Novel Drug Candidates Improve Ganglioside Accumulation and Neural Dysfunction in GM1 Gangliosidosis Models with Autophagy Activation.

GM1 gangliosidosis is a lysosomal storage disease caused by loss of lysosomal ß-galactosidase activity and characterized by progressive neurodegeneration due to massive accumulation of GM1 ganglioside in the brain. Here, we generated induced pluripotent stem cells (iPSCs) derived from patients with GM1 gangliosidosis, and the resultant neurons showed impaired neurotransmitter release as a presynaptic function and accumulation of GM1 ganglioside. Treatment of normal neurons with GM1 ganglioside also disturbed presynaptic function. A high-content drug-screening system was then established and identified two compounds as drug candidates for GM1 gangliosidosis. Treatment of the patient-derived neurons with the candidate agents activated autophagy pathways, reducing GM1 ganglioside accumulation in vitro and in vivo, and restoring the presynaptic dysfunction. Our findings thus demonstrated the potential value of patient-derived iPSC lines as cellular models of GM1 gangliosidosis and revealed two potential therapeutic agents for future clinical application.

Origin of IgM(+)IgG(+) lymphocytes in the bursa of Fabricius.

IgM(+)IgG(+) B cells were detected, by immunofluorescence staining of single cells, in the bursa of Fabricius after hatching. To study the role of maternal IgG (MIgG) in this emergence of IgM(+)IgG(+) B cells, MIgG-free chicks were established from surgically bursectomized hens. Deprivation of MIgG in chicks completely prevented the appearance of IgM(+)IgG(+) B cells in the bursa 1 week after hatching. However, introduction of fluorescein-isothiocyanate-labeled MIgG to MIgG-free chick embryos on day 18 of incubation retrieved IgM(+)IgG(+) B cells in the bursa 1 week after hatching. Thus, IgM(+)IgG(+) B cells are induced by the binding of MIgG to IgM(+) B cells in the bursa after hatching. Nevertheless, no binding of MIgG to IgM(+) B cells was observed in the bursa of chick embryos in which B-cell proliferation and differentiation were independent of external antigens (Ags). Additionally, the binding of MIgG to IgM(+) B cells after hatching was prevented by the isolation of the bursa from environmental stimuli by bursal duct ligation. Therefore, Ag stimulation from the external environment to the bursa is indispensable for the binding of MIgG to IgM(+) B cells in the bursa. Taken together, the data demonstrate that IgM(+)IgG(+) B cells are generated by Ag-dependent binding of MIgG to IgM(+) B cells in the bursa after hatching.

Dominant negative form of alpha4 inhibits the BCR crosslinking-induced phosphorylation of Bcl-xL and apoptosis in an immature B cell line WEHI-231.

We previously demonstrated that c-Jun N-terminal kinase (JNK) phosphorylates serine 62 of Bcl-xL to induce the degradation of Bcl-xL and apoptosis in WEHI-231 cells upon BCR crosslinking. In order to elucidate the regulatory mechanisms underlying the phosphorylation of Bcl-xL, we prepared an assay system in which JNK phosphorylated Bcl-xL in HEK293T cells. Consequently, we found that a signal transduction molecule, alpha4, enhanced the phosphorylation of Bcl-xL by JNK, while the co-expression of C-terminal alpha4 (220-340) diminished the phosphorylation of Bcl-xL induced by JNK. Furthermore, full-length alpha4 associated with both JNK and Bcl-xL, whereas C-terminal alpha4 (220-340) associated only with Bcl-xL, not JNK. In addition, WEHI-231 cells transfected with the cDNA of C-terminal alpha4 (220-340) exhibited decreased phosphorylation of Bcl-xL and stronger resistance to apoptosis induced by BCR crosslinking. These results indicate that alpha4 is an important regulatory molecule of apoptosis induced by BCR crosslinking in WEHI-231 cells and that C-terminal alpha4 (220-340) functions as a dominant negative form.

Protein phosphatase 6 controls BCR-induced apoptosis of WEHI-231 cells by regulating ubiquitination of Bcl-xL.

Crosslinking BCR in the immature B cell line WEHI-231 causes apoptosis. We found that Bcl-xL was degraded by polyubiquitination upon BCR crosslinking and in this study explored the mechanism that controls the degradation of Bcl-xL. Ser(62) of Bcl-xL was phosphorylated by JNK to trigger polyubiquitination, and this was opposed by serine/threonine protein phosphatase 6 (PP6) that physically associated with Bcl-xL. We show BCR crosslinking decreased PP6 activity to allow Ser(62) phosphorylation of Bcl-xL. CD40 crosslinking rescues BCR-induced apoptosis, and we found PP6 associated with CD40 and PP6 activation in response to CD40. Our data suggest that PP6 activity is regulated to control apoptosis by modulating Ser(62) phosphorylation of Bcl-xL, which results in its polyubiquitination and degradation.

The origin of IgG-containing cells in the bursa of Fabricius.

The bursa of Fabricius of the chicken is known as a primary lymphoid organ for B-cell development. Morphologically, the origin of IgG-containing cells in the bursa has not been clear until now, because abundant maternal IgG (MIgG) is transported to the chick embryo and distributed to the bursal tissue around hatching. Thus, it has been difficult to find out whether these cells themselves biosynthesize IgG or if they acquire MIgG via attachment to their surface. Our present study employing in situ hybridization clarified that IgG-containing cells in the medulla of bursal follicles did not biosynthesize IgG. To study the role of MIgG in the development of those IgG-containing cells, MIgG-free chicks were established from surgically bursectomized hen (SBx-hen). We found that, on the one hand, deprivation of MIgG from chicks completely inhibited the development of IgG-containing cells in the medulla after hatching. On the other hand, administration of MIgG to MIgG-free chicks recovered the emergence of those cells. In addition, we observed that those cells did not bear a B-cell marker and possessed dendrites with aggregated IgG. These results demonstrate that IgG-containing cells in the medulla are reticular cells that capture aggregated MIgG. Moreover, we show that the isolation of the bursa from environmental stimuli by bursal duct ligation (BDL) suppressed the development of IgG-containing cells after hatching. Thus, it is implied that environmental stimulations play a key role in MIgG aggregations and dendritic distributions of aggregated MIgG in the medulla after hatching.

CaMKII phosphorylates serine 10 of p27 and confers apoptosis resistance to HeLa cells.

Protein phosphatase (PP) 6 is a serine threonine phosphatase which belongs to the PP2A subfamily of protein phosphatases. PP6 has been implicated in the control of apoptosis. A dominant negative form PP6 (DN-PP6) mutant cDNA was prepared and transfected into HeLa cells to investigate the regulation of apoptosis. HeLa cells expressing DN-PP6 showed increased resistance to apoptosis induced by TNF and cycloheximide. CaMKII phosphorylation and the expression of p27 were increased in DN-PP6 transfectants. Transient expression or activation of CaMKII increased the expression of p27. Furthermore, CaMKII phosphorylated serine 10 of p27, which induces the translocation of p27 from nucleus to cytoplasm and increases the stability of p27. Overexpression of wild type but not the S10A mutant p27 cDNA increased the expression of Bcl-xL and conferred apoptosis resistance to HeLa cells. These results indicated that PP6 and CaMKII regulated apoptosis by controlling the expression level of p27.

Characteristic cellular composition of germinal centers.

We established the method of isolating individually encapsulated germinal centers (GCs) from immunized spleen and analyzed single cell suspension of GCs by flowcytometry. In GCs, the high frequency of sIgG+ cells (29%) and sIgA+ cells (5%) was detected. Two-color flowcytometry analysis showed that GCs contained 27% of sIgM-IgG+ cells, in which isotype switch from IgM to IgG had occurred, and 5% of Bu1-IgG+ cells, which were differentiating into plasma cells. On the other hand, sIgM-IgG+ and Bu1-IgG+ cells were not detected in the bursa, which contained 95% of B cells and only 1% of T cells. CD4+ but not CD8+ T cells were detected in the light zone of GCs and these CD4+ T cells are supposed to play a key role in isotype switching and differentiation into plasma cells in GCs. These results clearly demonstrate that GCs provide a site for isotype switching and differentiation into plasma cells.

Regulation of CaMKII by alpha4/PP2Ac contributes to learning and memory.

Ca(2+)-dependent CaMKIIalpha activation with autophosphorylation plays an essential role in learning and memory. The regulation of CaMKIIalpha by dephosphorylation by protein phosphatase 1 (PP1) has been demonstrated. We addressed whether the protein phosphatase 2A (PP2A) that is abundant in the brain could be involved in the regulation of CaMKIIalpha. CaMKIIalpha was associated with the catalytic subunit of PP2A (PP2Ac) and alpha4, a regulator of PP2A. To investigate whether alpha4 plays an important role in the CNS, we established a neuron specific Cre transgenic mouse and a neuron specific alpha4 deficient mouse (N-alpha4 KO mouse). This N-alpha4 KO mouse showed impaired learning and memory in a water maze and also shuttle-box avoidance test. The activity of CaMKIIalpha also increased in hippocampus. An overexpression of alpha4 in the neuronal cell line demonstrated the activity of CaMKIIalpha to be regulated by alpha4. alpha4 and PP2Ac were localized in the cytoplasm but not in the postsynaptic density (PSD), thus suggesting that the dephosphorylation of CaMKIIalpha by alpha4/PP2Ac occurred in the cytoplasm. These results suggest that alpha4 and PP2A may thus play an important role in CaMKIIalpha regulation and thereby also influence learning.

The heterodimer of alpha4 and PP2Ac is associated with S6 kinase1 in B cells.

Alpha4 is a signal transduction molecule that is required for B cell activation. Alpha4 associates with the catalytic subunit of protein phosphatase 2A (PP2Ac) and regulates its enzymatic activity. We examined the interaction of alpha4/PP2Ac with S6 kinase1 (S6K1) as a potential downstream signal transduction molecule because both alpha4/PP2Ac association and S6K1 activity were rapamycin-sensitive. Stimulation of spleen B cells with lipopolysaccharide induced the interaction of alpha4/PP2Ac and S6K1. Pull-down assay demonstrated that alpha4 interacts with S6K1 through PP2Ac. S6K1 and alpha4 bind to the different regions of PP2Ac as S6K1 to the region from amino acid 88th to 309th of PP2Ac and alpha4 to the two separated regions of the amino-terminal (from amino acid 19th to 22nd) and the middle (from 150th to 164th) portions of PP2Ac. These results suggest that alpha4 regulates S6K1 activity through PP2Ac in B cell activation.

T cell-specific gene targeting reveals that alpha4 is required for early T cell development.

Alpha4-mediated signaling is involved in a variety of functions in mammalian cells. To determine whether this is true for immunocompetent cells, we generated mutant (Lck-alpha4-) mice in which the alpha4 gene was deleted in a T cell-specific manner using the Cre/loxP system. These mice showed impaired early T cell development. Thymi at most ages were small and their architecture was disorganized. This defect was not due to increased thymocyte apoptosis but to decreased cell proliferation. T cell development was found to be severely arrested at the CD4/CD8 double-negative 3 stage and the thymus contained very few double-positive or single-positive (SP) mature thymocytes. The mutant thymocytes showed impaired proliferative responses to anti-CD3 monoclonal antibody (mAb) stimulation or to the cytokines IL-2, IL-1 or TNF. In the spleen, the numbers of mature SP T cells were decreased and their proliferative responses to anti-CD3 plus IL-2 or to anti-CD28 mAb were impaired. A severe impairment of CD3-induced expression of CD25 was also observed. These data suggest that alpha4 plays a critical role in the proliferation of thymocytes, which is necessary for early T cell development.

BCR signal through alpha 4 is involved in S6 kinase activation and required for B cell maturation including isotype switching and V region somatic hypermutation.

alpha 4 potentially mediates BCR signals through a rapamycin-sensitive TOR pathway. To investigate a potential role for alpha 4 in B cell activation, the alpha 4 gene was disrupted conditionally in B cells by mating male CD19-Cre mice with female alpha 4-floxed mice. CD19-Cre+/alpha 4flox mice showed loss of alpha 4 protein in B lineage cells and a decreased number of phenotypically normal mature B cells. Compared to normal B cells, alpha 4(-) B cells showed a decreased proliferation in response to the B cell stimulants (anti-IgM antibody plus IL-4, anti-CD40 mAb and lipopolysaccharide), and a reduced S6 kinase activation and rapamycin sensitivity. While CD19-Cre+/alpha 4flox mice showed impaired antibody responses to both T cell-independent and T cell-dependent (TD) antigens, the TD antigen response was markedly impaired as demonstrated by reduced isotype switching, reduced germinal center formation and reduced V region somatic hypermutation. These results show that alpha 4 plays a pivotal role in antigen-specific signal transduction during B cell activation and differentiation in vivo.