New boundaries and dissociation of the mouse hippocampus along the dorsal-ventral axis based on glutamatergic, GABAergic and catecholaminergic receptor densities.

In rodents, gene-expression, neuronal tuning, connectivity and neurogenesis studies have postulated that the dorsal, the intermediate and the ventral hippocampal formation (HF) are distinct entities. These findings are underpinned by behavioral studies showing a dissociable role of dorsal and ventral HF in learning, memory, stress and emotional processing. However, up to now, the molecular basis of such differences in relation to discrete boundaries is largely unknown. Therefore, we analyzed binding site densities for glutamatergic AMPA, NMDA, kainate and mGluR2/3 , GABAergic GABAA (including benzodiazepine binding sites), GABAB , dopaminergic D1/5 and noradrenergic α1 and α2 receptors as key modulators for signal transmission in hippocampal functions, using quantitative in vitro receptor autoradiography along the dorsal-ventral axis of the mouse HF. Beside general different receptor profiles of the dentate gyrus (DG) and Cornu Ammonis fields (CA1, CA2, CA3, CA4/hilus), we detected substantial differences between dorsal, intermediate and ventral subdivisions and individual layers for all investigated receptor types, except GABAB . For example, striking higher densities of α2 receptors were detected in the ventral DG, while the dorsal DG possesses higher numbers of kainate, NMDA, GABAA and D1/5 receptors. CA1 dorsal and intermediate subdivisions showed higher AMPA, NMDA, mGluR2/3 , GABAA , D1/5 receptors, while kainate receptors are higher expressed in ventral CA1, and noradrenergic α1 and α2 receptors in the intermediate region of CA1. CA2 dorsal was distinguished by higher kainate, α1 and α2 receptors in the intermediate region, while CA3 showed a more complex dissociation. Our findings resulted not only in a clear segmentation of the mouse hippocampus along the dorsal-ventral axis, but also provides insights into the neurochemical basis and likely associated physiological processes in hippocampal functions. Therein, the presented data has a high impact for future studies modeling and investigating dorsal, intermediate and ventral hippocampal dysfunction in relation to neurodegenerative diseases or psychiatric disorders.

Prodigiosin Sensitizes Sensitive and Resistant Urothelial Carcinoma Cells to Cisplatin Treatment.

Cisplatin-based treatment is the standard of care therapy for urothelial carcinomas. However, complex cisplatin resistance mechanisms limit the success of this approach. Both apoptosis and autophagy have been shown to contribute to this resistance. Prodigiosin, a secondary metabolite from various bacteria, exerts different biological activities including the modulation of these two cellular stress response pathways. We analyzed the effect of prodigiosin on protein levels of different autophagy- and apoptosis-related proteins in cisplatin-sensitive and -resistant urothelial carcinoma cells (UCCs). Furthermore, we investigated the effect on cell viability of prodigiosin alone or in combination with cisplatin. We made use of four different pairs of cisplatin-sensitive and -resistant UCCs. We found that prodigiosin blocked autophagy in UCCs and re-sensitized cisplatin-resistant cells to apoptotic cell death. Furthermore, we found that prodigiosin is a potent anticancer agent with nanomolar IC50 values in all tested UCCs. In combination studies, we observed that prodigiosin sensitized both cisplatin-sensitive and -resistant urothelial carcinoma cell lines to cisplatin treatment with synergistic effects in most tested cell lines. These effects of prodigiosin are at least partially mediated by altering lysosomal function, since we detected reduced activities of cathepsin B and L. We propose that prodigiosin is a promising candidate for the therapy of cisplatin-resistant urothelial carcinomas, either as a single agent or in combinatory therapeutic approaches.

Anthraquinones and autophagy - Three rings to rule them all?

In order to overcome therapy resistance in cancer, scientists search in nature for novel lead structures for the development of improved chemotherapeutics. Anthraquinones belong to a class of tricyclic organic natural compounds with promising anti-cancer effects. Anthraquinone derivatives are rich in structural diversity, and exhibit pleiotropic properties, among which the modulation of autophagy seems promising in the context of overcoming cancer-therapy resistance. Among the most promising derivatives in this regard are emodin, aloe emodin, rhein, physcion, chrysophanol and altersolanol A. On the molecular level, these compounds target autophagy via different upstream pathways including the AKT/mTOR-axis and transcription of autophagy-related proteins. The role of autophagy is pro-survival as well as cell death-promoting, depending on derivatives and their cell type specificity. This review summarizes observed effects of anthraquinone derivatives on autophagy and discusses targeted pathways and crosstalks. A cumulative knowledge about this topic paves the way for further research on modes of action, and aids to find a therapeutic window of anthraquinones in cancer-therapy.

Fin56-induced ferroptosis is supported by autophagy-mediated GPX4 degradation and functions synergistically with mTOR inhibition to kill bladder cancer cells.

Ferroptosis is a form of regulated cell death that emerges to be relevant for therapy-resistant and dedifferentiating cancers. Although several lines of evidence suggest that ferroptosis is a type of autophagy-dependent cell death, the underlying molecular mechanisms remain unclear. Fin56, a type 3 ferroptosis inducer, triggers ferroptosis by promoting glutathione peroxidase 4 (GPX4) protein degradation via a not fully understood pathway. Here, we determined that Fin56 induces ferroptosis and autophagy in bladder cancer cells and that Fin56-triggered ferroptosis mechanistically depends on the autophagic machinery. Furthermore, we found that autophagy inhibition at different stages attenuates Fin56-induced oxidative stress and GPX4 degradation. Moreover, we investigated the effects of Fin56 in combination with Torin 2, a potent mTOR inhibitor used to activate autophagy, on cell viability. We found that Fin56 synergizes with Torin 2 in cytotoxicity against bladder cancer cells. Collectively, our findings not only support the concept that ferroptosis is a type of autophagy-dependent cell death but imply that the combined application of ferroptosis inducers and mTOR inhibitors is a promising approach to improve therapeutic options in the treatment of bladder cancer.

TNF-induced necroptosis initiates early autophagy events via RIPK3-dependent AMPK activation, but inhibits late autophagy.

Macroautophagy/autophagy and necroptosis represent two opposing cellular s tress responses. Whereas autophagy primarily fulfills a cyto-protective function, necroptosis is a form of regulated cell death induced via death receptors. Here, we aimed at investigating the molecular crosstalk between these two pathways. We observed that RIPK3 directly associates with AMPK and phosphorylates its catalytic subunit PRKAA1/2 at T183/T172. Activated AMPK then phosphorylates the autophagy-regulating proteins ULK1 and BECN1. However, the lysosomal degradation of autophagosomes is blocked by TNF-induced necroptosis. Specifically, we observed dysregulated SNARE complexes upon TNF treatment; e.g., reduced levels of full-length STX17. In summary, we identified RIPK3 as an AMPK-activating kinase and thus a direct link between autophagy- and necroptosis-regulating kinases.Abbreviations: ACACA/ACC: acetyl-CoA carboxylase alpha; AMPK: AMP-activated protein kinase; ATG: autophagy-related; BECN1: beclin 1; GFP: green fluorescent protein; EBSS: Earle's balanced salt solution; Hs: Homo sapiens; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MLKL: mixed lineage kinase domain like pseudokinase; Mm: Mus musculus; MTOR: mechanistic target of rapamycin kinase; MVB: multivesicular body; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4/VPS15: phosphoinositide-3-kinase regulatory subunit 4; PLA: proximity ligation assay; PRKAA1: protein kinase AMP-activated catalytic subunit alpha 1; PRKAA2: protein kinase AMP-activated catalytic subunit alpha 2; PRKAB2: protein kinase AMP-activated non-catalytic subunit beta 2; PRKAG1: protein kinase AMP-activated non-catalytic subunit gamma 1; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; RIPK1: receptor interacting serine/threonine kinase 1; RIPK3: receptor interacting serine/threonine kinase 3; SNAP29: synaptosome associated protein 29; SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein receptor; SQSTM1/p62: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; STX7: syntaxin 7; STX17: syntaxin 17; TAX1BP1: Tax1 binding protein 1; TNF: tumor necrosis factor; ULK1: unc-51 like autophagy activating kinase 1; VAMP8: vesicle associated membrane protein 8; WT: wild-type.

High-throughput screening for natural compound-based autophagy modulators reveals novel chemotherapeutic mode of action for arzanol.

Autophagy is an intracellular recycling pathway with implications for intracellular homeostasis and cell survival. Its pharmacological modulation can aid chemotherapy by sensitizing cancer cells toward approved drugs and overcoming chemoresistance. Recent translational data on autophagy modulators show promising results in reducing tumor growth and metastasis, but also reveal a need for more specific compounds and novel lead structures. Here, we searched for such autophagy-modulating compounds in a flow cytometry-based high-throughput screening of an in-house natural compound library. We successfully identified novel inducers and inhibitors of the autophagic pathway. Among these, we identified arzanol as an autophagy-modulating drug that causes the accumulation of ATG16L1-positive structures, while it also induces the accumulation of lipidated LC3. Surprisingly, we observed a reduction of the size of autophagosomes compared to the bafilomycin control and a pronounced accumulation of p62/SQSTM1 in response to arzanol treatment in HeLa cells. We, therefore, speculate that arzanol acts both as an inducer of early autophagosome biogenesis and as an inhibitor of later autophagy events. We further show that arzanol is able to sensitize RT-112 bladder cancer cells towards cisplatin (CDDP). Its anticancer activity was confirmed in monotherapy against both CDDP-sensitive and -resistant bladder cancer cells. We classified arzanol as a novel mitotoxin that induces the fragmentation of mitochondria, and we identified a series of targets for arzanol that involve proteins of the class of mitochondria-associated quinone-binding oxidoreductases. Collectively, our results suggest arzanol as a valuable tool for autophagy research and as a lead compound for drug development in cancer therapy.

FIP200 controls the TBK1 activation threshold at SQSTM1/p62-positive condensates.

The protein kinase TBK1 is a central regulator of innate immune responses and autophagy, and ablation of either function has been linked to neuroinflammatory or degenerative diseases. Autophagy is an intracellular process that recycles old or damaged proteins and organelles. In recent years, the TBK1-dependent regulation of autophagy pathways has been characterized. However, the autophagy-dependent regulation of TBK1 activity awaits further clarification. Here, we observed that TBK1 is recruited to SQSTM1/p62-containing aggregates via the selective autophagy receptor TAX1BP1. In these aggregates, TBK1 phosphorylates SQSTM1/p62 at serine 403 and thus presumably regulates the efficient engulfment and clearance of these structures. We found that TBK1 activation is strongly increased if FIP200, a component of the autophagy-inducing ULK1 complex, is not present or cannot bind to TAX1BP1. Given our collective findings, we hypothesize that FIP200 ensures the inducible activation of TBK1 at SQSTM1/p62 condensates.

Carbamoyl-Phosphate Synthase 1 as a Novel Target of Phomoxanthone A, a Bioactive Fungal Metabolite.

Phomoxanthone A, a bioactive xanthone dimer isolated from the endophytic fungus Phomopsis sp., is a mitochondrial toxin weakening cellular respiration and electron transport chain activity by a fast breakup of the mitochondrial assembly. Here, a multi-disciplinary strategy has been developed and applied for identifying phomoxanthone A target(s) to fully address its mechanism of action, based on drug affinity response target stability and targeted limited proteolysis. Both approaches point to the identification of carbamoyl-phosphate synthase 1 as a major phomoxanthone A target in mitochondria cell lysates, giving also detailed insights into the ligand/target interaction sites by molecular docking and assessing an interesting phomoxanthone A stimulating activity on carbamoyl-phosphate synthase 1. Thus, phomoxanthone A can be regarded as an inspiring molecule for the development of new leads in counteracting hyperammonemia states.

The Autophagy-Initiating Kinase ULK1 Controls RIPK1-Mediated Cell Death.

Autophagy, apoptosis, and necroptosis are stress responses governing the ultimate fate of a cell. However, the crosstalk between these cellular stress responses is not entirely understood. Especially, it is not clear whether the autophagy-initiating kinase ULK1 and the cell-death-regulating kinase RIPK1 are involved in this potential crosstalk. Here, we identify RIPK1 as a substrate of ULK1. ULK1-dependent phosphorylation of RIPK1 reduces complex IIb/necrosome assembly and tumor necrosis factor (TNF)-induced cell death, whereas deprivation of ULK1 enhances TNF-induced cell death. We observe that ULK1 phosphorylates multiple sites of RIPK1, but it appears that especially phosphorylation of S357 within the intermediate domain of RIPK1 mediates this cell-death-inhibiting effect. We propose that ULK1 is a regulator of RIPK1-mediated cell death.

Targeting urothelial carcinoma cells by combining cisplatin with a specific inhibitor of the autophagy-inducing class III PtdIns3K complex.

BACKGROUND: Cisplatin-based regimens are routinely employed for the treatment of urothelial carcinoma. However, therapeutic success is hampered by the primary presence of or the development of cisplatin resistance. This chemoresistance is executed by multiple cellular pathways. In recent years, the cellular process of autophagy has been identified as a prosurvival pathway of cancer cells. On the one hand, autophagy enables cancer cells to survive conditions of low oxygen or nutrient supply, frequently found in tumors. On the other hand, autophagy supports chemoresistance of cancer cells. Here, we aimed at investigating the involvement of autophagy for cisplatin resistance in different urothelial carcinoma cell lines. MATERIALS & METHODS: We analyzed the expression levels of different autophagy-related proteins in cisplatin-sensitive and cisplatin-resistant urothelial carcinoma cell lines. Furthermore, we performed cell viability assays and caspase activity assays with cells treated with cisplatin, non-specific or specific autophagy inhibitors (chloroquine, 3-methyladenine, SAR405) or combinations thereof. RESULTS: We found that autophagy-related proteins are up-regulated in different cisplatin-resistant urothelial carcinoma cells compared to the sensitive parental cell lines. Furthermore, inhibition of autophagy, in general, or of the autophagy-inducing class III PtdIns3K complex, in particular, sensitized both sensitive and resistant urothelial carcinoma cells to cisplatin-induced cytotoxic effects. CONCLUSION: We propose that targeting the autophagic machinery might represent a suitable approach to complement or even increase cisplatin efficacy in order to overcome cisplatin resistance in urothelial carcinoma.

Systematic analysis of ATG13 domain requirements for autophagy induction.

Macroautophagy/autophagy is an evolutionarily conserved cellular process whose induction is regulated by the ULK1 protein kinase complex. The subunit ATG13 functions as an adaptor protein by recruiting ULK1, RB1CC1 and ATG101 to a core ULK1 complex. Furthermore, ATG13 directly binds both phospholipids and members of the Atg8 family. The central involvement of ATG13 in complex formation makes it an attractive target for autophagy regulation. Here, we analyzed known interactions of ATG13 with proteins and lipids for their potential modulation of ULK1 complex formation and autophagy induction. Targeting the ATG101-ATG13 interaction showed the strongest autophagy-inhibitory effect, whereas the inhibition of binding to ULK1 or RB1CC1 had only minor effects, emphasizing that mutations interfering with ULK1 complex assembly do not necessarily result in a blockade of autophagy. Furthermore, inhibition of ATG13 binding to phospholipids or Atg8 proteins had only mild effects on autophagy. Generally, the observed phenotypes were more severe when autophagy was induced by MTORC1/2 inhibition compared to amino acid starvation. Collectively, these data establish the interaction between ATG13 and ATG101 as a promising target in disease-settings where the inhibition of autophagy is desired.

The mycotoxin phomoxanthone A disturbs the form and function of the inner mitochondrial membrane.

Mitochondria are cellular organelles with crucial functions in the generation and distribution of ATP, the buffering of cytosolic Ca2+ and the initiation of apoptosis. Compounds that interfere with these functions are termed mitochondrial toxins, many of which are derived from microbes, such as antimycin A, oligomycin A, and ionomycin. Here, we identify the mycotoxin phomoxanthone A (PXA), derived from the endophytic fungus Phomopsis longicolla, as a mitochondrial toxin. We show that PXA elicits a strong release of Ca2+ from the mitochondria but not from the ER. In addition, PXA depolarises the mitochondria similarly to protonophoric uncouplers such as CCCP, yet unlike these, it does not increase but rather inhibits cellular respiration and electron transport chain activity. The respiration-dependent mitochondrial network structure rapidly collapses into fragments upon PXA treatment. Surprisingly, this fragmentation is independent from the canonical mitochondrial fission and fusion mediators DRP1 and OPA1, and exclusively affects the inner mitochondrial membrane, leading to cristae disruption, release of pro-apoptotic proteins, and apoptosis. Taken together, our results suggest that PXA is a mitochondrial toxin with a novel mode of action that might prove a useful tool for the study of mitochondrial ion homoeostasis and membrane dynamics.

A systems study reveals concurrent activation of AMPK and mTOR by amino acids.

Amino acids (aa) are not only building blocks for proteins, but also signalling molecules, with the mammalian target of rapamycin complex 1 (mTORC1) acting as a key mediator. However, little is known about whether aa, independently of mTORC1, activate other kinases of the mTOR signalling network. To delineate aa-stimulated mTOR network dynamics, we here combine a computational-experimental approach with text mining-enhanced quantitative proteomics. We report that AMP-activated protein kinase (AMPK), phosphatidylinositide 3-kinase (PI3K) and mTOR complex 2 (mTORC2) are acutely activated by aa-readdition in an mTORC1-independent manner. AMPK activation by aa is mediated by Ca2+/calmodulin-dependent protein kinase kinase ß (CaMKKß). In response, AMPK impinges on the autophagy regulators Unc-51-like kinase-1 (ULK1) and c-Jun. AMPK is widely recognized as an mTORC1 antagonist that is activated by starvation. We find that aa acutely activate AMPK concurrently with mTOR. We show that AMPK under aa sufficiency acts to sustain autophagy. This may be required to maintain protein homoeostasis and deliver metabolite intermediates for biosynthetic processes.