Manganese disrupts the maturation and degradation of axonal autophagosome leading to hippocampal synaptic toxicity in mice via the activation of LRRK2 on phosphorylation of Rab10.

Manganese (Mn) overexposure induces hippocampal synaptotoxicity by the accumulation of dysfunctional synaptic vesicles (SVs). Leucine-rich repeat kinase 2 (LRRK2) kinase activity is involved in regulating axonal transport (autophagosomal maturation) and lysosomal function. Nevertheless, it remains unclear whether Mn-induced synaptotoxicity is associated with the LRRK2-mediated disruption of autophagosomal maturation in axonal transport and the impairment of lysosomes in hippocampal neurons. Here, we established models of manganism in C57BL/6 mice and hippocampal neuronal HT22 cells to verify the role of LRRK2-mediated Rab10 phosphorylation in the Mn-induced dysfunction of autophagy- lysosomal fusion. Our results proved that Mn-induced the disorder of axonal transport and that lysosome impairments were associated with the increased recruitment of phospho-Rab10 at the axon and lysosomes. Next, we established Lrrk2-KD and LRRK2 kinase- specific inhibitor (GNE-0877, GNE) pre-treated HT22 cells to inhibit Lrrk2 gene expression and kinase activity, respectively. In Mn-treated Lrrk2-KD or GNE-pretreated normal neurons, our results indicated that lysosomal pH and integrity and autophagic flow were restored, indicating by decreased levels of phospho-Rab10 on lysosomes and JNK-interacting proteins (JIP4). In addition, GNE pretreatment could provide protection against Mn-induced synaptotoxicity in vivo, which was evidenced by the partial recovery in synaptic plasticity and synaptic damage. Thus, the Mn-induced abnormal activation of LRRK2 affected lysosomes and the recruitment of phospho-Rab10 by JIP4, which disrupted autophagosomal maturation in proximal axons and resulted in the hippocampal synaptic toxicity of mice.

Manganese-induced α-synuclein overexpression promotes the accumulation of dysfunctional synaptic vesicles and hippocampal synaptotoxicity by suppressing Rab26-dependent autophagy in presynaptic neurons.

Manganese (Mn) overexposure induces learning and memory impairments in mice by disrupting the functions of synapses and synaptic vesicles (SVs) in the hippocampus, which is associated with α-synuclein (α-Syn) overexpression. Rab26-dependent autophagy is a key signaling step required for impaired SV clearance; however, it is unclear whether Mn-induced α-Syn overexpression is linked to dysregulated Rab26-dependent autophagy in presynaptic neurons. In this study, we developed manganism models in male C57BL/6 mice and hippocampal primary neurons to observe the associations between Mn-induced α-Syn overexpression and impaired SV accumulation. The results of the in vivo experiments showed that 100 and 200 µmol/kg Mn exposure significantly impaired memory and synaptic plasticity in the mice, which was related to the accumulation of impaired SVs in the hippocampus. Consistent with the in vivo outcomes, the level of in vitro injured SVs in the 50 and 100 µmol/L Mn-exposed neuron group were higher than that in the control group. Moreover, 100 µmol/L Mn suppressed the initiation of Rab26-dependent autophagy at the synapse. Then, we transfected neurons with LV-α-Syn short hairpin RNA (shRNA) and exposed the neurons to Mn for an additional 24 h. Surprisingly, the area of colocalization between Rab26 and Atg16L1 and the expression level of LC3II-positive SVs were both higher in Mn-exposed LV-α-Syn shRNA-transfected neurons than those in Mn-treated normal or Mn-treated LV-scrambled shRNA-transfected neurons. Thus, Mn-induced α-Syn overexpression was responsible for the dysregulation of Rab26-dependent autophagy, thereby promoting the accumulation of injured SVs, and causing synaptotoxicity and cognitive and memory deficits in mice.

Comparative evaluation of immunization with recombinant protein and plasmid DNA vaccines of fusion antigen ROP2 and SAG1 from Toxoplasma gondii in mice: cellular and humoral immune responses.

The aim of this work was to evaluate immune responses in BALB/c mice vaccinated subcutaneously by recombinant protein, or intramuscularly by plasmid DNA with fusion antigen of rhoptry protein 2 (ROP2) and major surface protein 1 (SAG1) from Toxoplasma gondii (T. gondii). BALB/c mice were immunized with one of three different antigen formulations respectively, which were rROP2-SAG1, pcROP2-SAG1, and pcROP2-SAG1 boosted with rROP2-SAG1. The production of IgG, IgG subclasses, lymphoproliferation, and level of gamma interferon (IFN-γ) were detected after vaccination. The animals vaccinated with rROP2-SAG1 quickly developed specific anti-TLA (T. gondii lysate antigen) antibodies, which continued to rise after immunization. However, production of IgG against TLA in mice vaccinated with pcROP2-SAG1 was relatively slow and maintained a high level after reaching plateau. There are more vigorous specific lymphoproliferative responses observed in mice of group rROP2-SAG1 than in pcROP2-SAG1. Immune responses in mice of group pcROP2-SAG1 boosted with rROP2-SAG1 were similar to the protein immunization group. Three immunization procedures resulted in a similar level of IFN-γ production. Our results indicate that BALB/c mice vaccinated by three immunization procedures induce similar humoral and cellular immunity against infection of T. gondii. Mice immunized with recombinant protein rROP2-SAG1 produce more humoral immune responses than mice immunized with other antigen formulations.

[Immune response elicited by the recombinant protein and plasmid DNA of complex antigen ROP2-SAG1 from Toxoplasma gondii].

OBJECTIVE: To study the immune response elicited by the recombinant protein vaccine and DNA vaccine of the complex antigen ROP2-SAG1 from Toxoplasma gondii. METHODS: Sixty female BALB/c mice were randomly divided into 4 groups (15 per group). Mice in rROP2-SAGI group were immunized subcutaneously with 2.5 microg rROP2-SAG1 protein formulated in Freund's adjuvant. Mice in control group received only adjuvant emulsified with normal saline. Mice in recombinant plasmid pcROP2-SAG1 and control plasmid pcDNA3.1 groups were each injected intramuscularly with 100 microg of pcROP2-SAG1 and pcDNA3.1, respectively. All the mice received three immunizations at 2-week intervals. Serum samples were collected at 25, 45, and 70 days after immunization for determining antibody IgG, and at 2 weeks after the last immunization IgG1 and IgG2a were detected all by ELISA. Cell counting kit-8 (CCK-8) was used to determine the splenocyte proliferation, and the supernatant of cultured splenocytes was collected for the detection of IFN-gamma by ELISA. RESULTS: The level of IgG continued to rise in rROP2-SAG1 group after immunization, and similarly in pcROP2-SAG1 group. At 2 weeks after the last immunization, level of IgG1 (1.538 +/- 0.183) was higher than that of IgG2a (0.618 +/- 0.122) (P < 0.05) in rROP2-SAG1 group. Whereas no significant difference between IgG1 (1.107 +/- 0.137) and IgG2a (0.830 +/- 0.185) was observed in pcROP2-SAG1 group (P > 0.05). Compared with the pcROP2-SAG1 group (A450 = 0.123 +/- 0.018), more significant proliferation response of splenocytes was observed in rROP2-SAG1 group (0.348 +/- 0.042) (P < 0.05). There was no significant difference (P > 0.05) of IFN-gamma and IL-2 in the supematant of cultured splenocytes between the groups of rROP2-SAG1 and pcROP2-SAG1. CONCLUSION: The antibody level and splenocyte proliferation have been significantly higher in mice immunized with recombinant protein rROP2-SAG1 than those with recombinant plasmid pcROP2-SAG1.