of alpha-syn aggregation and inclusion formation, with important implications on the selective degeneration of these neurons in Parkinson's disease.

Rotenone and CCCP inhibit tyrosine hydroxylation in rat striatal tissue slices.

Complex I inhibition has been implicated in the neurotoxicity of MPTP and rotenone, which reproduce a neurochemical and neuropathological feature of Parkinson's disease in experimental animals. Previous studies performed in rat striatal slices have shown that dopaminergic neurotoxins, MPTP and manganese, inhibit tyrosine hydroxylation, a rate-limiting step of dopamine biosynthesis. In this study, we examined the effect of mitochondrial toxins such as rotenone and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) on tyrosine hydroxylation in rat striatal slices. Rotenone and CCCP inhibited DOPA formation with an accompanying decrease in ATP and increase in lactate of rat striatal slices during 1h incubation. Furthermore, rotenone reduced dopamine (DA), dihydroxyphenyl acetic acid (DOPAC) and homovanillic acid (HVA) levels in PC12 cells after 20 h incubation. These results suggest that tyrosine hydroxylation is inhibited in dopaminergic neurons soon after exposure to sub-micromolar concentrations of rotenone and CCCP, leading to dopamine depletion.