The impact of VPS35 D620N mutation on alternative autophagy and its reversal by estrogen in Parkinson's disease.

VPS35 plays a key role in neurodegenerative processes in Alzheimer's disease and Parkinson's disease (PD). Many genetic studies have shown a close relationship between autophagy and PD pathophysiology, and specifically, the PD-causing D620N mutation in VPS35 has been shown to impair autophagy. However, the molecular mechanisms underlying neuronal cell death and impaired autophagy in PD are debated. Notably, increasing evidence suggests that Rab9-dependent "alternative" autophagy, which is driven by a different molecular mechanism that driving ATG5-dependent "conventional" autophagy, also contributes to neurodegenerative process. In this study, we investigated the relationship between alternative autophagy and VPS35 D620N mutant-related PD pathogenesis. We isolated iPSCs from the blood mononuclear cell population of two PD patients carrying the VPS35 D620N mutant. In addition, we used CRISPR-Cas9 to generate SH-SY5Y cells carrying the D620N variant of VPS35. We first revealed that the number of autophagic vacuoles was significantly decreased in ATG5-knockout Mouse Embryonic Fibroblast or ATG5-knockdown patient-derived dopaminergic neurons carrying the VPS35 D620N mutant compared with that of the wild type VPS35 control cells. Furthermore, estrogen, which activates alternative autophagy pathways, increased the number of autophagic vacuoles in ATG5-knockdown VPS35 D620N mutant dopaminergic neurons. Estrogen induces Rab9 phosphorylation, mediated through Ulk1 phosphorylation, ultimately regulating alternative autophagy. Moreover, estrogen reduced the apoptosis rate of VPS35 D620N neurons, and this effect of estrogen was diminished under alternative autophagy knockdown conditions. In conclusion, alternative autophagy might be important for maintaining neuronal homeostasis and may be associated with the neuroprotective effect of estrogen in PD with VPS35 D620N.

Generation of four iPSC lines from a family harboring a 1p36-35 haplotype linked with bipolar disorder and recurrent depressive disorder: Three-generation patients and a healthy sibling.

Induced pluripotent stem cells (iPSCs) obtained from genetically characterized patients benefit the biological study of bipolar disorder (BD). Here, we present iPSC lines from three-generation patients with BD and recurrent depressive disorder (RDD) and a healthy control sibling in a family. All patients shared the specified haplotype in the 1p36-35, previously reported as the susceptibility locus of mood disorders. iPSCs were generated with the reprogramming factors OTC3/4, l-MYC, LIN28, SOX2, KLF4, and p53 shRNA through non-integrated episomal vectors. All iPSC lines strongly expressed pluripotency markers and proved the ability to differentiate into three germ lineages in vitro.

Generation and characterization of motor neuron progenitors and motor neurons using metachromatic leukodystrophy-induced pluripotent stem cells.

The pathological consequences leading to primary storage, autophagy impairment, impaired mitochondrial dynamics, and endoplasmic reticulum (ER) stress on neural cell dysfunction and apoptosis in metachromatic leukodystrophy (MLD) have been poorly elucidated. In the present study, we generated 2 cell lines of patient-specific-induced pluripotent stem cells (iPSCs) and modeled the progression of pathological events during the differentiation of iPSCs to motor neuron progenitors (MNPs) and mature motor neurons (MNs). The iPS cells were generated from two late-infantile MLD patient-derived skin fibroblasts using electroporation or the Sendai virus. Olig2+ MNPs were generated from both iPSC lines using a combination of small molecules in a chemically defined neural medium. Furthermore, the MNPs could be differentiated into mature MNs, which was confirmed by RT-PCR and MN markers, including SMI32 and ChAT. The population of MNs was approximately 50% under the culture conditions. Pathological observation of MLD patient-derived iPSCs revealed lysosomal accumulation and impaired autophagy. In addition, both MNPs and MNs derived from MLD-iPSCs showed increased lysosomal accumulation, dysfunctional autophagy, impaired mitophagy, endoplasmic reticulum (ER) stress or unfolded protein response (UPR) activation, and premature cellular death.

The inhibition of malignant melanoma cell invasion of bone by the TLR7 agonist R848 is dependent upon pro-inflammatory cytokines produced by bone marrow macrophages.

Distant metastasis remarkably worsens the prognoses of malignant melanoma patients. Toll-like receptors (TLRs) recognize molecules derived from many types of pathogens and activate the innate intravital immune system. In this study, we examined the effects of R848, a TLR7 ligand, on bone invasion by malignant melanoma cells. Mice underwent transplantation with cells of a malignant melanoma cell line B16F10, and were also administered R848 every three days. Hindlimbs were obtained 13 days after transplantation and invasion of bone marrow by B16F10 cells was evaluated. ELISA was used to determine the concentrations of cytokines in mouse serum and in the culture medium from bone marrow macrophages (BMMs) in the presence or absence of R848. In addition, MTS assays were used to examine the effects of media from BMM cultures on the proliferation of B16F10 cells. The rate of infiltration by B16F10 cells and the area of invasion were significantly reduced with R848 administration. Furthermore, serum levels of IL-6, IL-12, and IFN-γ were significantly increased in mice administered R848, with the same trend observed in the culture medium of BMMs treated with R848. In addition, B16F10 cell proliferation was suppressed by the addition of medium from cultured BMMs treated with R848. Neutralization by antibodies against IL-6, IL-12, and IFN-γ abrogated the suppression of proliferation of B16F10 cells by culture medium from BMMs treated with R848. Our results suggest that R848 drives the production of IL-6, IL-12, and IFN-γ in BMMs, which reduces proliferation and bone invasion by B16F10 cells.

Down-regulation of Irf8 by Lyz2-cre/loxP accelerates osteoclast differentiation in vitro.

Interferon regulatory factor 8 (Irf8) is a transcription factor that negatively regulates osteoclast differentiation and Irf8 global knockout (Irf8 -/-) mice have been shown to have reduced bone volume resulting from increased osteoclast numbers. However, detailed analysis of the functions of Irf8 in osteoclast precursors with a monocyte/macrophage linage is difficult, because the population and properties of hematopoietic cells in Irf8 -/- mice are severely altered. Therefore, to clearly elucidate the functions of Irf8 during osteoclastogenesis, we established myeloid cell-specific Irf8 conditional knockout (Irf8 fl/fl ;Lyz2 cre/+) mice. We found that trabecular bone volume in the Irf8 fl/fl ;Lyz2 cre/+ mice was not significantly affected, while exposure to M-CSF and RANKL significantly increased TRAP activity in vitro in osteoclasts that underwent osteoclastogenesis from bone marrow-derived macrophages (BMMs) induced from bone marrow cells (BMCs) of those mice by addition of M-CSF. Our results also showed that expression of Irf8 mRNA and protein in BMMs obtained from Irf8 fl/fl ;Lyz2 cre/+ mice and cultured with M-CSF was reduced. These findings predicted that Lyz2/Lyz2-cre expression is induced when BMCs differentiate into BMMs in cultures with M-CSF. In osteoclast differentiation cultures, Lyz2 was gradually increased by M-CSF during the first 3 days of culture, then rapidly decreased by the addition of RANKL with M-CSF during the next 3 days. Furthermore, BMCs differentiated into osteoclasts while maintaining a low level of Lyz2 expression when cultured simultaneously with both M-CSF and RANKL from the initiation of culture. These findings suggest that Lyz2-cre expression is induced along with differentiation to BMMs by BMCs obtained from Irf8 fl/fl ;Lyz2 cre/+ mice and cultured with M-CSF. In addition, Irf8 was down-regulated by activation of the cre/loxP recombination system in BMMs and osteoclastogenesis was accelerated. Based on our results, we propose the existence in vivo of a new lineage of osteoclast precursors among BMCs, which differentiate into osteoclasts without up-regulation of Lyz2 expression.