Expression of the LRRK2 gene in the midbrain dopaminergic neurons of the substantia nigra

A hallmark of Parkinson's disease (PD) is the progressive loss of the A9 midbrain dopaminergic (mDA) neurons in the substantia nigra pars compacta. Recently, multiple causative mutations have been identified in the leucine-rich repeat kinase 2 (LRRK2) gene for both familial and sporadic PD cases. Therefore, to investigate functional roles of LRRK2 in normal and/or diseased brain, it is critical to define LRRK2 expression in mDA neurons. To address whether LRRK2 mRNA and protein are expressed in mDA neurons, we purified DA neurons from the tyrosine hydroxylase (TH)-GFP transgenic mouse using FACS-sorting and analyzed the expression of LRRK2 and other mDA markers. We observed that all mDA markers tested in this study (TH, Pitx3, DAT, Nurr1 and Lmx1a) are robustly expressed only in GFP(+) cells, but not in GFP(-) cells. Notably, LRRK2 was expressed in both GFP(+) and GFP(-) cells. Consistent with this, our immunohistochemical analyses showed that LRRK2 is expressed in TH-positive mDA neurons as well as in surrounding TH-negative cells in the rat brain. Importantly, in the midbrain region, LRRK2 protein was preferentially expressed in A9 DA neurons of the substantia nigra, compared to A10 DA neurons of the ventral tegmental area. However, LRRK2 was also highly expressed in the cortical and hippocampal regions. Taken together, our results suggest that LRRK2 may have direct functional role(s) in the neurophysiology of A9 DA neurons and that dysfunction of these neurons by mutant LRRK2 may directly cause their selective degeneration.     

脑内炎症对黑质多巴胺能神经元的选择性损伤作用

目的 探讨脑内炎症反应对中脑黑质多巴胺能神经元的选择性变性作用.方法 健康SD雄性大鼠10只,随机分为实验组和对照组,实验组行脂多糖(LPS)右侧脑室定位注射,对照组注射生理盐水.注射后48周用免疫组织化学或组织化学法观察黑质多巴胺能、中缝核5-羟色胺能及基底核胆碱能3种不同类型神经元的变性及上述不同脑区小胶质细胞的激活情况.结果 免疫组织化学染色显示,LPS组在黑质、海马、纹状体、中缝核部位均可见到OX6阳性小胶质细胞,说明不同脑区均出现炎症反应.不同类型神经元染色结果显示,LPS组黑质多巴胺能神经元胞体变小、染色变浅、突起减少甚至消失,神经元数量比对照组减少40.1%(P<0.01);5-羟色胺能神经元及胆碱能神经元形态及数量均无明显改变.结论脑室注射LPS导致的脑内炎症反应可选择性引起黑质多巴胺能神经元变性损伤.     

Role of cellular FKBP52 protein in intracellular trafficking of recombinant adeno-associated virus 2 vectors

We have reported that tyrosine-phosphorylated forms of a cellular protein, FKBP52, inhibit the second-strand DNA synthesis of adeno-associated virus 2 (AAV), leading to inefficient transgene expression from recombinant AAV vectors. To further explore the role of FKBP52 in AAV-mediated transduction, we established murine embryo fibroblasts (MEFs) cultures from FKBP52 wild-type (WT), heterozygous (HE), and knockout (KO) mice. Conventional AAV vectors failed to transduce WT MEFs efficiently, and the transduction efficiency was not significantly increased in HE or KO MEFs. AAV vectors failed to traffic efficiently to the nucleus in these cells. Treatment with hydroxyurea (HU) increased the transduction efficiency of conventional AAV vectors by approximately 25-fold in WT MEFs, but only by approximately 4-fold in KO MEFs. The use of self-complementary AAV (scAAV) vectors, which bypass the requirement of viral second-strand DNA synthesis, revealed that HU treatment increased the transduction efficiency approximately 23-fold in WT MEFs, but only approximately 4-fold in KO MEFs, indicating that the lack of HU treatment-mediated increase in KO MEFs was not due to failure of AAV to undergo viral second-strand DNA synthesis. Following HU treatment, approximately 59% of AAV genomes were present in the nuclear fraction from WT MEFs, but only approximately 28% in KO MEFs, indicating that the pathway by which HU treatment mediates nuclear transport of AAV was impaired in KO MEFs. When KO MEFs were stably transfected with an FKBP52 expression plasmid, HU treatment-mediated increase in the transduction efficiency was restored in these cells, which correlated directly with improved intracellular trafficking. Intact AAV particles were also shown to interact with FKBP52 as well as with dynein, a known cellular protein involved in AAV trafficking. These studies suggest that FKBP52, being a cellular chaperone protein, facilitates intracellular trafficking of AAV, which has implications in the optimal use of recombinant AAV vectors in human gene therapy.     

Alterations in the histaminergic system in the substantia nigra and striatum of Parkinson's patients: a postmortem study

Earlier studies showed neuronal histamine production in the hypothalamic tuberomamillary nucleus to be unchanged in Parkinson's disease (PD), whereas the histamine levels and innervation in the substantia nigra (SN) increased. In the present study we used quantitative polymerase chain reaction (qPCR) to assess the changes in the histaminergic system in the SN, caudate nucleus (CN), and putamen (PU) in 7 PD patients and 7 controls. The messenger RNA (mRNA) expression of the histamine receptor-3 (H(3)R), which was localized immunocytochemically in the large pigmented neurons, was significantly decreased in the SN in PD, while histamine receptor-4 (H(4)R)-mRNA expression showed a significant increase in caudate nucleus and PU. In addition, significantly increased mRNA levels of histamine methyltransferase (HMT), a key enzyme involved in histamine metabolism, were found in the SN and in the PU in PD. Moreover, in the SN, the histamine methyltransferase-mRNA showed a strong negative correlation with PD disease duration. Our observations imply the presence of local changes in the histaminergic system that may contribute to PD pathology, and may thus provide a rationale for possible novel therapeutic strategies.     

Rafts, anchors and viruses--a role for glycosylphosphatidylinositol anchored proteins in the modification of enveloped viruses and viral vectors

Lipid rafts have been proposed as sites for the assembly of a number of viruses and are considered to play a major role in pseudotyping events. As a consequence, host glycosylphosphatidylinositol (GPI) anchored proteins commonly associated with lipid rafts can be found being incorporated into viral lipid envelopes with beneficial consequences for viral replication. In this review we will look at the link between lipid rafts, GPI-anchored proteins and retroviral particles and how these relationships can be exploited for the modification of enveloped viruses.     

激活小胶质细胞对帕金森病模型大鼠黑质多巴胺能神经元的影响

目的 探讨脂多糖(LPS)激活小胶质细胞(MG)对黑质损伤多巴胺(DA)能神经元细胞存活的影响.方法 采用立体定向技术向大鼠单侧黑质(SNpc)内注入LPS激活MG后,隔48h于原位注射高、低剂量鱼藤酮(BT)分别建立重度及轻度损伤模型;采用免疫组化法观察黑质酪氨酸羟化酶(TH)阳性细胞和离子钙结合转接分子1(Ib α...     

携带vIL-10的逆转录病毒重组体的构建及体外表达研究

目的构建携带vIL-10表达序列的重组逆转录病毒rRV-vIL-10并检测该重组病毒对体外培养的兔滑膜细胞的转染情况和目的基因的表达水平。方法①设计带有酶切位点的引物,对含有目的基因的质粒进行PCR扩增并纯化目的基因片段。②双酶切目的基因vIL-10与逆转录病毒载体pLXSN,将pLXSN与vIL-10进行定向克隆连接,筛出阳性克隆并进行鉴定。③扩增逆转录病毒重组体rRV-vIL-10,与辅助质粒pVSVG通过磷酸钙-共沉淀法共转染GP-293包装细胞,收集病毒并测定滴度。④将获得的重组逆转录病毒rRV-vIL-10转染体外培养的兔滑膜成纤维样细胞。细胞免疫组化测定目的蛋白的表达。结果①成功构建了携带治疗基因的重组逆转录病毒rRV-vIL-10,病毒滴度为5×106cfu/ml。②rRV-vIL-10能有效转染体外培养的兔滑膜成纤维样细胞,细胞免疫组化法可检测到vIL-10的表达。结论①成功构建了携带治疗基因vIL-10的重组逆转录病毒rRV-vIL-10。②以逆转录病毒为载体可以成功地将vIL-10基因导入体外培养的兔滑膜成纤维样细胞并表达vIL-10蛋白。     

Effect of antibodies against AMPA glutamate receptors on brain neurons in primary cultures of the cerebellum and hippocampus

Rabbit antibodies against GluR₁ subunit of AMPA glutamate receptors in a concentration of 1 μg/ml significantly increased intracellular Ca²⁺ concentration and decreased mitochondrial potential in hippocampal neurons, i.e. produced changes typical of the influence of glutamate in toxic concentrations. In cerebellar neurons rabbit antibodies potentiated glutamate-induced increase in intracellular Ca²⁺ concentration and significantly decreased the mitochondrial potential (compared to the level observed after application of glutamate alone). The exposure of cultured cerebellar neurons to antibodies in a concentration of 0.1 μg/ml for 24 h was followed by a 50% decrease in ATP concentration and development of neuronal necrosis. Our results attest to an important role of autoimmune damage to neurons during hyperstimulation of glutamate receptors. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 7, pp. 59–62, July, 2006     

Degeneration of dopaminergic neurons in the substantia nigra of zitter mutant rat and protection by chronic intake of Vitamin E

Dopaminergic cell death in the ventral and dorsal tiers of substantia nigra pars copmacta (SNc) and their prevention by anti-oxidant diet was immunohistochemically studied in the zitter mutant rats, which are characterized by abnormal metabolism of superoxide. Similar to previous reports, the number of SNc neurons in Nissl-stained section decreased with age. Tyrosine hydroxylase (TH) immunohistochemistry demonstrated that the dopaminergic neurons in the ventral tier of SNc degenerated early, whereas the dorsal tier gradually degenerated with age. Thus, the ventral tier dopaminergic neurons are affected first, but the dorsal tier neurons do become impact by the zi/zi mutation. Following 9-month period after weaning, zitter rats supplemented with 500 mg d,l--tocophenol (VE(+))/kg diet exhibited a significant increased of surviving TH-immunoreactive neurons in both the tiers of SNc as compared with the zi/zi rats with control and VE(−) diets. These results suggest that VE supplement may slow the dopaminergic cell loss in zitter mutant rat, and further support that degeneration of the dopaminergic neurons in this mutant rat is caused by oxidant stress. Thus, the zitter rat may represent a good model for studying the dopaminergic cell death by superoxide species.     

The conserved glycine residues in the transmembrane domain of the Semliki Forest virus fusion protein are not required for assembly and fusion

The alphavirus Semliki Forest virus (SFV) infects cells via a low pH-triggered fusion reaction mediated by the viral E1 protein. Both the E1 fusion peptide and transmembrane (TM) domain are essential for membrane fusion, but the functional requirements for the TM domain are poorly understood. Here we explored the role of the five TM domain glycine residues, including the highly conserved glycine pair at E1 residues 415/416. SFV mutants with alanine substitutions for individual or all five glycine residues (5G/A) showed growth kinetics and fusion pH dependence similar to those of wild-type SFV. Mutants with increasing substitution of glycine residues showed an increasingly more stringent requirement for cholesterol during fusion. The 5G/A mutant showed decreased fusion kinetics and extent in fluorescent lipid mixing assays. TM domain glycine residues thus are not required for efficient SFV fusion or assembly but can cause subtle effects on the properties of membrane fusion.     

Tropism and toxicity of adeno-associated viral vector serotypes 1, 2, 5, 6, 7, 8, and 9 in rat neurons and glia in vitro

Recombinant adeno-associated viral (rAAV) vectors are frequently used for gene delivery to the central nervous system and are capable of transducing neurons and glia in vitro. In this study, seven serotypes of a rAAV vector expressing green fluorescent protein (GFP) were characterized for tropism and toxicity in primary cortical cells derived from embryonic rat brain. At 2 days after transduction, serotypes 1 and 5 through 8 expressed GFP predominately in glia, but by 6 days post-transduction expression was neuronal except for AAV5. AAV2 and 9 produced minimal GFP expression. Using cell viability assays, toxicity was observed at higher multiplicities of infection (MOI) for all serotypes except AAV2 and 9. The toxicity of AAV1 and 5-8 affected mostly glia as indicated by a loss of glial-marker immunoreactivity. A frameshift mutation in the GFP gene reduced overall toxicity for serotypes 1, 5 and 6, but not 7 and 8 suggesting that the toxicity was not solely due to the overexpression of GFP. Collectively, a differential tropism and toxicity was observed among the AAV serotypes on primary cortical cultures with an overall preferential glial transduction and toxicity.     

Evidence for in vitro and in vivo autocatalytic processingof the primary translation product of beet necrotic yellowvein virus RNA 1 by a papain-like proteinase

Summary.  Beet necrotic yellow vein virus RNA 1 contains a single long ORF corresponding to the theoretical translation product of 237 kDa which contains the information necessary for replication of the viral genome. This ORF contains a putative papain-like proteinase domain which has been localized, on the basis of sequence alignments, between the helicase and polymerase domains. Here we show that the RNA 1 primary translation product can be cleaved autocatalytically in vitro into two species of 150 kDa and 66 kDa, the latter of which probably contains the entire polymerase domain. A 66 kDa protein was detected immunologically in infected C. quinoa protoplasts using an antiserum specific for the C-terminal region of the RNA 1 primary translation product, confirming that processing also occurs in vivo. Accepted October 30, 1996 Received September 10, 1996     

Lymphatic endothelium-specific hyaluronan receptor LYVE-1 is expressed by stabilin-1+, F4/80+, CD11b+ macrophages in malignant tumours and wound healing tissue in vivo and in bone marrow cultures in vitro: implications for the assessment of lymphangiogenesis

Lymphangiogenesis is a novel prognostic parameter for several cancers that is preferentially quantified by immunohistochemistry of the lymphatic endothelium-specific hyaluronan receptor LYVE-1. Recently, the specificity of LYVE-1 was challenged by serendipitous observations of LYVE-1 expression in rare tissue macrophages. As expression of the hyaluronan receptor-like molecule stabilin-1 is shared by sinusoidal endothelium and macrophages, a thorough analysis of LYVE-1 expression was performed using macrophage-specific markers in vivo and in vitro. In murine tumour models and excisional wound healing, LYVE-1 expression occurred in a subset of CD11b(+), F4/80(+) tissue macrophages that preferentially co-expressed stabilin-1. Upon comparison of single- and double-labelling immunofluorescence, it became apparent that LYVE-1(+) macrophages mimic sprouting and collapsed lymphatic vessels. In vitro, LYVE-1 expression was induced in 25-40% of murine bone marrow-derived macrophages upon exposure to B16F1 melanoma-conditioned medium and IL-4/dexamethasone. By FACS analysis, 11.5% of bone marrow-derived macrophages were LYVE-1(+), stabilin-1(+) double-positive, while 9.9% were LYVE-1(+), stabilin-1(-) and 33.5% were LYVE-1(-), stabilin-1(+). Northern and western analyses confirmed expression of LYVE-1 mRNA and protein in bone marrow-derived macrophages. In the light of the current debate about true endothelial trans-differentiation versus endothelial mimicry of monocytes/macrophages, LYVE-1(+), stabilin-1(+) non-continuous endothelial-like macrophages will require further developmental and functional analyses. In conclusion, the findings imply that LYVE-1 staining must be supplemented by double labelling with macrophage markers in order to differentiate clearly between LYVE-1(+) lymphatics and LYVE-1(+) tumour-infiltrating macrophages. This improved approach will help to clarify the prognostic significance of lymphangiogenesis in malignant tumours.