Inhibition of lipid kinase PIKfyve reveals a role for phosphatase Inpp4b in the regulation of PI(3)P-mediated lysosome dynamics through VPS34 activity.

Lysosome membranes contain diverse phosphoinositide (PtdIns) lipids that coordinate lysosome function and dynamics. The PtdIns repertoire on lysosomes is tightly regulated by the actions of diverse PtdIns kinases and phosphatases; however, specific roles for PtdIns in lysosomal functions and dynamics are currently unclear and require further investigation. It was previously shown that PIKfyve, a lipid kinase that synthesizes PtdIns(3,5)P2 from PtdIns(3)P, controls lysosome "fusion-fission" cycle dynamics, autophagosome turnover, and endocytic cargo delivery. Furthermore, INPP4B, a PtdIns 4-phosphatase that hydrolyzes PtdIns(3,4)P2 to form PtdIns(3)P, is emerging as a cancer-associated protein with roles in lysosomal biogenesis and other lysosomal functions. Here, we investigated the consequences of disrupting PIKfyve function in Inpp4b-deficient mouse embryonic fibroblasts. Through confocal fluorescence imaging, we observed the formation of massively enlarged lysosomes, accompanied by exacerbated reduction of endocytic trafficking, disrupted lysosome fusion-fission dynamics, and inhibition of autophagy. Finally, HPLC scintillation quantification of 3H-myo-inositol labeled PtdIns and PtdIns immunofluorescence staining, we observed that lysosomal PtdIns(3)P levels were significantly elevated in Inpp4b-deficient cells due to the hyperactivation of phosphatidylinositol 3-kinase catalytic subunit VPS34 enzymatic activity. In conclusion, our study identifies a novel signaling axis that maintains normal lysosomal homeostasis and dynamics, which includes the catalytic functions of Inpp4b, PIKfyve, and VPS34.

Reactive oxygen species prevent lysosome coalescence during PIKfyve inhibition.

Lysosomes are terminal, degradative organelles of the endosomal pathway that undergo repeated fusion-fission cycles with themselves, endosomes, phagosomes, and autophagosomes. Lysosome number and size depends on balanced fusion and fission rates. Thus, conditions that favour fusion over fission can reduce lysosome numbers while enlarging their size. Conversely, favouring fission over fusion may cause lysosome fragmentation and increase their numbers. PIKfyve is a phosphoinositide kinase that generates phosphatidylinositol-3,5-bisphosphate to modulate lysosomal functions. PIKfyve inhibition causes an increase in lysosome size and reduction in lysosome number, consistent with lysosome coalescence. This is thought to proceed through reduced lysosome reformation and/or fission after fusion with endosomes or other lysosomes. Previously, we observed that photo-damage during live-cell imaging prevented lysosome coalescence during PIKfyve inhibition. Thus, we postulated that lysosome fusion and/or fission dynamics are affected by reactive oxygen species (ROS). Here, we show that ROS generated by various independent mechanisms all impaired lysosome coalescence during PIKfyve inhibition and promoted lysosome fragmentation during PIKfyve re-activation. However, depending on the ROS species or mode of production, lysosome dynamics were affected distinctly. H2O2 impaired lysosome motility and reduced lysosome fusion with phagosomes, suggesting that H2O2 reduces lysosome fusogenecity. In comparison, inhibitors of oxidative phosphorylation, thiol groups, glutathione, or thioredoxin, did not impair lysosome motility but instead promoted clearance of actin puncta on lysosomes formed during PIKfyve inhibition. Additionally, actin depolymerizing agents prevented lysosome coalescence during PIKfyve inhibition. Thus, we discovered that ROS can generally prevent lysosome coalescence during PIKfyve inhibition using distinct mechanisms depending on the type of ROS.

The PH domain from the Toxoplasma gondii PH-containing protein-1 (TgPH1) serves as an ectopic reporter of phosphatidylinositol 3-phosphate in mammalian cells.

Phosphoinositide (PtdInsP) lipids recruit effector proteins to membranes to mediate a variety of functions including signal transduction and membrane trafficking. Each PtdInsP binds to a specific set of effectors through characteristic protein domains such as the PH, FYVE and PX domains. Domains with high affinity for a single PtdInsP species are useful as probes to visualize the distribution and dynamics of that PtdInsP. The endolysosomal system is governed by two primary PtdInsPs: phosphatidylinositol 3-phosphate [PtdIns(3)P] and phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2], which are thought to localize and control early endosomes and lysosomes/late endosomes, respectively. While PtdIns(3)P has been analysed with mammalian-derived PX and FYVE domains, PtdIns(3,5)P2 indicators remain controversial. Thus, complementary probes against these PtdInsPs are needed, including those originating from non-mammalian proteins. Here, we characterized in mammalian cells the dynamics of the PH domain from PH-containing protein-1 from the parasite Toxoplasma gondii (TgPH1), which was previously shown to bind PtdIns(3,5)P2 in vitro. However, we show that TgPH1 retains membrane-binding in PIKfyve-inhibited cells, suggesting that TgPH1 is not a viable PtdIns(3,5)P2 marker in mammalian cells. Instead, PtdIns(3)P depletion using pharmacological and enzyme-based assays dissociated TgPH1 from membranes. Indeed, TgPH1 co-localized with Rab5-positive early endosomes. In addition, TgPH1 co-localized and behaved similarly to the PX domain of p40phox and FYVE domain of EEA1, which are commonly used as PtdIns(3)P indicators. Collectively, TgPH1 offers a complementary reporter for PtdIns(3)P derived from a non-mammalian protein and that is distinct from commonly employed PX and FYVE domain-based probes.

Lysosome enlargement during inhibition of the lipid kinase PIKfyve proceeds through lysosome coalescence.

Lysosomes receive and degrade cargo from endocytosis, phagocytosis and autophagy. They also play an important role in sensing and instructing cells on their metabolic state. The lipid kinase PIKfyve generates phosphatidylinositol-3,5-bisphosphate to modulate lysosome function. PIKfyve inhibition leads to impaired degradative capacity, ion dysregulation, abated autophagic flux and a massive enlargement of lysosomes. Collectively, this leads to various physiological defects, including embryonic lethality, neurodegeneration and overt inflammation. The reasons for such drastic lysosome enlargement remain unclear. Here, we examined whether biosynthesis and/or fusion-fission dynamics contribute to swelling. First, we show that PIKfyve inhibition activates TFEB, TFE3 and MITF, enhancing lysosome gene expression. However, this did not augment lysosomal protein levels during acute PIKfyve inhibition, and deletion of TFEB and/or related proteins did not impair lysosome swelling. Instead, PIKfyve inhibition led to fewer but enlarged lysosomes, suggesting that an imbalance favouring lysosome fusion over fission causes lysosome enlargement. Indeed, conditions that abated fusion curtailed lysosome swelling in PIKfyve-inhibited cells.

Amyloid-ß Inhibits PDGFß Receptor Activation and Prevents PDGF-BBInduced Neuroprotection.

BACKGROUND: PDGFß receptors and their ligand, PDGF-BB, are upregulated in vivo after neuronal insults such as ischemia. When applied exogenously, PDGF-BB is neuroprotective against excitotoxicity and HIV proteins. OBJECTIVE: Given this growth factor's neuroprotective ability, we sought to determine if PDGF-BB would be neuroprotective against amyloid-ß (1-42), one of the pathological agents associated with Alzheimer's disease (AD). METHODS AND RESULTS: In both primary hippocampal neurons and the human-derived neuroblastoma cell line, SH-SY5Y, amyloid-ß treatment for 24 h decreased surviving cell number in a concentrationdependent manner. Pretreatment with PDGF-BB failed to provide any neuroprotection against amyloid-ß in primary neurons and only very limited protective effects in SH-SY5Y cells. In addition to its neuroprotective action, PDGF promotes cell growth and division in several systems, and the application of PDGFBB alone to serum-starved SH-SY5Y cells resulted in an increase in cell number. Amyloid-ß attenuated the mitogenic effects of PDGF-BB, inhibited PDGF-BB-induced PDGFß receptor phosphorylation, and attenuated the ability of PDGF-BB to protect neurons against NMDA-induced excitotoxicity. Despite the ability of amyloid-ß to inhibit PDGFß receptor activation, immunoprecipitation experiments failed to detect a physical interaction between amyloid-ß and PDGF-BB or the PDGFß receptor. However, G protein-coupled receptor transactivation of the PDGFß receptor (an exclusively intracellular signaling pathway) remained unaffected by the presence of amyloid-ß. CONCLUSIONS: As the PDGF system is upregulated upon neuronal damage, the ability of amyloid-ß to inhibit this endogenous neuroprotective system should be further investigated in the context of AD pathophysiology.