Clinical and genetic characteristics of children with dopa-responsive dystonia caused by tyrosine hydroxylase gene variations / 中华儿科杂志

Objective: To explore the clinical and genetic characteristics of children with dopa-responsive dystonia (DRD) caused by tyrosine hydroxylase (TH) gene variations. Methods: Clinical data of 9 children with DRD caused by TH gene variations diagnosed in the Department of Children Rehabilitation, the Third Affiliated Hospital of Zhengzhou University from January 2017 to August 2022 were retrospectively collected and analyzed, including the general conditions, clinical manifestations, laboratory tests, gene variations and follow-up data. Results: Of the 9 children with DRD caused by TH gene variations, 3 were males and 6 were females. The age at diagnosis was 12.0 (8.0, 15.0) months. The initial symptoms of the 8 severe patients were motor delay or degression. Clinical symptoms of the severe patients included motor delay (8 cases), truncal hypotonia (8 cases), limb muscle hypotonia (7 cases), hypokinesia (6 cases), decreased facial expression (4 cases), tremor (3 cases), limb dystonia (3 cases), diurnal fluctuation (2 cases), ptosis (2 cases), limb muscle hypertonia (1 case) and drooling (1 case). The initial symptom of the very severe patient was motor delay. Clinical symptoms of the very severe patient included motor delay, truncal hypotonia, oculogyric crises, status dystonicus, hypokinesia, decreased facial expression, and decreased sleep. Eleven TH gene variants were found, including 5 missense variants, 3 splice site variants, 2 nonsense variants, and 1 insertion variant, as well as 2 novel variants (c.941C>A (p.T314K), c.316_317insCGT (p.F106delinsSF)). Nine patients were followed up for 40 (29, 43) months, and no one was lost to follow-up. Seven of the 8 severe patients were treated by levodopa and benserazide hydrochloride tablets and 1 severe patient was treated by levodopa tablets. All the severe patients responded well to levodopa and benserazide hydrochloride tablets or levodopa tablets. Although the weight of the patients increased and the drug dosage was not increased, the curative effect remained stable and there was no obvious adverse reaction. One severe patient developed dyskinesia in the early stage of treatment with levodopa and benserazide hydrochloride tablets and it disappeared after oral administration of benzhexol hydrochloride tablets. Until the last follow-up, motor development of 7 severe patients returned to normal and 1 severe patient still had motor delay due to receiving levodopa and benserazide hydrochloride tablets for only 2 months. The very severe patient was extremely sensitive to levodopa and benserazide hydrochloride tablets and no improvement was observed in this patient. Conclusions: Most of the DRD caused by TH gene variations are severe form. The clinical manifestations are varied and easily misdiagnosed. Patients of the severe patients responded well to levodopa and benserazide hydrochloride tablets or levodopa tablets, and it takes a long time before full effects of treatment become established. Long-term effect is stable without increasing the drug dosage, and no obvious side effect is observed.

MASH1 induces neuron transdifferentiation of adrenal medulla chromaffin cells / 中南大学学报(医学版)

OBJECTIVES@#Nerve growth factor (NGF) induces neuron transdifferentiation of adrenal medulla chromaffin cells (AMCCs) and consequently downregulates the secretion of epinephrine (EPI), which may be involved in the pathogenesis of bronchial asthma. Mammalian achaete scute-homologous 1 (MASH1), a key regulator of neurogenesis in the nervous system, has been proved to be elevated in AMCCs with neuron transdifferentiation in vivo. This study aims to explore the role of MASH1 in the process of neuron transdifferentiation of AMCCs and the mechanisms.@*METHODS@#Rat AMCCs were isolated and cultured. AMCCs were transfected with siMASH1 or MASH1 overexpression plasmid, then were stimulated with NGF and/or dexamethasone, PD98059 (a MAPK kinase-1 inhibitor) for 48 hours. Morphological changes were observed using light and electron microscope. Phenylethanolamine-N-methyltransferase (PNMT, the key enzyme for epinephrine synthesis) and tyrosine hydroxylase were detected by immunofluorescence. Western blotting was used to test the protein levels of PNMT, MASH1, peripherin (neuronal markers), extracellular regulated protein kinases (ERK), phosphorylated extracellular regulated protein kinases (pERK), and JMJD3. Real-time RT-PCR was applied to analyze the mRNA levels of MASH1 and JMJD3. EPI levels in the cellular supernatant were measured using ELISA.@*RESULTS@#Cells with both tyrosine hydroxylase and PNMT positive by immunofluorescence were proved to be AMCCs. Exposure to NGF, AMCCs exhibited neurite-like processes concomitant with increases in pERK/ERK, peripherin, and MASH1 levels (all P<0.05). Additionally, impairment of endocrine phenotype was proved by a signifcant decrease in the PNMT level and the secretion of EPI from AMCCs (all P<0.01). MASH1 interference reversed the effect of NGF, causing increases in the levels of PNMT and EPI, conversely reduced the peripherin level and cell processes (all P<0.01). MASH1 overexpression significantly increased the number of cell processes and peripherin level, while decreased the levels of PNMT and EPI (all P<0.01). Compared with the NGF group, the levels of MASH1, JMJD3 protein and mRNA in AMCCs in the NGF+PD98059 group were decreased (all P<0.05). After treatment with PD98059 and dexamethasone, the effect of NGF on promoting the transdifferentiation of AMCCs was inhibited, and the number of cell processes and EPI levels were decreased (both P<0.05). In addition, the activity of the pERK/MASH1 pathway activated by NGF was also inhibited.@*CONCLUSIONS@#MASH1 is the key factor in neuron transdifferentiation of AMCCs. NGF-induced neuron transdifferentiation is probably mediated via pERK/MASH1 signaling.

Inhibition connexin 43 by mimetic peptide Gap27 mediates protective effects on 6-hydroxydopamine induced Parkinson's disease mouse model / 北京大学学报(医学版)

OBJECTIVE@#To explore whether the using of mimetic peptide Gap27, a selective inhibitor of connexin 43 (Cx43), could block the death of dopamine neurons and influence the expression of Cx43 in 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease mouse models.@*METHODS@#Eighteen C57BL/6 mice were randomly divided into control group, 6-OHDA group and 6-OHDA+Gap27 group, with 6 mice in each group. Bilateral substantia nigra stereotactic injection was performed. The control group was injected with ascorbate solution, 6-OHDA group was injected with 6-OHDA solution, and 6-OHDA+Gap27 group was injected with 6-OHDA and Gap27 mixed solution. Immuno-histochemical staining was used to detect the number of dopamine neurons, quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of Cx43 messenger ribonucleic acid (mRNA), immuno-fluorescence staining was used to detect the distribution of Cx43 protein, the contents of Cx43 protein and Cx43 phosphorylation at serine 368 (Cx43-ps368) in mouse midbrain were detected by Western blot.@*RESULTS@#After injection of 6-OHDA, numerous dopamine neurons in substantia nigra died as Cx43 content increased, Cx43-ps368 content decreased. Mixing Gap27 while injecting 6-OHDA could reduce the number of death dopamine neurons and weaken the changes of Cx43 and Cx43-ps368 content caused by 6-OHDA. The number of tyrosine hydroxylase (TH) immunoreactive positive neurons in 6-OHDA group decreased to 27.7% ± 0.02% of the control group (P < 0.01); The number of TH immunoreactive positive neurons in 6-OHDA+Gap27 group was (1.64±0.16) times higher than that in 6-OHDA group (P < 0.05); The content of total Cx43 protein in 6-OHDA group was (1.44±0.07) times higher than that in 6-OHDA+Gap27 group (P < 0.05) while (1.68±0.07) times higher than that in control group (P < 0.01). In 6-OHDA group, the content of Cx43-ps368 protein and its proportion in total Cx43 protein were significantly lower than that in 6-OHDA+Gap27 group (P < 0.05).@*CONCLUSION@#In 6-OHDA mouse models, mimetic peptide Gap27 played a protective role in reducing the damage to substantia nigra dopamine neurons, which was induced by 6-OHDA. The overexpression of Cx43 protein might have neurotoxicity to dopamine neuron. Meanwhile, decreasing Cx43 protein level and keeping Cx43-ps368 protein level may be the protective mechanisms of Gap27.

Dynamic changes of locus coeruleus damage in Parkinson's disease-like mice induced by paraquat / 中华劳动卫生职业病杂志

Objective: To observe the dynamic changes of brainstem locus coeruleus (LC) damage in Parkinson' s disease (PD) -like mice by paraquat (PQ) . Methods: In October 2019, 36 male C57BL/6 mice were randomly divided into the exposure group and the control group, with 18 mice in each group. The mice in the exposure group were given intraperitoneal injection of 15 mg/kg PQ, and the mice in the control group were given intraperitoneal injection of 0.9% saline, twice a week for 8 weeks. Neurobehavioral changes (pole climbing test, swimming test, open field test, tail hanging test, high plus maze test and water maze test) were observed at 4 weeks, 6 weeks and 8 weeks, respectively, and the changes of motor ability, emotion and cognitive function were evaluated. The brain tissue of mice were taken and stained with Hematoxylin-Eosin (HE) to observe the pathological changes of LC. Nissl staining was used to detect the changes of neuronal Nissl bodies in LC. Immunohistochemistry (IHC) staining was used to detect the expression of neuron nuclear antigen (NeuN) , dopamine (DA) neurons and norepinephrine (NE) neuron markers tyrosine hydroxylase (TH) , α-synuclein (α-syn) in substantia nigra (SN) and LC. The expression levels of NeuN, TH and α-syn in the midbrain and brainstem were detected by Western blotting. TUNEL staining was used to detect neuronal apoptosis in LC. Results: Compared with the 4th week of PQ exposure group, the time of pole climbing and swimming immobility were gradually increased, the ratio of open arm residence time of high plus maze test and the number of times of the platform and the residence time of platform quadrant in water maze test were gradually decreased (P<0.05) in the exposure group with the progress of exposure time. The results of HE and Nissl staining showed that the neurons in LC gradually arranged loosely, the nucleus were deeply stained, the cytoplasm was pyknosis, and the number of Nissl bodies gradually decreased (P<0.05) in the exposure group with the progress of exposure time. IHC results showed that the number of NeuN and TH positive cells in SN and LC of mice were gradually decreased, and the positive expression of α-syn was gradually increased (P<0.05) in the exposure group with the progress of exposure time. Western blotting results showed that the expression levels of NeuN and TH in the midbrain and brainstem were gradually decreased, and the expression level of α-syn was gradually increased (P<0.05) in the exposure group with the progress of exposure time. TUNEL staining showed that the apoptosis rates of neurons in LC were gradually increased (P<0.05) in the exposure group with the progress of exposure time. Conclusion: PQ induces progressive damage in the LC area of PD-like mice, which may be caused by the abnormal accumulation of pathological α-syn in the LC area.

Preliminary study on time-dependent changes of intestinal tract and brain-gut axis in mice model of Parkinson's disease induced by paraquat / 中华劳动卫生职业病杂志

Objective: To observe the intestinal time-dependent changes in Parkinson's disease (PD) mouse model constructed by intraperitoneal injection of paraquat (PQ) and to establish the brain-gut axis connection initially. Methods: In October 2019, 48 mice were randomly divided into treated group and control groups: treated 4-week (P-4) group, treated 6-week (P-6) group, treated 8-week (P-8) group, control 4-week (C-4) group, control 6-week (C-6) group, and control 8-week (C-8) group. The treated group was injected with 15 mg/kg PQ solution and the control group was injected with 0.9% saline (0.2 ml/20 g) by intraperitoneal injection twice a week. After the initial state (0 weeks) and the treatment at the end of 4, 6 and 8 weeks, the mood changes and motor functions of mice were assessed by neurobehavioral tests (open field test, pole climbing test, tail suspension test and elevated plus maze test) . And the number of fecal pellets for 1 h and water content were calculated to assess the functional status of the gastrointestinal tract. Western blotting experiments were performed to detect the expression levels of α-synuclein (α-syn) and tyrosine hydroxylase (TH) in the nigrostriatal region of the mouse brain, the tight junction markers zonula occludens-1 (ZO-1) and Occludin, the inflammatory markers of integrin αM subunit (CD11b) , inducible nitric oxide synthase (iNOS) , high mobility group box 1 (HMGB1) , interleukin-1β (IL-1β) , and the neuronal markers βⅢ-tubulin and α-syn protein in the colon.Immunohistochemical staining was performed to detect the expression levels of colonic tight junction proteins ZO-1 and Occludin. Immunofluorescence staining was performed to detect the expression levels of TH in the substantia nigra region of the midbrain, and the co-localization of colonic intestine neuronal marker (βⅢ-tubulin) and Ser129 α-syn in the colonic. Results: Compared with the initial state (0 weeks) and C-8 group, mice in the P-8 group had significantly higher pole climbing test scores and resting time, and significantly lower total active distance, mean active speed, percentage of open arm entry and 1 h fecal instances (P<0.05) . After poisoning, the 1 h fecal water content of model mice first increased and then decreased, the P-4 and P-6 groups were significantly higher than the simultaneous point control group, and the P-8 groups were significantly lower than the initial state (P<0.05) . Compared with control, P-4 and P-6 groups, the expression levels of ZO-1 and Occludin in the P-8 group were significantly decreased (P<0.05) . Compared with control group, the expression levels of CD11b and IL-1β in the P-4 group were significantly increased (P<0.05) . Compared with control and P-4 group, the expression levels of CD11b, iNOS, HMGB1 and IL-1β in the P-6 and P-8 groups were significantly increased (P<0.05) . Compared with the control and P-4 groups, the expression levels of βⅢ-tubulin in the colon of mice in the P-8 group were significantly decreased, and the expression levels of α-syn and Ser129 α-syn were significantly increased (P<0.05) . The expression level of Ser129 α-syn in the colon of model mice was negatively correlated with the expression level of βⅢ-tubulin (r(s)=-0.9149, 95%CI: -0.9771--0.7085, P<0.001) . Ser129 α-syn and βⅢ-tubulin co-localization in the colonic intermuscular plexus region increased gradually with the time of exposure. Compared with the control, P-4 and P-6 groups, the expression level of TH in the nigrostriatal region of the brain was significantly decreased, and the expression levels of α-syn and Ser129 α-syn were significantly increased in the P-8 group (P<0.05) . Correlation analysis showed that the relative expression level of Ser129 α-syn in the nigrostriatal region of the brain was negatively correlated with the expression level of TH in the model mice (r(s)=-0.9716, 95% CI: -0.9925--0.8953, P<0.001) . Conclusion: The PD mouse model is successfully established by PQ, and the intestinal function of the model mice is reduced in a time-dependent manner. And on this basis, it is preliminary determined that the abnormal aggregation of α-syn may be an important substance connecting the brain-gut axis.

Tyrosine hydroxylase and ß2-adrenergic receptor expression in leukocytes of spontaneously hypertensive rats: putative peripheral markers of central sympathetic activity

The sympathetic nervous system (SNS) plays a fundamental role in the pathophysiology of cardiovascular diseases, including primary arterial hypertension. In this study, we aimed to investigate whether the expression of the rate-limiting enzyme in catecholamine synthesis, tyrosine hydroxylase (TH), and the β2-adrenergic receptor (β2-AR) in immune cells from peripheral blood, reflect central SNS activity in spontaneously hypertensive rats (SHR). TH expression in the lower brainstem and adrenal glands and β2-AR expression in the lower brainstem were analyzed by western blot analyses. In the leukocytes, TH and β2-AR expression was evaluated by flow cytometry before and after chronic treatment with the centrally-acting sympathoinhibitory drug clonidine. Western blot analyses showed increased TH and β2-AR expression in the lower brainstem and increased TH in adrenal glands from SHR compared to normotensive Wistar Kyoto rats (WKY). Lower brainstem from SHR treated with clonidine presented reduced TH and β2-AR levels, and adrenal glands had decreased TH expression compared to SHR treated with vehicle. Flow cytometry showed that the percentage of leukocytes that express β2-AR is higher in SHR than in WKY. However, the percentage of leukocytes that expressed TH was higher in WKY than in SHR. Moreover, chronic treatment with clonidine normalized the levels of TH and β2-AR in leukocytes from SHR to similar levels of those of WKY. Our study demonstrated that the percentage of leukocytes expressing TH and β2-AR was altered in arterial hypertension and can be modulated by central sympathetic inhibition with clonidine treatment.

Clinical and genetic analysis of a case with infantile Parkinsonism with motor delay due to tyrosine hydroxylase deficiency / 中华医学遗传学杂志

OBJECTIVE@#To explore the clinical characteristics and genetic variants in a child with tyrosine hydroxylase-deficient infantile Parkinsonism with motor delay.@*METHODS@#Clinical feature of the patient was summarized. Genomic DNA was extracted from peripheral blood samples taken from the child and her family members. All exons of GCH1, TH and SPR genes were subjected to targeted capture and next-generation sequencing. Suspected variants were verified by Sanger sequencing.@*RESULTS@#The child could not sit alone at 7 month and 11 days. Physical examination suggested motor retardation and hypotonia, limb stiffness, head nodding, slight torticollis, and language and intellectual developmental delays. She developed involuntary shaking of limbs at 3 month old, which lasted approximately 10 seconds and aggregated with excitement and before sleeping. Cranial MRI revealed widening of subarachnoid space on the temporomandibular and particularly temporal sides. Genetic testing revealed that she has carried a nonsense c.457C>T (p.R153X) variant, which was known to be pathogenic, and a novel missense c.720C>G (p.I240M) variant of the TH gene. The two variants were derived from her father and mother, respectively.@*CONCLUSION@#The child was diagnosed as tyrosine hydroxylase-deficient infantile Parkinsonism with motor delay due to compound heterozygous variants of the TH gene. Above finding has enriched the spectrum of TH gene variants.

Neuroprotective Effect of β-Lapachone in MPTP-Induced Parkinson's Disease Mouse Model: Involvement of Astroglial p-AMPK/Nrf2/HO-1 Signaling Pathways

Parkinson's disease is a neurodegenerative disease characterized by the progressive loss of dopaminergic neurons within the substantia nigra pars compacta. In the present study, we investigated whether β-Lapachone (β-LAP), a natural naphthoquinone compound isolated from the lapacho tree (Tabebuia avellanedae), elicits neuroprotective effects in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease mouse model. β-LAP reduced the tyrosine hydroxylase (TH)-immuno-reactive fiber loss induced by MPTP in the dorsolateral striatum, and alleviated motor dysfunction as determined by the rotarod test. In addition, β-LAP protected against MPTP-induced loss of TH positive neurons, and upregulated B-cell lymphoma 2 protein (Bcl-2) expression in the substantia nigra. Based on previous reports on the neuroprotective role of nuclear factor-E2-related factor-2 (Nrf2) in neurodegenerative diseases, we investigated whether β-LAP induces upregulation of the Nrf2-hemeoxygenae-1 (HO-1) signaling pathway molecules in MPTP-injected mouse brains. Western blot and immunohistochemical analyses indicated that β-LAP increased HO-1 expression in glial fibrillary acidic protein-positive astrocytes. Moreover, β-LAP increased the nuclear translocation and DNA binding activity of Nrf2, and the phosphorylation of upstream adenosine monophosphate-activated protein kinase (AMPK). β-LAP also increased the localization of p-AMPK and Nrf2 in astrocytes. Collectively, our data suggest that β-LAP exerts neuroprotective effect in MPTP-injected mice by upregulating the p-AMPK/Nrf2/HO-1 signaling pathways in astrocytes.

Delayed Treatment of Capsaicin Produces Partial Motor Recovery by Enhancing Dopamine Function in MPP⁺-lesioned Rats via Ciliary Neurotrophic Factor

Transient receptor potential vanilloid subtype 1 (TRPV1) on astrocytes prevents ongoing degeneration of nigrostriatal dopamine (DA) neurons in MPP⁺-lesioned rats via ciliary neurotrophic factor (CNTF). The present study determined whether such a beneficial effect of astrocytic TRPV1 could be achieved after completion of injury of DA neurons, rather than ongoing injury, which seems more relevant to therapeutics. To test this, the MPP⁺-lesioned rat model utilized here exhibited approximately 70~80% degeneration of nigrostriatal DA neurons that was completed at 2 weeks post medial forebrain bundle injection of MPP⁺. TRPV1 agonist, capsaicin (CAP), was intraperitoneally administered. CNTF receptor alpha neutralizing antibody (CNTFRαNAb) was nigral injected to evaluate the role of CNTF endogenously produced by astrocyte through TRPV1 activation on DA neurons. Delayed treatment of CAP produced a significant reduction in amphetamine-induced rotational asymmetry. Accompanying this behavioral recovery, CAP treatment increased CNTF levels and tyrosine hydroxylase (TH) activity in the substantia nigra pars compacta (SNpc), and levels of DA and its metabolites in the striatum compared to controls. Interestingly, behavioral recovery and increases in biochemical indices were not reflected in trophic changes of the DA system. Instead, behavioral recovery was temporal and dependent on the continuous presence of CAP treatment. The results suggest that delayed treatment of CAP increases nigral TH enzyme activity and striatal levels of DA and its metabolites by CNTF endogenously derived from CAP-activated astrocytes through TRPV1, leading to functional recovery. Consequently, these findings may be useful in the treatment of DA imbalances associated with Parkinson's disease.

Change of Extracellular Glutamate Level in Striatum during Deep Brain Stimulation of the Entopeduncular Nucleus in Rats

OBJECTIVE: Globus pallidus interna (GPi) is acknowledged as an essential treatment for advanced Parkinson’s disease (PD). Nonetheless, the neurotransmitter study about its results is undiscovered. The goal of this research was to examine influences of entopeduncular nucleus (EPN) stimulation, identical to human GPi, in no-lesioned (NL) rat and 6-hydroxydopamine (6-HD)-lesioned rat on glutamate change in the striatum. METHODS: Extracellular glutamate level changes in striatum of NL category, NL with deep brain stimulation (DBS) category, 6-HD category, and 6-HD with DBS category were examined using microdialysis and high-pressure liquid chromatography. Tyrosine hydroxylase (TH) immunoreactivities in substantia nigra and striatum of the four categories were also analyzed. RESULTS: Extracellular glutamate levels in the striatum of NL with DBS category and 6-HD with DBS category were significantly increased by EPN stimulation compared to those in the NL category and 6-HD category. EPN stimulation had no significant effect on the expression of TH in NL or 6-HD category. CONCLUSION: Clinical results of GPi DBS are not only limited to direct inhibitory outflow to thalamus. They also include extensive alteration within basal ganglia.

HDAC Inhibition by Valproic Acid Induces Neuroprotection and Improvement of PD-like Behaviors in LRRK2 R1441G Transgenic Mice

Parkinson’s disease (PD) is one of the late-onset neurodegenerative movement disorder. Major pathological markers of PD include progressive loss of dopaminergic neurons, Lewy body formation, genetic mutations, and environmental factors. Epigenetic regulation of specific gene expression via impaired histone acetylation is associated with neuronal dysfunction in various neurodegenerative diseases. In this study, we hypothesized that histone deacetylase (HDAC) inhibitor, valproic acid (VPA), can improve motor function by enhancing cell survival in PD genetic model mice with LRRK2 R1441G mutation. To address this question, we administered VPA in LRRK2 R1441G transgenic mice to determine whether VPA affects 1) histone acetylation and HDAC expression, 2) dopaminergic neuron survival, 3) inflammatory responses, 4) motor or non-motor symptoms. As results, VPA administration increased histone acetylation level and the number of tyrosine hydroxylase (TH) positive neurons in substantia nigra of LRRK2 R1441G mice. VPA reduced iba-1 positive activated microglia and the mRNA levels of pro-inflammatory marker genes in LRRK2 R1441G mice. In addition, VPA induced the improvement of PD-like motor and non-motor behavior in LRRK2 R1441G mice. These data suggest that the inhibition of HDAC can be further studied as potential future therapeutics for PD.

7,8,4′-Trihydroxyisoflavone, a Metabolized Product of Daidzein, Attenuates 6-Hydroxydopamine-Induced Neurotoxicity in SH-SY5Y Cells

Daidzein isolated from soybean (Glycine max) has been widely studied for its antioxidant and anti-inflammatory activities. However, the protective effects of 7,8,4′-trihydroxyisoflavone (THIF), a major metabolite of daidzein, on 6-hydroxydopamine (OHDA)-induced neurotoxicity are not well understood. In the current study, 7,8,4′-THIF significantly inhibited neuronal cell death and lactate dehydrogenase (LDH) release induced by 6-OHDA in SH-SY5Y cells, which were used as an in vitro model of Parkinson's disease (PD). Moreover, pretreatment with 7,8,4′-THIF significantly increased the levels of superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) and decreased malondialdehyde (MDA) activity in 6-OHDA-induced SH-SY5Y cells. In addition, 7,8,4′-THIF significantly recovered 6-OHDA-induced cleaved caspase-3, cleaved caspase-9, cleaved poly-ADP-ribose polymerase (PARP), increased Bax, and decreased Bcl-2 levels. Additionally, 7,8,4′-THIF significantly restored the expression levels of phosphorylated c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 1/2 (ERK 1/2), phosphatidylinositol 3-kinases (PI3K)/Akt, and glycogen synthase kinase-3 beta (GSK-3β) in 6-OHDA-induced SH-SY5Y cells. Further, 7,8,4′-THIF significantly increased the reduced tyrosine hydroxylase (TH) level induced by 6-OHDA in SH-SY5Y cells. Collectively, these results suggest that 7,8,4′-THIF protects against 6-OHDA-induced neuronal cell death in cellular PD models. Also, these effects are mediated partly by inhibiting activation of the MAPK and PI3K/Akt/GSK-3β pathways.

Change of Extracellular Glutamate Level in Striatum during Deep Brain Stimulation of the Entopeduncular Nucleus in Rats

OBJECTIVE: Globus pallidus interna (GPi) is acknowledged as an essential treatment for advanced Parkinson’s disease (PD). Nonetheless, the neurotransmitter study about its results is undiscovered. The goal of this research was to examine influences of entopeduncular nucleus (EPN) stimulation, identical to human GPi, in no-lesioned (NL) rat and 6-hydroxydopamine (6-HD)-lesioned rat on glutamate change in the striatum.METHODS: Extracellular glutamate level changes in striatum of NL category, NL with deep brain stimulation (DBS) category, 6-HD category, and 6-HD with DBS category were examined using microdialysis and high-pressure liquid chromatography. Tyrosine hydroxylase (TH) immunoreactivities in substantia nigra and striatum of the four categories were also analyzed.RESULTS: Extracellular glutamate levels in the striatum of NL with DBS category and 6-HD with DBS category were significantly increased by EPN stimulation compared to those in the NL category and 6-HD category. EPN stimulation had no significant effect on the expression of TH in NL or 6-HD category.CONCLUSION: Clinical results of GPi DBS are not only limited to direct inhibitory outflow to thalamus. They also include extensive alteration within basal ganglia.

Quantification of Tyrosine Hydroxylase and ErbB4 in the Locus Coeruleus of Mood Disorder Patients Using a Multispectral Method to Prevent Interference with Immunocytochemical Signals by Neuromelanin / 神经科学通报·英文版

The locus coeruleus (LC) has been studied in major depressive disorder (MDD) and bipolar disorder (BD). A major problem of immunocytochemical studies in the human LC is interference with the staining of the immunocytochemical end-product by the omnipresent natural brown pigment neuromelanin. Here, we used a multispectral method to untangle the two colors: blue immunocytochemical staining and brown neuromelanin. We found significantly increased tyrosine hydroxylase (TH) in the LC of MDD patients-thus validating the method-but not in BD patients, and we did not find significant changes in the receptor tyrosine-protein kinase ErbB4 in the LC in MDD or BD patients. We observed clear co-localization of ErbB4, TH, and neuromelanin in the LC neurons. The different stress-related molecular changes in the LC may contribute to the different clinical symptoms in MDD and BD.

Involvement of catecholamines in the myocardium of rats submitted to experimental model of portal hypertension / Envolvimento das catecolaminas no miocárdio de ratos submetidos a modelo experimental de hipertensão portal

ABSTRACT Background: The role of autonomic nervous system in the development and maintenance of portal hypertension is not fully elucidated. It is known that the gene expression of norepinephrine in the superior mesenteric artery varies with time, and it may contribute for splanchnic vasodilation and its consequent hemodynamic repercussions. It is still not known exactly how the adrenergic expression behaves at the heart level in the initial stages of this process. Aim: To evaluate the immunohistochemical expression of the enzyme tyrosine hydroxylase (tyrosine 3-monooxygenase), involved in the synthesis of norepinephrine, in the myocardium of rats submitted to partial ligation of the portal vein. Methods: Twenty-four Wistar rats were divided into two groups: Sham Operated and Portal Hypertension. The partial ligation was performed in the Portal Hypertension group, and after 1/6/24 h and 3/5/14 days the animals were euthanized. Immunohistochemical analysis was performed to quantify the expression of the stained enzyme using the ImageJ program. Results: The Portal Hypertension group expressed percentages between 4.6-6% of the marked area, while the Sham Operated group varied between 4-5%. Although there was no statistical significance, the percentage stained in the Portal Hypertension group followed an increasing pattern in the first 6 h and a decreasing pattern after 24 h, which was not observed in the Sham Operated group. Conclusion: The expression of noradrenaline in rat myocardium during the first two weeks after partial ligation of the portal vein, with tyrosine hydroxylase as marker, did not show differences between groups over time.

RESUMO Racional: O papel do sistema nervoso autônomo na hipertensão portal não está completamente elucidado. Sabe-se que, nessa condição, a expressão gênica da norepinefrina na artéria mesentérica superior modifica-se com o tempo, podendo ser importante contribuinte para a vasodilatação esplâncnica e suas repercussões hemodinâmicas. Apesar dos estudos sobre as repercussões cardiovasculares na hipertensão portal, ainda não se sabe como a expressão adrenérgica se comporta a nível cardíaco nas etapas iniciais desse processo. Objetivo: Avaliar a expressão imunoistoquímica da enzima tirosina hidroxilase (tirosina 3-mono-oxigenase), relacionada à síntese da norepinefrina, no miocárdio de ratos submetidos à ligadura parcial da veia porta. Métodos: Foram utilizados 24 ratos, distribuídos em dois grupos: Sham Operated e Hipertensão Portal. A ligadura parcial da veia porta foi realizada apenas no grupo Hipertensão Portal e, após 1/6/24 h e 3/5/14 dias, os animais foram eutanasiados. Foi feita a análise imunoistoquímica para quantificar a expressão da enzima corada, utilizando o programa ImageJ. Resultados: No grupo Hipertensão Portal, o miocárdio expressou percentuais entre 4,6-6% de área marcada, enquanto que no grupo Sham Operated variou entre 4-5%, sem significância estatística. Apenas no grupo Hipertensão Portal, a porcentagem corada pela enzima seguiu padrão crescente nas primeiras 6 h e decrescente após 24 h. Conclusão: A expressão da noradrenalina no miocárdio de ratos durante as primeiras duas semanas após a ligadura parcial da veia porta, tendo como marcador a enzima tirosina hidroxilase, não apresentou diferenças entre grupos ao longo do tempo.

Dexmedetomidine Ameliorates Sleep Deprivation-Induced Depressive Behaviors in Mice / 대한배뇨장애요실금학회지

PURPOSE: Sleep deprivation induces depressive symptoms. Dexmedetomidine is a α2-adrenoreceptor agonist and this drug possesses sedative, anxiolytic, analgesic, and anesthetic-sparing effect. In this study, the action of dexmedetomidine on sleep deprivation-induced depressive behaviors was investigated using mice. METHODS: For the inducing of sleep deprivation, the mice were placed inside a water cage containing 15 platforms and filled with water up to 1 cm below the platform surface for 7 days. One day after sleep deprivation, dexmedetomidine at the respective dosage (0.5, 1, and 2 μg/kg) was intraperitoneally treated into the mice, one time per a day during 6 days. Then, forced swimming test and tail suspension test were conducted. Immunohistochemistry for tyrosine hydroxylase (TH), 5-hydroxytryptamine (5-HT; serotonin), tryptophan hydroxylase (TPH) and western blot for D1 dopamine receptor were also performed. RESULTS: Sleep deprivation increased the immobility latency in the forced swimming test and tail suspension test. The expressions of TPH, 5-HT, and D1 dopamine receptor were decreased, whereas, TH expression was increased by sleep deprivation. Dexmedetomidine decreased the immobility latency and increased the expressions of TPH, 5-HT, and D1 dopamine receptor, whereas, HT expression was decreased by dexmedetomidine treatment. CONCLUSIONS: In our results, dexmedetomidine alleviated sleep deprivation-induced depressive behaviors by increasing 5-HT synthesis and by decreasing dopamine production with up-regulation of D1 dopamine receptor.

Interleukin-4 Contributes to Degeneration of Dopamine Neurons in the Lipopolysaccharide-treated Substantia Nigra in vivo

The present study investigated the effects of interleukin (IL)-4 on dopamine (DA) neurons in the substantia nigra (SN) in vivo of lipopolysaccharide (LPS)-treated rat. Tyrosine hydroxylase immunohistochemistry showed a significant loss of nigral DA neurons at 3 and 7 day post-LPS. In parallel, IL-4 immunoreactivity was upregulated as early as 1 day, reached a peak at 3 day and remained elevated at 7 day post-LPS. IL-4 immunoreactivity was detected exclusively in microglia. IL-4 neutralizing antibody (NA) significantly increased survival of DA neurons in LPS-treated SN in vivo by inhibiting microglial activation and production of proinflammatory mediator such as IL-1β as assessed by immunihistochemical, RT-PCR and ELISA analysis, respectively. Accompanying neuroprotection are IL-4NA effects on decreased disruption of blood-brain barrier and astrocytes. The present data suggest that endogenously expressed IL-4 from reactive microglia may be involved in the neuropathological processes of degeneration of DA neurons occurring in Parkinson's disease.

Astrocyte Specificity and Coverage of hGFAP-CreERT2 Tg(GFAP-Cre/ERT2)13Kdmc Mouse Line in Various Brain Regions

Astrocyte is the most abundant cell type in the central nervous system and its importance has been increasingly recognized in the brain pathophysiology. To study in vivo function of astrocyte, astrocyte-specific gene-targeting is regarded as a powerful approach. Especially, hGFAP-CreERT2, which expresses tamoxifen-inducible Cre recombinase under the human GFAP promoter, has been developed and characterized from several research groups. However, one of these mouse lines, [Tg(GFAP-Cre/ERT2)13Kdmc] from Ken McCarthy group has not been quantitatively analyzed, despite its frequent use. Here, we performed comprehensive characterization of this mouse line with quantitative analysis. By crossing this mouse line with Ai14 (RCL-tdTomato), a very sensitive Cre reporter mouse line, we visualized the Cre-expressing cells in various brain regions. For quantitative analysis, we immunostained S100β as an astrocytic marker and NeuN, tyrosine hydroxylase or calbindin as a neuronal marker in different brain regions. We calculated ‘astrocyte specificity’ as the proportion of co-labelled S100β and tdTomato positive cells in the total number of tdTomato positive cells and the ‘astrocyte coverage’ as the proportion of co-labelled S100β and tdTomato positive cells in the total number of S100β positive cells. Interestingly, we found varying degree of astrocyte specificity and coverage in each brain region. In cortex, hypothalamus, substantia nigra pars compacta and cerebellar Purkinje layer, we observed high astrocyte specificity (over 89%) and relatively high astrocyte coverage (over 70%). In striatum, hippocampal CA1 layer, dentate gyrus and cerebellar granule layer, we observed high astrocyte specificity (over 80%), but relative low astrocyte coverage (50–60%). However, thalamus and amygdala showed low astrocyte specificity (about 65%) and significant neuron specificity (over 30%). This hGFAP-CreERT2 mouse line can be useful for genetic modulations of target gene either in gain-of-function or loss-of-function studies in the brain regions with high astrocyte specificity and coverage. However, the use of this mouse line should be restricted to gain-of-function studies in the brain regions with high astrocyte specificity but low coverage. In conclusion, hGFAP-CreERT2 mouse line could be a powerful tool for gene-targeting of astrocytes in cortex, striatum, hippocampus, hypothalamus, substantia nigra pars compacta and cerebellum, but not in thalamus and amygdala.

Berberine alleviates symptoms of anxiety by enhancing dopamine expression in rats with post-traumatic stress disorder

Post-traumatic stress disorder (PTSD) is a trauma-induced psychiatric disorder characterized by impaired fear extermination, hyperarousal, anxiety, depression, and amnesic symptoms that may involve the release of monoamines in the fear circuit. The present study measured several anxiety-related behavioral responses to examine the effects of berberine (BER) on symptoms of anxiety in rats after single prolonged stress (SPS) exposure, and to determine if BER reversed the dopamine (DA) dysfunction. Rats received BER (10, 20, or 30 mg/kg, intraperitoneally, once daily) for 14 days after SPS exposure. BER administration significantly increased the time spent in the open arms and reduced grooming behavior during the elevated plus maze test, and increased the time spent in the central zone and the number of central zone crossings in the open field test. BER restored neurochemical abnormalities and the SPS-induced decrease in DA tissue levels in the hippocampus and striatum. The increased DA concentration during BER treatment may partly be attributed to mRNA expression of tyrosine hydroxylase and the DA transporter in the hippocampus, while BER exerted no significant effects on vesicular monoamine transporter mRNA expression in the hippocampus of rats with PTSD. These results suggest that BER had anxiolytic-like effects on behavioral and biochemical measures associated with anxiety. These findings support a role for reduced anxiety altered DAergic transmission and reduced anxiety in rats with PTSD. Thus, BER may be a useful agent to treat or alleviate psychiatric disorders like those observed in patients with PTSD.

Ameliorative Effects of Ombuoside on Dopamine Biosynthesis in PC12 Cells

This study investigated the effects of ombuoside, a flavonol glycoside, on dopamine biosynthesis in PC12 cells. Ombuoside at concentrations of 1, 5, and 10 µM increased intracellular dopamine levels at 1 – 24 h. Ombuoside (1, 5, and 10 µM) also significantly increased the phosphorylation of tyrosine hydroxylase (TH) (Ser40) and cyclic AMP-response element binding protein (CREB) (Ser133) at 0.5 – 6 h. In addition, ombuoside (1, 5, and 10 µM) combined with L-DOPA (20, 100, and 200 µM) further increased intracellular dopamine levels for 24 h compared to L-DOPA alone. These results suggest that ombuoside regulates dopamine biosynthesis by modulating TH and CREB activation in PC12 cells.