Phosphorylation of amyloid precursor protein by mutant LRRK2 promotes AICD activity and neurotoxicity in Parkinson's disease.

Mutations in LRRK2, which encodes leucine-rich repeat kinase 2, are the most common genetic cause of familial and sporadic Parkinson's disease (PD), a degenerative disease of the central nervous system that causes impaired motor function and, in advanced stages, dementia. Dementia is a common symptom of another neurodegenerative disease, Alzheimer's disease, and research suggests that there may be pathophysiological and genetic links between the two diseases. Aggregates of ß amyloid [a protein produced through cleavage of amyloid precursor protein (APP)] are seen in both diseases and in PD patients carrying G2019S-mutant LRRK2. Using patient-derived cells, brain tissue, and PD model mice, we found that LRRK2 interacted with and phosphorylated APP at Thr668 within its intracellular domain (AICD). Phosphorylation of APP at Thr668 promoted AICD transcriptional activity and correlated with increased nuclear abundance of AICD and decreased abundance of a dopaminergic neuron marker in cultures and brain tissue. The AICD regulates the transcription of genes involved in cytoskeletal dynamics and apoptosis. Overexpression of AICD, but not a phosphodeficient mutant (AICDT668A), increased the loss of dopaminergic neurons in older mice expressing LRRK2G2019S Moreover, the amount of Thr668-phosphorylated APP was substantially greater in postmortem brain tissue and dopaminergic neurons (generated by reprogramming skin cells) from LRRK2G2019S patients than in those from healthy individuals. LRRK2 inhibitors reduced the phosphorylation of APP at Thr668 in the patient-derived dopaminergic neurons and in the midbrains of LRRK2G2019S mice. Thus, APP is a substrate of LRRK2, and its phosphorylation promotes AICD function and neurotoxicity in PD.

Ribosomal S6 Kinase 2 (RSK2) maintains genomic stability by activating the Atm/p53-dependent DNA damage pathway.

Ribosomal S6 Kinase 2 (RSK2) is a member of the p90(RSK) family of serine/threonine kinases, which are widely expressed and respond to many growth factors, peptide hormones, and neurotransmitters. Loss-of function mutations in the RPS6KA3 gene, which encodes the RSK2 protein, have been implicated in Coffin-Lowry Syndrome (CLS), an X-linked mental retardation disorder associated with cognitive deficits and behavioral impairments. However, the cellular and molecular mechanisms underlying this neurological disorder are not known. Recent evidence suggests that defective DNA damage signaling might be associated with neurological disorders, but the role of RSK2 in the DNA damage pathway remains to be elucidated. Here, we show that Adriamycin-induced DNA damage leads to the phosphorylation of RSK2 at Ser227 and Thr577 in the chromatin fraction, promotes RSK2 nuclear translocation, and enhances RSK2 and Atm interactions in the nuclear fraction. Furthermore, using RSK2 knockout mouse fibroblasts and RSK2-deficient cells from CLS patients, we demonstrate that ablation of RSK2 impairs the phosphorylation of Atm at Ser1981 and the phosphorylation of p53 at Ser18 (mouse) or Ser15 (human) in response to genotoxic stress. We also show that RSK2 affects p53-mediated downstream cellular events in response to DNA damage, that RSK2 knockout relieves cell cycle arrest at the G2/M phase, and that an increased number of γH2AX foci, which are associated with defects in DNA repair, are present in RSK2-deficient cells. Taken together, our findings demonstrated that RSK2 plays an important role in the DNA damage pathway that maintains genomic stability by mediating cell cycle progression and DNA repair.

[Sinerem labeling and MRI tracking of neural stem cells in vivo and in vitro].

OBJECTIVE: To label rat neural stem cells (NSCs) with the complex of Sinerem, the ultrasmall superparamagnetic iron oxide (USPIO), and poly-L-lysine (PLL), and evaluate the feasibility of tracking the labeled cells with magnetic resonance imaging (MRI) in vitro and in vivo. METHODS: Sinerem was incubated with PLL to obtain the complex of Sinerem-PLL. The mesenchymal stem cells (MSCs) isolated from the bone marrow of SD rats were cultured and induced to differentiate into the neural stem cells. The second-passage cells were cultured overnight with the Sinerem-PLL complex, after which Prussian blue staining and transmission electron microscopy were performed to observe the nanoparticles in the cytoplasm. Cell apoptosis assay was performed to assess the cell viability 1 day, 1 week, and 2 weeks after the labeling. Cell tracking with 4.7 MR system was carried out in vivo and in vitro using T(2)WI and T(2)*WI sequences. RESULTS: The NSCs could be effectively labeled with Sinerem-PLL complex with the labeling efficiency exceeding 95%. Prussian blue staining showed numerous blue iron particles in the cytoplasm, and under transmission electron microscope, these particles accumulated in the endosomes/lysosomes. The labeling did not significantly affect the cell viability and proliferation. Remarkable low signal density changes of the labeled cells was seen on T(2)WI and T(2)*WI in vivo and in vitro. CONCLUSION: NSCs can be effectively labeled with Sinerem-PLL complex, and MRI can be used to track the labeled cells in vivo and in vitro.

[Effect of superparamagnetic iron oxide labeling on neural stem cell survival and proliferation].

OBJECTIVE: To study the effect of superparamgnetic iron oxides (ferumoxides) on the survival and proliferation of neural stem cells (NSCs) and determine the optimal ferumoxides concentration for labeling. METHODS: Bone marrow stromal cells (BMSCs) were obtained from rat femoral marrow and cultured in vitro to induce their differentiation into NSCs. Ferumoxides labeling of the NSCs was performed with different final concentrations of ferumoxides, and the labeling efficiency and viability of the labeled NSCs were evaluated by Prussian blue staining, MTT assay, flow cytometry and transmission electron microscope. RESULTS: The NSCs could be effectively labeled with ferumoxides with a labeling efficiency of around 90%. Prussian blue staining showed numerous fine granules with blue staining in the cytoplasm of the labeled NSCs, and the intensity of the blue staining was in positive correlation with the ferumoxide concentration for labeling. Transmission electron microscopy of the labeled NSCs revealed the presence of numerous vesicles spreading in the cytoplasm and filled with electron-dense magnetic iron particles. The ferumoxides vesicles increased with the labeling concentration of ferumoxides, and at the final concentration exceeding 25 microg/ml, ferumoxides vesicles in the NSCs gave rise to conglomeration which hampered observation of the cellular ultrastructure by transmission electron microscope. The results of flow cytometry and MTT assay demonstrated that the cell viability, proliferation, differentiation and apoptosis of the labeled cells were affected by ferumoxides at the concentration above 25 microg/ml, but such effects could be minimal at lower concentrations. CONCLUSION: Ferumoxides might be feasible for in vitro labeling of the NSCs with the optimal concentration of 25 microg/ml.

[Detection of HPV 58 and cloning of its E7 gene in cervical cancer].

OBJECTIVE: To detect HPV 58, a common type of human papillomavirus (HPV), clone and express its E7 gene from biopsy specimens of cervical cancer. METHODS: HPV 58 from 58 biopsy tissues of cervical cancer was detected by GP5+/GP6+ PCR followed by template-directed dye-terminator incorporation assay with fluorescence polarization detection (TDI-FP). HPV 58 E7 gene was amplified from one HPV 58-positive sample, and then cloned into pGEM-T Easy vector. The recombinant plasmid, HPV58E7-pGEM-T was confirmed by sequencing. Subsequently, E7 gene was cloned into prokaryotic expression vector pRSET-A. The constructed pRSET-58E7 plasmids were transfected into BL21(DE3) cells, and induced to express 58 E7 protein by IPTG. RESULTS: Among the 58 biopsy tissues of cervical cancer, 10 were HPV 58-positive, accounting for 19.2% of 52 HPV-positive cases. HPV 58 E7 gene was amplified from one HPV 58-positive sample. The constructed plasmids were identified containing HPV58 E7 gene by restriction enzyme analysis and sequencing. SDS-PAGE analysis showed that HPV58 E7 His6 fusion protein of M(r) 16 x 10(3) was expressed by pRSET-58E7 after induction by IPTG. The fusion protein accounted for 30% of total bacterial proteins. CONCLUSION: HPV 58 is not uncommon in Chinese women with cervical cancer in Shaanxi province. Constructed HPV58 E7 recombinant plasmids can be effectively expressed in E.coli, which may provide a tool in diagnosis and vaccine design for HPV of HPV58-associated tumors.

Detection and genotyping of human papillomavirus DNA in cervical cancer tissues with fluorescence polarization.

To evaluate the type-specific prevalence of eight common types of human papillomavirus (HPV) in patients with cervical cancer living in Shanxi, China, with fluorescence polarization detection, crude DNA extracted from 137 samples of early-stage cervical cancer (within stage IIa) and chronic cervicitis was subjected to HPV L1 consensus GP5+/GP6+ system. Then, the HPV-positive products identified by GP5 + /GP6+ PCR were genotyped based on template-directed dye-terminator incorporation assay with fluorescence polarization detection (TDI-FP): the PCR products were respectively hybridized with designed type-specific probes within the GP5+/GP6+ amplicons for eight common HPV types (HPV 6, 11, 18, 16, 31, 33, 35, and 58), and specific fluorescence-labeled ddNTPs (TAMRA-ddTTP or R110-ddGTP) were directly incorporated to the ends of the corresponding probes under directing of the corresponding template in PCR products, which was reflected and read by high FP values for TAMRA or R110. HPV DNA was detected in 38.89% (28/72) cases of chronic cervicitis, and 87.69% (57/65) cases of cervical cancer. There was a significant difference in HPV prevalence between these two groups. The four commonly identified types in patients with cervical cancer were HPV 16 (45.6%), HPV 18 (22.8%), HPV 58 (17.5%), and HPV 31 (7.02%), and in those with chronic cervicitis were HPV 16 (35.7%), HPV 11 (32.1%), HPV 6 (21.4%), and HPV 18 (10.7%). 57.14% of HPV types detected in patients with chronic cervicitis were high-risk types. HPV 16 was the most common viral type identified in both groups. Type-specific prevalence of HPV DNA has some characteristics in patients with chronic cervicitis and cervical cancer living in Shanxi, China and the worldwide uncommon type HPV 58 is relatively common in both kinds of cases. The high prevalence of HPV 58 in Chinese women should been considered in diagnosis and vaccine designs of HPV.