Mouse models for hereditary spastic paraplegia uncover a role of PI4K2A in autophagic lysosome reformation

Hereditary spastic paraplegia (HSP) denotes genetically heterogeneous disorders characterized by leg spasticity due to degeneration of corticospinal axons. SPG11 and SPG15 have a similar clinical course and together are the most prevalent autosomal recessive HSP entity. The respective proteins play a role for macroautophagy/autophagy and autophagic lysosome reformation (ALR). Here, we report that spg11 and zfyve26 KO mice developed motor impairments within the same course of time. This correlated with enhanced accumulation of autofluorescent material in neurons and progressive neuron loss. In agreement with defective ALR, tubulation events were diminished in starved KO mouse embryonic fibroblasts (MEFs) and lysosomes decreased in neurons of KO brain sections. Confirming that both proteins act in the same molecular pathway, the pathologies were not aggravated upon simultaneous disruption of both. We further show that PI4K2A (phosphatidylinositol 4-kinase type 2 alpha), which phosphorylates phosphatidylinositol to phosphatidylinositol-4-phosphate (PtdIns4P), accumulated in autofluorescent deposits isolated from KO but not WT brains. Elevated PI4K2A abundance was already found at autolysosomes of neurons of presymptomatic KO mice. Immunolabelings further suggested higher levels of PtdIns4P at LAMP1-positive structures in starved KO MEFs. An increased association with LAMP1-positive structures was also observed for clathrin and DNM2/dynamin 2, which are important effectors of ALR recruited by phospholipids. Because PI4K2A overexpression impaired ALR, while its knockdown increased tubulation, we conclude that PI4K2A modulates phosphoinositide levels at autolysosomes and thus the recruitment of downstream effectors of ALR. Therefore, PI4K2A may play an important role in the pathogenesis of SPG11 and SPG15. Abbreviations: ALR: autophagic lysosome reformation; AP-5: adaptor protein complex 5; BFP: blue fluorescent protein; dKO: double knockout; EBSS: Earle’s balanced salt solution; FBA: foot base angle; GFP: green fluorescent protein; HSP: hereditary spastic paraplegia; KO: knockout; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; SQSTM1/p62: sequestosome 1; PI4K2A: phosphatidylinositol 4-kinase type 2 alpha; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns4P: phosphatidylinositol-4-phosphate; RFP: red fluorescent protein; SPG: spastic paraplegia gene; TGN: trans-Golgi network; WT: wild typ

Additional file 1 of The protein composition of exosomes released by prostate cancer cells is distinctly regulated by androgen receptor-antagonists and -agonist to stimulate growth of target cells

Supplementary Material 1

Senolytic compounds control a distinct fate of androgen receptor agonist- and antagonist-induced cellular senescent LNCaP prostate cancer cells

Background The benefit of inducing cellular senescence as a tumor suppressive strategy remains questionable due to the senescence-associated secretory phenotype. Hence, studies and development of senolytic compounds that induce cell death in senescent cells have recently emerged. Senescent cells are hypothesized to exhibit different upregulated pro-survival/anti-apoptotic networks depending on the senescent inducers. This might limit the effect of a particular senolytic compound that targets rather only a specific pathway. Interestingly, cellular senescence in prostate cancer (PCa) cells can be induced by either androgen receptor (AR) agonists at supraphysiological androgen level (SAL) used in bipolar androgen therapy or by AR antagonists. This challenges to define ligand-specific senolytic compounds. Results Here, we first induced cellular senescence by treating androgen-sensitive PCa LNCaP cells with either SAL or the AR antagonist Enzalutamide (ENZ). Subsequently, cells were incubated with the HSP90 inhibitor Ganetespib (GT), the Bcl-2 family inhibitor ABT263, or the Akt inhibitor MK2206 to analyze senolysis. GT and ABT263 are known senolytic compounds. We observed that GT exhibits senolytic activity specifically in SAL-pretreated PCa cells. Mechanistically, GT treatment results in reduction of AR, Akt, and phospho-S6 (p-S6) protein levels. Surprisingly, ABT263 lacks senolytic effect in both AR agonist- and antagonist-pretreated cells. ABT263 treatment does not affect AR, Akt, or S6 protein levels. Treatment with MK2206 does not reduce AR protein level and, as expected, potently inhibits Akt phosphorylation. However, ENZ-induced cellular senescent cells undergo apoptosis by MK2206, whereas SAL-treated cells are resistant. In line with this, we reveal that the pro-survival p-S6 level is higher in SAL-induced cellular senescent PCa cells compared to ENZ-treated cells. These data indicate a difference in the agonist- or antagonist-induced cellular senescence and suggest a novel role of MK2206 as a senolytic agent preferentially for AR antagonist-treated cells. Conclusion Taken together, our data suggest that both AR agonist and antagonist induce cellular senescence but differentially upregulate a pro-survival signaling which preferentially sensitize androgen-sensitive PCa LNCaP cells to a specific senolytic compound

Facing Sorrow as a Group Unites. Facing Sorrow in a Group Divides

Collective gatherings foster group cohesion through providing occasion for emotional sharing among participants. However, prior studies have failed to disentangle two processes that are involved in emotional sharing: 1) focusing shared attention on the same emotion-eliciting event and 2) actively sharing one's experiences and disclosing one's feelings to others. To date, it has remained untested if shared attention influences group cohesion independent of active emotional sharing. Our experiment investigated the effect of shared versus individual attention on cohesion in groups of strangers. We predicted that differences in group cohesion as called forth by shared vs. individual attention are most pronounced when experiencing highly arousing negative affect, in that the act of experiencing intensely negative affect with others buffers negative affect's otherwise detrimental effect on group cohesion. Two-hundred sixteen participants were assembled in groups of 3 to 4 people to either watch an emotion-eliciting film simultaneously on a common screen or to watch the same emotion-eliciting film clip on a laptop in front of each group member using earphones. The film clips were chosen to elicit either highly arousing negative affect or one of three other affective states representing the other poles in Russel's Circumplex model of affect. We examined self-reported affective and cognitive group cohesion and a behavioral measure of group cohesion. Results support our buffer-hypothesis, in that experiencing intense negative affect in unison leads to higher levels of group cohesion than experiencing this affect individually despite the group setting. The present study demonstrates that shared attention to intense negative emotional stimuli affects group cohesion independently of active emotional sharing