Proinflammatory activation of microglia in the cerebellum hyperexcites Purkinje cells to trigger ataxia.

Specific medications to combat cerebellar ataxias, a group of debilitating movement disorders characterized by difficulty with walking, balance and coordination, are still lacking. Notably, cerebellar microglial activation appears to be a common feature in different types of ataxic patients and rodent models. However, direct evidence that cerebellar microglial activation in vivo is sufficient to induce ataxia is still lacking. Here, by employing chemogenetic approaches to manipulate cerebellar microglia selectively and directly, we found that specific chemogenetic activation of microglia in the cerebellar vermis directly leads to ataxia symptoms in wild-type mice and aggravated ataxic motor deficits in 3-acetylpyridine (3-AP) mice, a classic mouse model of cerebellar ataxia. Mechanistically, cerebellar microglial proinflammatory activation induced by either chemogenetic M3D(Gq) stimulation or 3-AP modeling hyperexcites Purkinje cells (PCs), which consequently triggers ataxia. Blockade of microglia-derived TNF-α, one of the most important proinflammatory cytokines, attenuates the hyperactivity of PCs driven by microglia. Moreover, chemogenetic inhibition of cerebellar microglial activation or suppression of cerebellar microglial activation by PLX3397 and minocycline reduces the production of proinflammatory cytokines, including TNF-α, to effectively restore the overactivation of PCs and alleviate motor deficits in 3-AP mice. These results suggest that cerebellar microglial activation may aggravate the neuroinflammatory response and subsequently induce dysfunction of PCs, which in turn triggers ataxic motor deficits. Our findings thus reveal a causal relationship between proinflammatory activation of cerebellar microglia and ataxic motor symptoms, which may offer novel evidence for therapeutic intervention for cerebellar ataxias by targeting microglia and microglia-derived inflammatory mediators.

GSK-3ß is Dephosphorylated by PP2A in a Leu309 Methylation-Independent Manner.

Hyperphosphorylation of tau is pivotally involved in the pathogenesis of Alzheimer's disease (AD) and related tauopathies. Glycogen synthase kinase-3ß (GSK-3ß) and protein phosphate 2A (PP2A) are crucial enzymes to regulate tau phosphorylation. GSK-3ß activity is regulated by its inhibitory phosphorylation at Ser9. We previously reported the cross-talk between GSK-3ß and PP2A signaling and showed that PP2A could dephosphorylate GSK-3ß at Ser9. Here, we investigated the dephosphorylation of GSK-3ß in brain extracts in the presence of phosphatase inhibitors and found that a PP2A-like phosphatase activity was required for dephosphorylation of GSK-3ß at Ser9. PP2A interacted with GSK-3ß and suppressed its Ser9 phosphorylation in vitro and in HEK-293FT cells. Activity of PP2A negatively correlated to the level of phosphorylated GSK-3ß in kainic acid-induced excitotoxic mouse brain. Alteration of methylation of the catalytic subunit of PP2A (PP2Ac) at Leu309 did not affect GSK-3ß phosphorylation. These findings suggest that Leu309 methylation is not required for PP2A to dephosphorylate GSK-3ß at Ser9.

Rapid alteration of protein phosphorylation during postmortem: implication in the study of protein phosphorylation.

Protein phosphorylation is an important post-translational modification of proteins. Postmortem tissues are widely being utilized in the biomedical studies, but the effects of postmortem on protein phosphorylation have not been received enough attention. In the present study, we found here that most proteins in mouse brain, heart, liver, and kidney were rapidly dephosphorylated to various degrees during 20 sec to 10 min postmortem. Phosphorylation of tau at Thr212 and glycogen synthase kinase 3ß (GSK-3ß) at Ser9 was reduced by 50% in the brain with 40 sec postmortem, a regular time for tissue processing. During postmortem, phosphorylation of cAMP-dependent protein kinase (PKA) and AMP activated kinase (AMPK) was increased in the brain, but not in other organs. Perfusion of the brain with cold or room temperature phosphate-buffered saline (PBS) also caused significant alteration of protein phosphorylation. Cooling down and maintaining mouse brains in the ice-cold buffer prevented the alteration effectively. This study suggests that phosphorylation of proteins is rapidly changed during postmortem. Thus, immediate processing of tissues followed by cooling down in ice-cold buffer is vitally important and perfusion has to be avoided when protein phosphorylation is to be studied.

Cross talk between PI3K-AKT-GSK-3ß and PP2A pathways determines tau hyperphosphorylation.

Glycogen synthase kinase-3ß (GSK-3ß) and protein phosphatase 2A (PP2A) are the important enzymes controlling tau hyperphosphorylation. The relationship between these two enzymes and its impact on tau hyperphosphorylation are not well understood. In the present study, we determined the cross talk between PI3K-AKT-GSK-3ß and PP2A pathways and found that the former regulated the methylation of PP2Ac via GSK-3ß. Upregulation of GSK-3ß led to an increase in the methylation and activity of PP2Ac through suppression of protein phosphatase methylesterase-1 expression and phosphorylation of leucine carboxyl methyltransferase 1. PP2A also regulated GSK-3ß phosphorylation. Downregulation of PP2A enhanced Ser9 phosphorylation of GSK-3ß and inhibited its kinase activity. Thus, GSK-3ß and PP2A regulate each other and control tau phosphorylation both directly and indirectly through each other. Reduction of tau phosphorylation by inhibition of GSK-3ß may be more than offset by inhibition of PP2A through a shift in phosphatase methylesterase-1/leucine carboxyl methyltransferase 1 balance; PP2A regulates phosphorylation of tau at Ser262/356, a required site for tau pathology. These findings suggest targeting PP2A rather than GSK-3ß to inhibit tau pathology.

Expression, purification, and crystal structure of N-terminal domains of human ubiquitin-activating enzyme (E1).

Ubiquitin-activating enzyme (E1) is a key regulator in protein ubiquitination, which lies on the upstream of the ubiquitin-related pathways and determines the activation of the downstream enzyme cascade. Thus far, no structural information about the human ubiquitin-activating enzyme has been reported. We expressed and purified the N-terminal domains of human E1 and determined their crystal structures, which contain inactive adenylation domain (IAD) and the first catalytic cysteine half-domain (FCCH). This study presents the crystal structure of human E1 fragment for the first time. The main structure of both IAD and FCCH superimposed well with their corresponding domains in yeast Uba1, but their relative positions vary significantly. This work provides new structural insights in understanding the mechanisms of ubiquitin activation in humans.

IL-17RA is essential for optimal localization of follicular Th cells in the germinal center light zone to promote autoantibody-producing B cells.

Germinal centers (GCs) provide a microenvironment that promotes and regulates the interactions of B cells with follicular Th (TFH) cells. In this study, we show that there are significantly higher frequencies of CXCR5(+)ICOS(+) TFH cells in autoimmune BXD2 mice, and these cells express both IL-21R and IL-17RA. Although IL-17 and IL-21 are both important for the formation of spontaneous GCs and development of pathogenic autoantibodies, IL-21, but not IL-17, is required for the proper development of TFH cells in BXD2 mice. The total numbers of TFH cells and their ability to induce B cell responses in vitro were not affected by a deficiency of IL-17RA in BXD2-Il17ra(-/-) mice, the majority of CXCR5(+) TFH cells from BXD2-Il17ra(-/-) mice were, however, not localized in the GC light zone (LZ). Interruption of IL-17 signaling, either acutely by AdIL-17R:Fc or chronically by Il17ra(-/-), disrupted TFH-B interactions and abrogated the generation of autoantibody-forming B cells in BXD2 mice. IL-17 upregulated the expression of regulator of G-protein signaling 16 (RGS16) to promote the ability of TFH to form conjugates with B cells, which was abolished in TFH cells from BXD2-Rgs16(-/-) mice. The results suggests that IL-17 is an extrinsic stop signal that it acts on postdifferentiated IL-17RA(+) TFH to enable its interaction with responder B cells in the LZ niche. These data suggest a novel concept that TFH differentiation and its stabilization in the LZ are two separate checkpoints and that IL-21 and IL-17 act at each checkpoint to enable pathogenic GC development.

Calpain, Atg5 and Bak play important roles in the crosstalk between apoptosis and autophagy induced by influx of extracellular calcium.

Calcium (Ca(2+)) signals are involved in important checkpoints in cell death pathways and promote both apoptosis and autophagy. However, the relationship between autophagy and apoptosis in response to Ca(2+) level elevation is poorly understood. Here, we provided evidence that the influx of extracellular Ca(2+) triggered by Trichokonin VI (TK VI), an antimicrobial peptide, induced calpain-dependent apoptosis and autophagy in hepatocellular carcinoma (HCC) cells. Remarkably, TK VI preferentially induced apoptosis that was associated with calpain-mediated Bax and Atg5 cleavage, which resulted in the collapse of the mitochondrial membrane potential and cytochrome c release. Interestingly, truncated, but not full-length Atg5, associated with Bcl-xL and promoted the intrinsic pathway. Moreover, TK VI treatment induced reactive oxygen species (ROS) accumulation, an effect in which Bak might play a major role. This accumulation of ROS resulted in the subsequent disposal of damaged mitochondria within autophagosomes via Atg5-mediated and mitochondria-selective autophagy. Both the inhibition of calpain activity and Bax deficiency activated a switch that promoted an enhancement of autophagy. The inhibition of both apoptosis and autophagy significantly attenuated the TK VI cytotoxicity, indicating that the two processes had stimulatory effects during TK VI-meditated cell death. These results suggested that calpain, Bak and Atg5 were molecular links between autophagy and apoptosis and revealed novel aspects of the crosstalk between these two processes. The potential of TK VI is proposed as a promising anticancer agent for its well-characterized activity of Ca(2+) agonist and as a possible novel therapeutic strategy that acts on cancer cell mitochondria.

Antimicrobial peptaibols, novel suppressors of tumor cells, targeted calcium-mediated apoptosis and autophagy in human hepatocellular carcinoma cells.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers in the world which is highly chemoresistant to currently available chemotherapeutic agents. Thus, novel therapeutic targets are needed to be sought for the successful treatment of HCC. Peptaibols, a family of peptides synthesized non-ribosomally by the Trichoderma species and other fungi, exhibit antibiotic activities against bacteria and fungi. Few studies recently showed that peptaibols exerted cytotoxicity toward human lung epithelial and breast carcinoma cells. However, the mechanism involved in peptaibol-induced cell death remains poorly understood. RESULTS: Here, we showed that Trichokonin VI (TK VI), a peptaibol from Trichoderma pseudokoningii SMF2, induced growth inhibition of HCC cells in a dose-dependent manner. It did not obviously impair the viability of normal liver cells at lower concentration. Moreover, the suppression of cell viability resulted from the programmed cell death (PCD) with characteristics of apoptosis and autophagy. An influx of Ca2+ triggered the activation of mu-calpain and proceeded to the translocation of Bax to mitochondria and subsequent promotion of apoptosis. On the other hand, typically morphological characteristics consistent with autophagy were also observed by punctate distribution of MDC staining and the induction of LC3-II, including extensive autophagic vacuolization and enclosure of cell organelles by these autophagosomes. More significantly, specific depletion of Bak expression by small RNA interfering (siRNA) could partly attenuate TK VI-induced autophagy. However, siRNA against Bax led to increased autophagy. CONCLUSION: Taken together, these findings showed for the first time that peptaibols were novel regulators involved in both apoptosis and autophagy, suggesting that the class of peptaibols might serve as potential suppressors of tumor cells.

Marginal zone precursor B cells as cellular agents for type I IFN-promoted antigen transport in autoimmunity.

The pathogenic connection of type I IFN and its role in regulating the migration response of Ag delivery by B cells into lymphoid follicles in an autoimmune condition has not been well-identified. Here, we show that there was a significantly larger population of marginal zone precursor (MZ-P) B cells, defined as being IgM(hi)CD1d(hi)CD21(hi)CD23(hi) in the spleens of autoimmune BXD2 mice compared with B6 mice. MZ-P B cells were highly proliferative compared with marginal zone (MZ) and follicular (FO) B cells. The intrafollicular accumulation of MZ-P B cells in proximity to germinal centers (GCs) in BXD2 mice facilitated rapid Ag delivery to the GC area, whereas Ag-carrying MZ B cells, residing predominantly in the periphery, had a lower ability to carry Ag into the GCs. IFN-alpha, generated by plasmacytoid dendritic cells, induced the expression of CD69 and suppressed the sphingosine-1-phosphate-induced chemotactic response, promoting FO-oriented Ag transport by MZ-P B cells. Knockout of type I IFN receptor in BXD2 (BXD2-Ifnalphar(-/-)) mice substantially diffused the intrafollicular MZ-P B cell conglomeration and shifted their location to the FO-MZ border near the marginal sinus, making Ag delivery to the FO interior less efficient. The development of spontaneous GCs was decreased in BXD2-Ifnalphar(-/-) mice. Together, our results suggest that the MZ-P B cells are major Ag-delivery B cells and that the FO entry of these B cells is highly regulated by type I IFN-producing plasmacytoid dendritic cells in the marginal sinus in the spleens of autoimmune BXD2 mice.

Structural insight into the autoinhibition mechanism of AMP-activated protein kinase.

The AMP-activated protein kinase (AMPK) is characterized by its ability to bind to AMP, which enables it to adjust enzymatic activity by sensing the cellular energy status and maintain the balance between ATP production and consumption in eukaryotic cells. It also has important roles in the regulation of cell growth and proliferation, and in the establishment and maintenance of cell polarity. These important functions have rendered AMPK an important drug target for obesity, type 2 diabetes and cancer treatments. However, the regulatory mechanism of AMPK activity by AMP binding remains unsolved. Here we report the crystal structures of an unphosphorylated fragment of the AMPK alpha-subunit (KD-AID) from Schizosaccharomyces pombe that contains both the catalytic kinase domain and an autoinhibitory domain (AID), and of a phosphorylated kinase domain from Saccharomyces cerevisiae (Snf1-pKD). The AID binds, from the 'backside', to the hinge region of its kinase domain, forming contacts with both amino-terminal and carboxy-terminal lobes. Structural analyses indicate that AID binding might constrain the mobility of helix alphaC, hence resulting in an autoinhibited KD-AID with much lower kinase activity than that of the kinase domain alone. AMP activates AMPK both allosterically and by inhibiting dephosphorylation. Further in vitro kinetic studies demonstrate that disruption of the KD-AID interface reverses the autoinhibition and these AMPK heterotrimeric mutants no longer respond to the change in AMP concentration. The structural and biochemical data have shown the primary mechanism of AMPK autoinhibition and suggest a conformational switch model for AMPK activation by AMP.

Tip30-induced apoptosis requires translocation of Bax and involves mitochondrial release of cytochrome c and Smac/DIABLO in hepatocellular carcinoma cells.

TIP30 (Tat-interacting protein 30), a newly found proapoptotic factor, appears to be involved in multiple functions including metabolic suppression, apoptosis induction, and diminishing angiogenic properties. In the present study, we reported that mitochondrial events were required for apoptosis induced by TIP30 in hepatocellular carcinoma cells (HCC cells). Translocation of Bax was essential for TIP30-induced apoptosis, whereas overexpression of the anti-apoptotic protein Bcl-xL delayed both second mitochondria-derived activator of caspases (Smac/DIABLO) release and onset of apoptosis. Furthermore, TIP30-induced apoptosis was dependent on caspase activity because the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone (z-VAD-fmk) blocked DNA fragmentation. Release of Smac/DIABLO from the mitochondria through the TIP30-P53-Bax cascade was required to remove the inhibitory effect of XIAP (X-linked Inhibitor of Apoptosis) and allowed apoptosis to proceed. Our results showed for the first time that Bax-dependent release of Smac/DIABLO, cytochrome c and AIF from the mitochondria mediated the contribution of the mitochondrial pathway to TIP30-mediated apoptosis. Our data suggested that adenovirus-mediated overexpression of TIP30 was capable of inducing therapeutic programmed cell death in vitro by activating the mitochondrial pathway of apoptosis. On the basis of these studies, elucidating the mechanism by which TIP30 induces cell death might establish it as an anticancer approach.

Endofin acts as a Smad anchor for receptor activation in BMP signaling.

Signaling through receptors of the transforming growth factor beta (TGFbeta) superfamily is mediated by cytoplasmic Smad proteins. It has been demonstrated that Smad anchor for receptor activation (SARA) facilitates TGFbeta and activin/nodal signaling by recruiting and presenting Smad2/3 to the receptor complex. SARA does not bind Smad1 and hence does not enhance bone morphogenetic protein (BMP) signaling. Here we report for the first time that the endosome-associated FYVE-domain protein endofin acts as a Smad anchor for receptor activation in BMP signaling. We demonstrate that endofin binds Smad1 preferentially and enhances Smad1 phosphorylation and nuclear localization upon BMP stimulation. Silencing of endofin by RNAi resulted in a reduction in BMP-dependent Smad1 phosphorylation. Moreover, disruption of the membrane-anchoring FYVE motif by point mutation led to a reduction of BMP-responsive gene expression in cell culture and Xenopus ectodermal explants. Furthermore, we demonstrate that endofin contains a protein-phosphatase-binding motif, which functions to negatively modulate BMP signals through receptor dephosphorylation. Taken together, our results suggest that endofin plays an important role in both positive and negative feedback regulation of the BMP signaling pathway.