Prevention of intracellular degradation of I2020T mutant LRRK2 restores its protectivity against apoptosis

Leucine-rich repeat kinase 2 (LRRK2) is the causal gene for autosomal dominant familial Parkinson’s disease. We have previously reported a novel molecular feature characteristic to I2020T mutant LRRK2: higher susceptibility to post-translational degradation than the wild-type LRRK2. In the present study, we demonstrated that the protective effect of I2020T LRRK2 against hydrogen peroxide-induced apoptosis was impaired in comparison with the wild-type molecule. When the intracellular level of the protein had been allowed to recover by treatment with proteolysis inhibitors, the protective effect of I2020T LRRK2 against apoptosis was increased. We further confirmed that a decrease in the intracellular protein level of WT LRRK2 by knocking down resulted in a reduction of protectivity against apoptosis. These results suggest that higher susceptibility of I2020T mutant LRRK2 to intracellular degradation than the wild-type molecule may be one of the mechanisms involved in the neurodegeneration associated with this LRRK2 mutation.     

High volume hydrodynamic injection of plasmid DNA via the hepatic artery results in a high level of gene expression in rat hepatocellular carcinoma induced by diethylnitrosamine

BACKGROUND: Hydrodynamic injection of naked plasmid DNA (pDNA) via the tail vein is a safe and effective method of gene transfer to the liver. However, successful gene transfer has yet to be shown for hepatocellular carcinoma (HCC); therefore, we investigated the feasibility and efficacy of hydrodynamic injection via the tail vein and hepatic artery in a diethylnitrosamine (DEN)-induced HCC model in rats. METHODS: HCC was induced in Sprague-Dawley rats by 100 ppm DEN in drinking water. pCMV-SPORT-beta-galactosidase (beta-gal, 400 microg) was injected (i) via the tail vein in a volume of 0.1 ml/g in 30 s or (ii) via the hepatic artery in a volume of 5 or 10 ml at 1 ml/s, either with or without temporary occlusion of the inferior vena cava (IVC) and portal vein (PV). The liver was harvested 24 h after administration, and beta-gal expression was evaluated with X-gal staining and measurement of enzymatic activity in tissue homogenates. RESULTS: Hydrodynamic injection via the tail vein achieved transgene expression only in non-cancerous tissue (tumor: 0.16 +/- 0.04%, non-tumor: 5.07 +/- 1.66%). Hydrodynamic injection via the hepatic artery was tolerated, but failed to produce efficient transgene expression in tumor and non-tumor cells. On the other hand, concomitant use of temporary IVC/PV occlusion with hydrodynamic injection via the hepatic artery dramatically increased transgene expression in cancer cells, but tumor-selective gene transfer was not achieved with this procedure (tumor: 7.38 +/- 3.66%, non-tumor: 7.77 +/- 1.06%). CONCLUSIONS: High-volume hydrodynamic injection of a pDNA solution via the hepatic artery with IVC/PV occlusion achieved a high level of gene expression in a HCC rat model. This gene transfer technique may have potential in clinical gene therapy for HCC.     

Premeiotic endomitosis produces diploid eggs in the natural clone loach, Misgurnus anguillicaudatus (Teleostei: Cobitidae)

The natural clone loach produces unreduced eggs genetically identical to somatic cells of the mother fish and such diploid eggs normally develop as a clone without genetic contribution of sperm. Following the identification of clonal nature and diploidy of eggs, we conducted cytological studies to determine the mechanisms responsible for this unusual oogenesis. Cytolological observation of full-grown oocytes cultured in vitro revealed that oocytes of both the clone and the control loach underwent two successive meiotic divisions: formation of a bipolar spindle and metaphase in meiosis I and equal segregation of chromosomes, extrusion of the first polar body and the appearance of metaphase of meiosis II. However, spindle size of the clone was larger than that of the control. Bivalent chromosome number of germinal vesicle of oocytes was 25 in the control diploid, whereas 50 in the clone. The results suggest that chromosomes are duplicated by mitosis without cytokinesis before meiosis, i.e. premeiotic endomitosis and then oocytes differentiated from tetraploid oogonia undergo a quasinormal meiosis followed by two successive divisions to produce diploid eggs.     

Escherichia coli ferredoxin-NADP+ reductase and oxygen-insensitive nitroreductase are capable of functioning as ferric reductase and of driving the Fenton reaction

Two free flavin-independent enzymes were purified by detecting the NAD(P)H oxidation in the presence of Fe(III)-EDTA and t-butyl hydroperoxide from E. coli. The enzyme that requires NADH or NADPH as an electron donor was a 28 kDa protein, and N-terminal sequencing revealed it to be oxygen-insensitive nitroreductase (NfnB). The second enzyme that requires NADPH as an electron donor was a 30 kDa protein, and N-terminal sequencing revealed it to be ferredoxin-NADP⁺ reductase (Fpr). The chemical stoichiometry of the Fenton activities of both NfnB and Fpr in the presence of Fe(III)-EDTA, NAD(P)H and hydrogen peroxide was investigated. Both enzymes showed a one-electron reduction in the reaction forming hydroxyl radical from hydrogen peroxide. Also, the observed Fenton activities of both enzymes in the presence of synthetic chelate iron compounds were higher than their activities in the presence of natural chelate iron compounds. When the Fenton reaction occurs, the ferric iron must be reduced to ferrous iron. The ferric reductase activities of both NfnB and Fpr occurred with synthetic chelate iron compounds. Unlike NfnB, Fpr also showed the ferric reductase activity on an iron storage protein, ferritin, and various natural iron chelate compounds including siderophore. The Fenton and ferric reductase reactions of both NfnB and Fpr occurred in the absence of free flavin. Although the k _cat/K _m value of NfnB for Fe(III)-EDTA was not affected by free flavin, the k _cat/K _m value of Fpr for Fe(III)-EDTA was 12-times greater in the presence of free FAD than in the absence of free FAD.     

Dominant-negative effects of LRRK2 heterodimers: A possible mechanism of neurodegeneration in Parkinson’s disease caused by LRRK2 I2020T mutation

Leucine-rich repeat kinase 2 (LRRK2) is the molecule responsible for autosomal-dominant Parkinson’s disease (PD), PARK8, but the etiologic effects of its mutation remain unknown. In the present study, we investigated a novel mechanism for the neurodegeneration induced by I2020T mutant LRRK2. Using native gel electrophoresis and immunoprecipitation, we found that wild-type (WT) LRRK2 formed a heterodimer with I2020T LRRK2 in transfected cells, and that the heterodimer exhibited a markedly lower intracellular protein level than the WT/WT-homodimer. An increased amount of I2020T LRRK2 decreased the protein level of co-transfected WT LRRK2. A pulse-chase experiment revealed that the intracellular protein lifetime of WT LRRK2 was shortened by co-transfection with I2020T LRRK2. These results suggest that I2020T LRRK2 enhances the intracellular degradation of WT LRRK2 through WT/I2020T-heterodimer formation. Overexpression of WT LRRK2 in HEK293 cells increased the phosphorylation level of Akt1 (S473), a possible physiological substrate of LRRK2, and made cells resistant to hydrogen peroxide-induced apoptosis. However, both Akt1 phosphorylation and apoptosis resistance were reduced in WT/I2020T-expressing cells in comparison with WT/WT-expressing cells. Reduction of Akt1 phosphorylation and apoptosis resistance were also evident when a neuroblastoma SH-SY5Y clone overexpressing WT LRRK2 was transfected with the I2020T LRRK2. Altogether, these results suggest that the I2020T mutation enhances the intracellular degradation of LRRK2 through WT/I2020T-heterodimer formation, leading to reduced Akt1 phosphorylation and diminished protectivity against apoptosis. Our findings suggest the possibility of a dominant-negative mechanism of neurodegeneration in PD caused by I2020T LRRK2 mutation.     

Effect of Dephosphorylation on the Self-association and the Precipitation of β-Casein

The pyrolyzate of the nondialyzable melanoidin prepared from glucose-ammonia reaction system (kept in pH 5.3~6.0 during the reaction) was fractionated to volatile fraction and nonvolatile fraction. Among the volatile components, two pyridines and four alkylpyrazines were identified. On the other hand, one imidazole compound and two β-hydroxypyridines isolated from the nonvolatile fraction were identified as 4(5)-methylimidazole, 3-hydroxypyridine and 2-methyl-5-hydroxypyridine, respectively. It is inferred that these compounds are not produced by the fission of the main skeleton in the melanoidin molecule, but formed by pyrolysis of the heterocyclic compounds present as a small moiety in the melanoidin.     

Spontaneous regeneration of the central nervous system in gastropods

Of all organs in mammals including humans, the brain has the most limited regenerative capacity after injury or damage. In spite of extensive efforts to treat ischemic/stroke injury of the brain, thus far no reliable therapeutic method has been developed. However, some molluscan species show remarkable brain regenerative ability and can achieve full functional recovery following injury. The terrestrial pulmonates are equipped with a highly developed olfactory center, called the procerebrum, which is involved in olfactory discrimination and odor-aversion learning. Recent studies revealed that the procerebrum of the land slug can spontaneously recover structurally and functionally relatively soon after injury. Surprisingly, no exogenous interventions are required for this reconstitutive repair. The neurogenesis continues in the procerebrum in adult slugs as in the hippocampus and the olfactory bulb of mammals, and the reconstitutive regeneration seems to be mediated by enhanced neurogenesis. In this review, we discuss the relationship between neurogenesis and the regenerative ability of the brain, and also the evolutionary origin of the brain structures in which adult neurogenesis has been observed.     

<Emphasis Type="Italic">Synechocystis</Emphasis> ferredoxin-NADP<Superscript>+</Superscript> oxidoreductase is capable of functioning as ferric reductase and of driving the Fenton reaction in the absence or presence of free flavin

We purified free flavin-independent NADPH oxidoreductase from Synechocystis sp. PCC6803 based on NADPH oxidation activity elicited during reduction of t-butyl hydroperoxide in the presence of Fe(III)-EDTA. The N-terminal sequencing of the purified enzyme revealed it to be ferredoxin-NADP⁺ oxidoreductase (FNR _S ). The purified enzyme reacted with cytochrome c, ferricyanide and 2,6-dichloroindophenol (DCIP). The substrate specificity of the enzyme was similar to the known FNR. DNA degradation occurring in the presence of NADPH, Fe(III)-EDTA and hydrogen peroxide was potently enhanced by the purified enzyme, indicating that Synechocystis FNR _S may drive the Fenton reaction. The Fenton reaction by Synechocystis FNR _S in the presence of natural chelate iron compounds tended to be considerably lower than that in the presence of synthetic chelate iron compounds. The Synechocystis FNR _S is considered to reduce ferric iron to ferrous iron when it evokes the Fenton reaction. Although Synechocystis FNR _S was able to reduce iron compounds in the absence of free flavin, the ferric reduction by the enzyme was enhanced by the addition of free flavin. The enhancement was detected not only in the presence of natural chelate iron compounds but also synthetic chelate iron compounds.     

Target innervation is necessary for neuronal polyploidization in the terrestrial slug Limax

The brain of gastropod mollusks contains many giant neurons with polyploid genomic DNAs. Such DNAs are generated through repeated DNA endoreplication during body growth. However, it is not known what triggers DNA endoreplication in neurons. There are two possibilities: (1) DNAs are replicated in response to some unknown molecules in the hemolymph that reflect the nutritive status of the animal; or (2) DNAs are replicated in response to some unknown factors that are retrogradely transported through axons from the innervated target organs. We first tested whether hemolymph with rich nutrition could induce DNA endoreplication. We tested whether the transplanted brain exhibits enhanced DNA endoreplication like an endogenous brain does when transplanted into the homocoel of the body of a slug whose body growth is promoted by an increased food supply. However, no enhancement was observed in the frequency of DNA endoreplication when we compared the transplanted brains in the growth‐promoted and growth‐suppressed host slugs, suggesting that the humoral environment is irrelevant to triggering the body growth‐dependent DNA endoreplication. Next, we tested the requirement of target innervation by surgically dissecting a unilateral posterior pedal nerve of an endogenous brain. Substantially lower number of neurons exhibited DNA endoreplication in the pedal ganglion ipsilateral to the dissected nerve. These results support the view that enhanced DNA endoreplication is mediated by target innervation and is not brought about through the direct effect of humoral factors in the hemolymph during body growth. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 609–620, 2013