Neuromelanin in Parkinson’s Disease: Tyrosine Hydroxylase and Tyrosinase

Parkinson’s disease (PD) is an aging-related disease and the second most common neurodegenerative disease after Alzheimer’s disease. The main symptoms of PD are movement disorders accompanied with deficiency of neurotransmitter dopamine (DA) in the striatum due to cell death of the nigrostriatal DA neurons. Two main histopathological hallmarks exist in PD: cytosolic inclusion bodies termed Lewy bodies that mainly consist of α-synuclein protein, the oligomers of which produced by misfolding are regarded to be neurotoxic, causing DA cell death; and black pigments termed neuromelanin (NM) that are contained in DA neurons and markedly decrease in PD. The synthesis of human NM is regarded to be similar to that of melanin in melanocytes; melanin synthesis in skin is via DOPAquinone (DQ) by tyrosinase, whereas NM synthesis in DA neurons is via DAquinone (DAQ) by tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC). DA in cytoplasm is highly reactive and is assumed to be oxidized spontaneously or by an unidentified tyrosinase to DAQ and then, synthesized to NM. Intracellular NM accumulation above a specific threshold has been reported to be associated with DA neuron death and PD phenotypes. This review reports recent progress in the biosynthesis and pathophysiology of NM in PD.     

Production of salicylate hydroxylase from Pseudomonas putida UUC-1 and its application in the construction of a biosensor

The enzyme salicylate hydroxylase was produced from Pseudomonas putida UUC-1 in an 80 dm³ 16 h batch fermentation process yielding, ～ 9 K units per 60 dm³. This enzyme preparation has been studied for its application in the construction of biosensor systems, e.g. carbon past electrodes, screen-printed carbon electrodes, and disposable carbon enzyme electrodes, which will be used for the rapid estimation of salicylate in blood sample. The linear range of the disposable carbon enzyme electrode in response to salicylate was achieved to 1·8 mmol dm^?3.     

TGFβ-1 Induced Cross-Linking of the Extracellular Matrix of Primary Human Dermal Fibroblasts

Excessive cross-linking is a major factor in the resistance to the remodelling of the extracellular matrix (ECM) during fibrotic progression. The role of TGFβ signalling in impairing ECM remodelling has been demonstrated in various fibrotic models. We hypothesised that increased ECM cross-linking by TGFβ contributes to skin fibrosis in Systemic Sclerosis (SSc). Proteomics was used to identify cross-linking enzymes in the ECM of primary human dermal fibroblasts, and to compare their levels following treatment with TGFβ-1. A significant upregulation and enrichment of lysyl-oxidase-like 1, 2 and 4 and transglutaminase 2 were found. Western blotting confirmed the upregulation of lysyl hydroxylase 2 in the ECM. Increased transglutaminase activity in TGFβ-1 treated ECM was revealed from a cell-based assay. We employed a mass spectrometry-based method to identify alterations in the ECM cross-linking pattern caused by TGFβ-1. Cross-linking sites were identified in collagens I and V, fibrinogen and fibronectin. One cross-linking site in fibrinogen alpha was found only in TGFβ-treated samples. In conclusion, we have mapped novel cross-links between ECM proteins and demonstrated that activation of TGFβ signalling in cultured dermal fibroblasts upregulates multiple cross-linking enzymes in the ECM.     

Failure of Diphtheria Toxin Model to Induce Parkinson-Like Behavior in Mice

Rodent models of Parkinson’s disease are based on transgenic expression of mutant synuclein, deletion of PD genes, injections of MPTP or rotenone, or seeding of synuclein fibrils. The models show histopathologic features of PD such as Lewi bodies but mostly only subtle in vivo manifestations or systemic toxicity. The models only partly mimic a predominant loss of dopaminergic neurons in the substantia nigra. We therefore generated mice that express the transgenic diphtheria toxin receptor (DTR) specifically in DA neurons by crossing DAT-Cre mice with Rosa26 loxP-STOP-loxP DTR mice. After defining a well-tolerated DTx dose, DAT-DTR and DTR-flfl controls were subjected to non-toxic DTx treatment (5 × 100 pg/g) and subsequent histology and behavioral tests. DAT protein levels were reduced in the midbrain, and tyrosine hydroxylase-positive neurons were reduced in the substantia nigra, whereas the pan-neuronal marker NeuN was not affected. Despite the promising histologic results, there was no difference in motor function tests or open field behavior. These are tests in which double mutant Pink1^−/−SNCA^A53T Parkinson mice show behavioral abnormalities. Higher doses of DTx were toxic in both groups. The data suggest that DTx treatment in mice with Cre/loxP-driven DAT-DTR expression leads to partial ablation of DA-neurons but without PD-reminiscent behavioral correlates.     

酪氨酸羟化酶阳性神经元在扬子鳄、家鸡及大鼠脑桥的分布比较

目的: 观察并比较扬子鳄、家鸡及大鼠脑桥酪氨酸羟化酶(Tyrosine hydroxylase,TH)阳性神经元的形态和分布,为生物进化及扬子鳄种群遗传保护与神经系统疾病防治提供比较解剖学资料。方法: 扬子鳄6只、家鸡和SD大鼠各15只。三种动物再各分三组进行分次实验,每次扬子鳄2只,家鸡和SD大鼠各5只,同一时间内电击处死后,取脑桥组织,采用免疫组织化学方法结合黑马病理图像分析系统检测三种动物脑桥TH阳性神经元的分布特征。结果: 三种动物脑桥均见TH阳性神经元成团或散在分布,均可见椭圆形、圆形、梭形、多角形等,但三种动物间脑桥不同形态的细胞数量大多存在差异(P<0.01);三种动物脑桥TH阳性神经元均以中、小细胞为主,大细胞相对较少;与扬子鳄相比,家鸡及大鼠脑桥同节段TH阳性神经元数均明显增加,灰度值降低,均具有统计学差异(P＜0.01);与家鸡相比,大鼠脑桥TH阳性神经元数明显增加,灰度值降低,均具有统计学差异(P＜0.01)。结论: 扬子鳄、家鸡及大鼠脑桥TH神经元数量依次增多,细胞平均灰度值依次降低,不同形态的细胞比例存在一定差异,提示脑桥TH神经元的分布差异可能与不同物种脑桥执行其相关神经系统生理功能的不同有关。     

Toxic and Phenotypic Effects of AAV_Cre Used to Transduce Mesencephalic Dopaminergic Neurons

A popular approach to spatiotemporally target genes using the loxP/Cre recombination system is stereotaxic microinjection of adeno-associated virus (AAV) expressing Cre recombinase (AAV_Cre) in specific neuronal structures. Here, we report that AAV_Cre microinjection in the ventral tegmental area (VTA) of ErbB4 Cyt-1-floxed (ErbB4 Cyt-1^fl/fl) mice at titers commonly used in the literature (~10¹²–10¹³ GC/mL) can have neurotoxic effects on dopaminergic neurons and elicit behavioral abnormalities. However, these effects of AAV_Cre microinjection are independent of ErbB4 Cyt-1 recombination because they are also observed in microinjected wild-type (WT) controls. Mice microinjected with AAV_Cre (10¹²–10¹³ GC/mL) exhibit reductions of tyrosine hydroxylase (TH) and dopamine transporter (DAT) expression, loss of dopaminergic neurons, and they behaviorally become hyperactive, fail to habituate in the open field and exhibit sensorimotor gating deficits compared to controls microinjected with AAV_GFP. Importantly, these AAV_Cre non-specific effects are: (1) independent of serotype, (2) occur with vectors expressing either Cre or Cre-GFP fusion protein and (3) preventable by reducing viral titers by 1000-fold (10¹⁰ GC/mL), which retains sufficient recombination activity to target floxed genes. Our studies emphasize the importance of including AAV_Cre-injected WT controls in experiments because recombination-independent effects on gene expression, neurotoxicity and behaviors could be erroneously attributed to consequences of gene ablation.     

Synergistic Actions of Tailoring Enzymes in Pradimicin Biosynthesis

Three key tailoring enzymes in pradimicin biosynthesis: PdmJ, PdmW, and PdmN, were investigated. PdmW was characterized as the C‐6 hydroxylase by structural characterization of the corresponding product, 6‐hydroxy‐G‐2A. The efficiencies of the C‐5 and C‐6 hydroxylations, catalyzed respectively by PdmJ and PdmW, were low when they were expressed individually with the early biosynthetic enzymes that form G‐2A. When these two cytochrome P450 enzymes were co‐expressed, a dihydroxylated product, 5,6‐dihydroxy‐G‐2A, was efficiently produced, indicating that these two enzymes work synergistically in pradimicin biosynthesis. Heterologously expressed PdmN in Streptomyces coelicolor CH999 converted G‐2A to JX137a by ligating a unit of D ‐alanine to the carboxyl group. PdmN has relaxed substrate specificity toward both amino acid donors and acceptors. Through combinatorial biosynthesis, a series of new pradimicin analogues were produced.     

Strategic Modification of Gut Microbiota through Oral Bacteriotherapy Influences Hypoxia Inducible Factor-1α: Therapeutic Implication in Alzheimer’s Disease

Dysbiosis contributes to Alzheimer’s disease (AD) pathogenesis, and oral bacteriotherapy represents a promising preventative and therapeutic opportunity to remodel gut microbiota and to delay AD onset and progression by reducing neuroinflammation and amyloid and tau proteins aggregation. Specifically, SLAB51 multi-strain probiotic formulation positively influences multiple neuro-chemical pathways, but exact links between probiotics oral consumption and cerebral beneficial effects remain a gap of knowledge. Considering that cerebral blood oxygenation is particularly reduced in AD and that the decreased neurovascular function contributes to AD damages, hypoxia conditioning represents an encouraging strategy to cure diseases of the central nervous system. In this work, 8-week-old 3xTg-AD and wild-type mice were chronically supplemented with SLAB51 to evaluate effects on hypoxia-inducible factor-1α (HIF-1α), a key molecule regulating host-microbial crosstalk and a potential target in neurodegenerative pathologies. We report evidence that chronic supplementation with SLAB51 enhanced cerebral expression of HIF-1α and decreased levels of prolyl hydroxylase 2 (PHD2), an oxygen dependent regulator of HIF-1α degradation; moreover, it successfully counteracted the increase of inducible nitric oxide synthase (iNOS) brain expression and nitric oxide plasma levels in AD mice. Altogether, the results demonstrate an additional mechanism through which SLAB51 exerts neuroprotective and anti-inflammatory effects in this model of AD.