Cerebral blood flow and freezing of gait in Parkinson's disease.

OBJECTIVE: We investigated the relationship between freezing of gait (FOG) severity in Parkinson's disease (PD) and regional cerebral blood flow (rCBF) using single-photon emission computed tomography (SPECT) and evaluated the effect of selegiline therapy. METHOD: We evaluated 54 patients with PD (FOG positive: 21 patients, and FOG negative: 33 patients) with N-isopropyl-p-[I-123] iodoamphetamine ((123) I-IMP) SPECT and the Unified Parkinson's Disease Rating Scale (UPDRS) part III, Mini-Mental State Examination (MMSE), and Beck Depression Inventory. [Correction added on 18 April 2012, after online publication: In the preceding statement, 55 instead of 54 patients with PD were evaluated, and FOG negative consisted of 34 instead of 33 patients] Furthermore, we examined rCBF in FOG-negative patients treated with levodopa with or without selegiline. RESULTS: Z-values of bilateral Brodmann areas (BA) 10 and 11 and left BA32 showed significant increases in the FOG-positive group compared with the FOG-negative group. [Correction added on 18 April 2012, after online publication: In the preceding statement, Z-values was changed to Z-scores] There were significantly positive correlations between Z-values of these areas and FOG score, especially on both sides of BA11. [Correction added on 18 April 2012, after online publication: In the preceding statement, Z-values was changed to Z-scores] An increase in Z-values in bilateral BA10 and 11 and left BA32 in the levodopa-selegiline treatment group after 1 year was significantly inhibited compared with the levodopa treatment group. [Correction added on 18 April 2012, after online publication: In the preceding statement, left BA32 was changed to right BA32, and Z-values was changed to Z-scores] CONCLUSION: There was a close relationship between FOG severity in PD and an increase in rCBF in BA 10, 11 and 32. Furthermore, selegiline's FOG prevention effect may be related to maintaining rCBF in these same areas.

The relationship between depression and regional cerebral blood flow in Parkinson's disease and the effect of selegiline treatment.

OBJECTIVES: We examined the relationship between severity of depression in Parkinson's disease (PD) and regional cerebral blood flow (rCBF) using single photon emission computed tomography (SPECT) and the reaction to levodopa-selegiline combination therapy. MATERIALS AND METHODS: We evaluated 52 patients with PD and nine age-matched controls with SPECT and the Unified Parkinson's Disease Rating Scale (UPDRS) part III, Mini-Mental State Examination (MMSE), and Beck Depression Inventory (BDI) to evaluate depression severity and its connection with rCBF. Furthermore, we examined rCBF in patients with PD treated with levodopa with or without selegiline. RESULTS: A significant fall in rCBF was observed in the bilateral posterior cingulate, hippocampus, and cuneus and the superior parietal and primary visual areas in PD patients with minor depression and in all regions in those with major depression. Elevations in UPDRS part III and BDI scores and falls in MMSE scores were of significantly lower magnitude in the levodopa-selegiline group than in the levodopa group. Whole brain rCBF fell significantly less in the levodopa-selegiline group than in the levodopa group. CONCLUSIONS: These results indicate that selegiline controlled not only worsening of motor function and cognitive function in PD but also aggravation of minor depression, and restrained a fall in whole brain rCBF.

Biochemistry of postmortem brains in Parkinson's disease: historical overview and future prospects.

Biochemical studies on postmortem brains of patients with Parkinson's disease (PD) have greatly contributed to our understanding of the molecular pathogenesis of this disease. The discovery by 1960 of a dopamine deficiency in the nigro-striatal dopamine region of the PD brain was a landmark in research on PD. At that time we collaborated with Hirotaro Narabayashi and his colleagues in Japan and with Peter Riederer in Germany on the biochemistry of PD by using postmortem brain samples in their brain banks. We found that the activity, mRNA level, and protein content of tyrosine hydroxylase (TH), as well as the levels of the tetrahydrobiopterin (BH4) cofactor of TH and the activity of the BH4-synthesizing enzyme, GTP cyclohydrolase I (GCHI), were markedly decreased in the substantia nigra and striatum in the PD brain. In contrast, the molecular activity (enzyme activity/enzyme protein) of TH was increased, suggesting a compensatory increase in the enzyme activity. The mRNA levels of all four isoforms of human TH (hTH1-hTH4), produced by alternative mRNA splicing, were also markedly decreased. This finding is in contrast to a completely parallel decrease in the activity and protein content of dopamine beta-hydroxylase (DBH) without changes in its molecular activity in cerebrospinal fluid (CSF) in PD. We also found that the activities and/or the levels of the mRNA and protein of aromatic L-amino acid decarboxylase (AADC, DOPA decarboxylase), DBH, phenylethanolamine N-methyltransferase (PNMT), which synthesize dopamine, noradrenaline, and adrenaline, respectively, were also decreased in PD brains, indicating that all catecholamine systems were widely impaired in PD brains. Programmed cell death of the nigro-striatal dopamine neurons in PD has been suggested from the following findings on postmortem brains: (1) increased levels of pro-inflammatory cytokines such as TNF-alpha and IL-6; (2) increased levels of apoptosis-related factors such as TNF-alpha receptor R1 (p 55), soluble Fas and bcl-2, and increased activities of caspases 1 and 3; and (3) decreased levels of neurotrophins such as brain-derived nerve growth factor (BDNF). Immunohistochemical data and the mRNA levels of the above molecules in PD brains supported these biochemical data. We confirmed by double immunofluorescence staining the production of TNF-alpha and IL-6 in activated microglia in the putamen of PD patients. Owing to the recent development of highly sensitive and wide-range analytical methods for quantifying mRNAs and proteins, future assays of the levels of various mRNAs and proteins not only in micro-dissected brain tissues containing neurons and glial cells, but also in single cells from frozen brain slices isolated by laser capture micro-dissection, coupled with toluidine blue, Nissl staining or immunohistochemical staining, should further contribute to the elucidation of the molecular pathogenesis of PD and other neurodegenerative or neuropsychiatric diseases.

Effect of peripherally administered lipopolysaccharide (LPS) on GTP cyclohydrolase I, tetrahydrobiopterin and norepinephrine in the locus coeruleus in mice.

Lipopolysaccharide (LPS), an endotoxin released from the outer membranes of Gram-negative bacteria, triggers cells to synthesize and release inflammatory cytokines that may progress to septic shock in vivo. We found that LPS enhances tetrahydrobiopterin (BH4) biosynthesis by inducing the biosynthetic enzyme GTP cyclohydrolase I (GCH) in vitro in the mouse neuroblastoma cell line N1E-115. Furthermore, we observed that gene expression of GCH in the locus coeruleus (LC) in mice was enhanced by peripheral administration of LPS, resulting in increased concentrations of BH4, and norepinephrine, and its metabolite 4-hydroxy-3-methoxyphenylglycol (MHPG). These results suggest that tyrosine hydroxylase (TH) activity is increased by increased content of BH4 due to enhanced mRNA expression of GCH in the LC resulting in the increase in norepinephrine in the LC during endotoxemia. LPS in blood may act as a stressor to increase norepinephrine biosynthesis in the mouse LC.

Role of cytokines in inflammatory process in Parkinson's disease.

We investigated whether the cytokines produced in activated microglia in the substantia nigra (SN) and putamen in sporadic Parkinson's disease (PD) are neuroprotective or neurotoxic. In autopsy brains of PD, the number of MHC class II (CR3/43)-positive activated microglia, which were also ICAM-1 (CD 54)-, LFA-1 (CD 11a)-, TNF-alpha-, and IL-6-positive, increased in the SN and putamen during progress of PD. At the early stage activated microglia were mainly associated with tyrosine hydroxylase (TH)-positive neurites in the putamen, and at the advanced stage with damaged TH-positive neurons in the SN. The activated microglia in PD were observed not only in the nigro-striatal region, but also in various brain regions such as the hippocampus and cerebral cortex. We examined the distribution of activated microglia and the expression of cytokines and neurotrophins in the hippocampus of PD and Lewy body disease (LBD). The levels of IL-6 and TNF-alpha mRNAs increased both in PD and LBD, but those of BDNF mRNA and protein drastically decreased specifically in LBD, in which neuronal loss was observed not only in the nigro-striatum but also in the hippocampus. The results suggest activated microglia in the hippocampus to be probably neuroprotective in PD, but those to be neurotoxic in LBD. As an evidence supporting this hypothesis, two subsets of microglia were isolated from mouse brain by cell sorting: one subset with high production of reactive oxygen species (ROS) and the other with no production of ROS. When co-cultured with neuronal cells, one microglia clone with high ROS production was neurotoxic, but another clone with no ROS production neuroprotective. On the other hand, Sawada with coworkers found that a neuroprotective microglial clone in a culture experiment converted to a toxic microglial clone by transduction of the HIV-1 Nef protein with increasing NADPH oxidase activity. Taken together, all these results suggest that activated microglia may change in vivo from neuroprotective to neurotoxic subtsets as degeneration of dopamine neurons in the SN progresses in PD. We conclude that the cytokines from activated microglia in the SN and putamen may be initially neuroprotective, but may later become neurotoxic during the progress of PD. Toxic change of activated microglia may also occur in Alzheimer's disease and other neurodegenerative diseases in which inflammatory process is found.

Molecular mechanism of the relation of monoamine oxidase B and its inhibitors to Parkinson's disease: possible implications of glial cells.

Monoamine oxidases A and B (MAO A and MAO B) are the major enzymes that catalyze the oxidative deamination of monoamine neurotaransmitters such as dopamine (DA), noradrenaline, and serotonin in the central and peripheral nervous systems. MAO B is mainly localized in glial cells. MAO B also oxidizes the xenobiotic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to a parkinsonism-producing neurotoxin, 1-methyl-4-phenyl-pyridinium (MPP+). MAO B may be closely related to the pathogenesis of Parkinson's disease (PD), in which neuromelanin-containing DA neurons in the substantia nigra projecting to the striatum in the brain selectively degenerate. MAO B degrades the neurotransmitter DA that is deficient in the nigro-striatal region in PD, and forms H2O2 and toxic aldehyde metabolites of DA. H2O2 produces highly toxic reactive oxygen species (ROS) by Fenton reaction that is catalyzed by iron and neuromelanin. MAO B inhibitors such as L-(-)-deprenyl (selegiline) and rasagiline are effective for the treatment of PD. Concerning the mechanism of the clinical efficacy of MAO B inhibitors in PD, the inhibition of DA degradation (a symptomatic effect) and also the prevention of the formation of neurotoxic DA metabolites, i.e., ROS and dopamine derived aldehydes have been speculated. As another mechanism of clinical efficacy, MAO B inhibitors such as selegiline are speculated to have neuroprotective effects to prevent progress of PD. The possible mechanism of neuroprotection of MAO B inhibitors may be related not only to MAO B inhibition but also to induction and activation of multiple factors for anti-oxidative stress and anti-apoptosis: i.e., catalase, superoxide dismutase 1 and 2, thioredoxin, Bcl-2, the cellular poly(ADP-ribosyl)ation, and binding to glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Furthermore, it should be noted that selegiline increases production of neurotrophins such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and glial cell line-derived neurotrphic factor (GDNF), possibly from glial cells, to protect neurons from inflammatory process.

Inhibition of inflammatory cytokine secretion from mouse microglia cells by DHMEQ, an NF-kappaB inhibitor.

Activation of microglia has been implicated in various neurodegenerative disorders, and thus the inhibition of microglial activity may suppress these disorders. Earlier we designed and synthesized an NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ) that showed anti-inflammatory and anti-tumor activities in vivo. In the present research, we studied whether DHMEQ would inhibit the activation of mouse microglial cells. DHMEQ inhibited lipopolysaccharide (LPS)-induced activation of NF-kappaB in an electrophoresis mobility shift assay. It also inhibited LPS-induced secretions of TNF-alpha and IL-6 from mouse microglial cell line 6-1 cells.

Inflammatory process in Parkinson's disease: role for cytokines.

Parkinson's disease (PD) is a movement disorder caused by degeneration of the nigrostriatal dopamine (DA) neurons in the substantia nigra pars compacta and the resultant deficiency in the neurotransmitter DA at the nerve terminals in the striatum. We and other investigators found increased levels of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6, and decreased levels of neurotrophins such as brain-derived neurotrophic factor (BDNF) in the nigrostriatal region of postmortem brains and/or in the ventricular or lumbar cerebrospinal fluid (CSF) from patients with sporadic PD, and in animal models, such as 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)- and 6-hydroxydopamine (6-OHDA)-induced PD. These changes in cytokine and neurotrophin levels may be initiated by activated microglia, which may then promote apoptotic cell death and subsequent phagocytosis of DA neurons. Cytokines as pleiotropic factors, promote signals that either lead to cell death or exert neuroprotective effects. The discovery of toxic changes in trophic microglia by M. Sawada and co-workers is important to this point. Ultimately, microglial cells may regulate cellular changes that cause either harm or benefit by producing cytokines or neurotrophins depending upon the primary cause and the circumstances during the inflammatory process of PD.

Expression levels of Rab2, a G protein, and Bag-1, a Bcl-2 binding protein are controlled by withdrawal of nicotine from cultured pheochromocytoma PC12 cells.

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.

Changes in the concentrations of tetrahydrobiopterin, the cofactor of tyrosine hydroxylase, in blood under physical stress and in depression.

This paper studies changes in the concentrations of tetrahydrobiopterin (BH4), the cofactor of tyrosine hydroxylase, in blood under physical stress and in depression. BH4 was found to be transiently released from the sympathetic nerves under severe physical stress but continuously released in depression with an increased oxidation rate of BH4 to B.

Increase of transcriptional levels of egr-1 and nur77 genes due to both nicotine treatment and withdrawal in pheochromocytoma cells.

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.

Parkinson's disease: changes in apoptosis-related factors suggesting possible gene therapy.

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.

Delayed delivery of AAV-GDNF prevents nigral neurodegeneration and promotes functional recovery in a rat model of Parkinson's disease.

Glial cell line-derived neurotrophic factor (GDNF) is a strong candidate agent in the neuroprotective treatment of Parkinson's disease (PD). We investigated whether adeno-associated viral (AAV) vector-mediated delivery of a GDNF gene in a delayed manner could prevent progressive degeneration of dopaminergic (DA) neurons, while preserving a functional nigrostriatal pathway. Four weeks after a unilateral intrastriatal injection of 6-hydroxydopamine (6-OHDA), rats received injection of AAV vectors expressing GDNF tagged with FLAG peptide (AAV-GDNFflag) or beta-galactosidase (AAV-LacZ) into the lesioned striatum. Immunostaining for FLAG demonstrated retrograde transport of GDNFflag to the substantia nigra (SN). The density of tyrosine hydroxylase (TH)-positive DA fibers in the striatum and the number of TH-positive or cholera toxin subunit B (CTB, neuronal tracer)-labeled neurons in the SN were significantly greater in the AAV-GDNFflag group than in the AAV-LacZ group. Dopamine levels and those of its metabolites in the striatum were remarkably higher in the AAV-GDNFflag group compared with the control group. Consistent with anatomical and biochemical changes, significant behavioral recovery was observed from 4-20 weeks following AAV-GDNFflag injection. These data indicate that a delayed delivery of GDNF gene using AAV vector is efficacious even 4 weeks after the onset of progressive degeneration in a rat model of PD.

Inhibition of azoxymethane-induced aberrant crypt foci formation in rat colorectum by whole leaf Aloe arborescens Miller var. natalensis Berger.

We examined the modifying effect of whole-leaf Aloe arborescens Miller var. natalensis Berger (designated as 'ALOE') on azoxymethane (AOM)-induced aberrant crypt foci (ACF), putative preneoplastic lesions, in the rat colorectum. Male F344 rats (4 weeks old) were fed the basal diet, or experimental diets containing 1% or 5% ALOE for 5 weeks. One week later, all rats except those in the vehicle-treated groups were injected s.c. with AOM (15 mg/kg, once weekly for 3 weeks). At 9 weeks of age, all the rats were killed, and the colorectum and liver were evaluated for ACF and cytosolic quinone reductase (QR; a phase 2 enzyme), respectively. In rats given AOM and ALOE (1% or 5% in diet) the numbers of ACF/colorectum, aberrant crypts/colorectum, aberrant crypts/focus and large ACF/colorectum were significantly decreased compared with those of rats given AOM alone (all p < 0.01). No ACF were found in rats treated without AOM. In addition, ALOE significantly increased cytosolic QR activity in the liver (p < 0.01). These results indicated that ALOE inhibited the development of AOM-induced ACF in the rat colorectum, with increased QR activity in the liver, and therefore suggested that ALOE might have a chemopreventive effect against colon carcinogenesis at least in the initiation stage.

Manganese mimics the action of 1-methyl-4-phenylpyridinium ion, a dopaminergic neurotoxin, in rat striatal tissue slices.

Manganese and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) are known to induce neurological pathologies similar to that of parkinsonism. Previous studies performed in rat striatal slices have shown that MPTP and related compounds inhibit tyrosine hydroxylation, a rate-limiting step of dopamine biosynthesis. Here, we reported that manganese inhibited tyrosine hydroxylation in rat striatal slices. In addition, manganese caused increase in the levels of lactate indicating that aerobic glycolysis was inhibited in striatal slices. This inhibition was unique to manganese since other divalent cations, such as magnesium and zinc, did not increase lactate concentrations. These results suggest that the mechanisms by which manganese produces dysfunction of the nervous system are similar to those of MPTP.

A new splicing variant for human tyrosine hydroxylase in the adrenal medulla.

It has been reported that several mRNA isoforms of tyrosine 3-monooxygenase (tyrosine hydroxylase; TH) occur only in primates. New TH isoforms produced by skipping of exon 3 in the adrenal medulla of patients with progressive supranuclear palsy (PSP) have recently been reported, J. Neurochem. 67 (1996) 19. Here, we looked for the presence of new TH isoforms in control brains and adrenal medulla and in brains from patients with PSP. We found a novel type of TH mRNA in the adrenal medulla from one of the control subjects. The mRNA lacked exon 4, resulting in a premature stop codon at amino acid 147. This result suggests the importance of alternative splicing in the regulation of TH activity.