Autophagy

Plants, with their unique evolutionary trajectory and complex physiological adaptations, have developed organ-specific mechanisms to cope with various environmental stresses. Autophagy, an essential catabolic process, plays an important role in maintaining plant growth, immunity, and overall fitness. In this study, we reveal the spatio-temporal dynamics of autophagic responses in Arabidopsis thaliana roots and shoots under different stress conditions, including AZD8055 treatment and carbon and nitrogen depletion. Our findings demonstrate that roots exhibit stronger autophagic activity than shoots under all three conditions, highlighting the unique adaptations of these two organs. Furthermore, we dissect the selectivity of autophagy in targeting organelles, revealing immediate, delayed, and no uptake categories. Additionally, we observe organelles coexisting within autophagic bodies, shedding light on the complexity of cargo selection. This study enhances our understanding of plant specific autophagy dynamics, emphasizing its role in sustaining the source-sink functions and offering insights into plant adaptation to diverse stressors.

The endoplasmic reticulum (ER) and its muscle-specialized form, the sarcoplasmic reticulum (SR), are crucial organelles in muscle cells, involved notably in protein synthesis, calcium regulation and muscle contraction. A well-known process involved in ER remodeling and homeostasis is ER-phagy, also called reticulophagy, a selective form of autophagic process in which ER-phagy receptors mediate the delivery of ER portions to lysosomes for degradation. SH3KBP1 is an adaptor protein involved in membrane trafficking. Recently, it was shown to control ER morphology and SR formation in striated skeletal muscle. In this study, we demonstrate that SH3KBP1 can bind to LC3B and CKAP4 proteins, bridging ER to autophagosome membranes, and is degraded by autophagy, in developing muscle fibers. Moreover, SH3KBP1 down-regulation impacts basal autophagy efficiency and ER-phagy stimulation; it also impairs the turnover of numerous ER-resident proteins. Our work highlights a new role for SH3KBP1 as a soluble ER-phagy receptor in striated skeletal muscle.

Heat shock protein family B member 8 (HSPB8) is a chaperone involved in the chaperone-assisted selective autophagy (CASA) complex. HSPB8 in conjunction with cochaperone BAG3, promotes autophagy-mediated removal of misfolded proteins associated with various neurodegenerative diseases. Mutations in HSPB8, previously associated with Charcot Marie Tooth disease type 2L, have recently been linked to an autosomal dominant rimmed vacuolar myopathy (MFM13), and is considered a multisystem proteinopathy. Patients have distal and proximal limb girdle myopathy with muscle biopsy showing fatty replacement, endomysial fibrosis, and rimmed vacuoles leading to muscle atrophy and early demise. We have demonstrated reduced expression of HSPB8, altered autophagy and TDP-43 accumulation in patient fibroblasts. Using CRISPR technology, we generated a knock-in Hspb8 mouse model of the c.515dupC hot spot frameshift variant to study disease pathology. Overexpressed murine Hspb8 frameshift mutant (c.515dupC, fs) displays insolubility and aggregation propensity in Murine Neuroblastoma X Spinal Cord 34 (NSC-34) cells. Mutant Hspb8 mice developed late-onset muscle weakness beginning at 15 months. Muscle biochemical analyses revealed reduced HSPB8 levels, increased TDP-43, and altered autophagy markers, partially recapitulating the human phenotype. Fiber type analysis, neuromuscular junction integrity, and motor neurons show mild myopathy without neurodegeneration. Given the lack of available treatments, we evaluated trehalose, a natural disaccharide that induces HSPB8 and enhances autophagy. Administration of 2% trehalose in drinking water improves motor performance, restores HSPB8 expression, and ameliorates autophagic and TDP-43 pathology in mutant mice. These findings support the value of our preclinical models for translational studies, and autophagy enhancement as a potential therapeutic strategy for HSPB8-related myopathy.

Macroautophagy/autophagy is a process that degrades intracellular components and is strongly triggered by nitrogen starvation (-N). Some ATG (autophagy related) genes are activated at the transcriptional level in nitrogen starvation; however, a full understanding of transcriptional induction and the role of chromatin during this process remains unclear. To address this, we measured the occupancy of RNA polymerase II (Pol II), histone H3, and acetylated H4 (H4Ac) under nutrient-rich and -N conditions by ChIP-seq. We found that most genes are rapidly downregulated within 15-30 min, including ribosomal protein (RP) and biogenesis (RiBi) genes. Meanwhile, genes involved in amino acid (AA) biosynthesis are upregulated, along with many ATG genes. Unexpectedly, RP and RiBi genes were reinduced by 3 hours. Furthermore, many upregulated genes remained active during prolonged starvation. Histones are typically removed from promoters during transcription activation. Consistent with this, we found that most induced genes exhibited histone eviction and increased H4 acetylation at their promoters, suggesting a possible role for histone acetylation in their activation. In line with this, depleting Esa1, an essential H4 histone acetyltransferase, nearly abolished the induction of ribosomal biosynthetic genes and many AA biosynthetic genes. Sustained activation of many genes during prolonged starvation highlights the vital role of transcription in supporting autophagy and cell survival. This is the first comprehensive study to detail changes in chromatin, histone acetylation, and transcription during nitrogen starvation, highlighting the importance of Esa1 and H4Ac in this process.

Despite advances in the understanding of the cellular and molecular mechanisms involved in erythropoiesis, there are still unanswered questions regarding the coordination between autophagy, vesicular trafficking, and endocytic signaling during this process. The complexity of these events suggests the existence of regulatory mechanisms capable of integrating these pathways. In this context, low-density lipoprotein receptor-related protein 1 (LRP1) emerges as a potential modulator given its function as a multifunctional endocytic receptor and its involvement in the regulation of degradation and signaling processes in various cellular models. However, its role in modulating mitophagy, a particular type of autophagy, and its link to vesicular trafficking associated with multivesicular bodies (MVBs) and the release of exosomes during erythroid maturation has been poorly explored. In this regard, alpha-2-Macroglobulin (2M), the main physiological ligand of LRP1, has been identified in extracellular vesicles (EVs) in various pathophysiological contexts, suggesting that it may be involved in vesicular dynamics and cellular clearance. In this study, we demonstrate that activated 2M (2M*), induces autophagy and particularly mitophagy, in K562 cells, and that LRP1 is directly responsible for this activation. Furthermore, we observed that 2M* stimulates the interaction of autophagosomes with MVBs/amphisomes and that EVs from K562 cells are positive for LC3, supporting a close relationship between the endocytic pathway and the autophagic pathway mediated by the 2M-LRP1 interaction. Taken together, these findings expand our understanding of erythroid biology and provide a conceptual foundation for exploring altered mechanisms in erythropoietic diseases and for the development of diagnostic and therapeutic strategies.

Effective lysosome function is essential for health, and declines with ageing and disease. Upon lysosome damage, cells mount a complex stress response to restore homeostasis. Membrane ATG8ylation plays a central role, orchestrating lysosome repair, replacement and removal, either directly at the damaged membrane, via CASM (conjugation of ATG8s to single membranes), or at nascent autophagosomes, during lysophagy. Here, we identify a novel role for CASM in driving an antioxidant response to lysosome stress. Following damage, CASM regulates both lysosome ubiquitination and p62 recruitment. Membrane-associated p62 undergoes S349-phosphorylation, which permits KEAP1 sequestration, thereby releasing master transcription factor, NRF2. Liberated NRF2 translocates to the nucleus and promotes transcription of antioxidant and detoxifying genes, co-ordinating a cytoprotective response, in a CASM-dependent manner. These findings position CASM as a major upstream response to lysosome stress and uncover the p62/KEAP1/NRF2 axis as a novel effector pathway, termed SOLAR (SQSTM1/p62 Oligomer-mediated lysosome antioxidant response).

Mutations in the molecular motor protein KIF1A result in a spectrum of neurodevelopmental and neurodegenerative disorders termed KIF1A-Associated Neurological Disorder (KAND). KIF1A mutations variably disrupt synaptic vesicle trafficking, but the effects of KIF1A mutations on other trafficking pathways remain unexplored. Autophagy is a conserved pathway required for neuronal homeostasis. We investigated the role of KIF1A in autophagy using gene-edited human IPSC-derived neurons. KIF1A loss inhibited the trafficking of ATG9, a transmembrane lipid scramblase necessary for autophagosome biogenesis. This deficit significantly reduced autophagosome biogenesis and the density of axonal autophagosomes. KIF1A loss also depleted lysosomes from the axon, inhibiting autophagosome maturation. In neurons gene-edited to heterozygously express a pathogenic variant linked to a Rett-like syndrome in KAND patients, we also noted significant deficits in autophagy and lysosomal trafficking. Together, these results suggest that KIF1A-mediated transport is critical to neuronal autophagy and that deficits in autophagy may contribute to pathogenesis in KAND. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/740140v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1f3180corg.highwire.dtl.DTLVardef@afe5dcorg.highwire.dtl.DTLVardef@1be676org.highwire.dtl.DTLVardef@1b75c6b_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cellular senescence combines stable proliferative arrest with extensive changes in secretory, metabolic and organelle programs. Procyanidin C1 (PCC1) has dose-dependent senomorphic and senolytic activity, but the structure of its transcriptome-wide response is not well defined. We reanalyzed published RNA-sequencing counts from bleomycin-induced senescent PSC27 human stromal cells treated with 50 {micro}M PCC1. The primary contrast contained three independent biological replicates per condition. PCC1 altered 8,420 genes at adjusted P<0.05 and absolute log2 fold change[&ge;]1. Ribosomal genes showed the clearest coordinated response, followed by lysosomal genes. Analysis across the full ontologies also identified lysosomal-membrane, proton-transport, late-endosome, lipid-localization, cholesterol and fatty-acid programs. Prespecified analyses revealed two trajectories relative to senescence. Lipid-transport, lipid-binding and plasma-membrane programs decreased during senescence and increased after PCC1, whereas lysosomal-membrane, mitochondrial-membrane, cholesterol and ion-transport programs increased in both contrasts. Senescence-associated outputs were more selective. A prespecified SASP-effector score decreased, but a broad Reactome SASP set did not pass false-discovery correction. AP-1-family expression shifted, and an HSP90/HSF1/proteostasis panel increased. Together, these data support a model in which PCC1 induces coordinated transcriptomic remodeling in senescent stromal cells, encompassing membrane-lipid remodeling, changes in cellular infrastructure, and selective modulation of senescence-associated outputs.

Autophagy is a central pathway for cellular homeostasis, mediating degradation and recycling of cytoplasmic components through lysosomal processing. Although autophagy has been implicated in axon growth and neuronal injury responses, its role in axon regeneration remains incompletely understood. In parallel, integrin trafficking and focal adhesion dynamics are critical determinants of axonal growth, and increasing evidence indicates that autophagy regulates focal adhesion turnover in non-neuronal cells; however, whether this mechanism operates in neurons remains unknown. Here, we investigated the dynamics of autophagy during axon growth and regeneration in adult sensory neurons and examined its relationship with integrin trafficking. Using live imaging, we analyzed autophagic vesicles and integrin-containing compartments in axons under basal conditions and following axotomy. We find that axonal injury induces marked short- and long-term alterations in distal axon and growth cone autophagic vesicle dynamics, integrin trafficking, and autophagy-associated integrin turnover. Importantly, these changes correlate with axonal growth and regenerative capacity. Furthermore, we identify a functional interplay between autophagy and integrins, suggesting bidirectional regulation in which autophagy contributes to adhesion receptor recycling, while integrins feedback to modulate autophagy. Finally, pharmacological modulation indicates that autophagy plays a critical role in both axonal growth and regeneration, and that rapamycin enhances regenerative responses, likely through modulation of autophagy-dependent integrin recycling rather than a general increase in autophagic activity. Together, these findings reveal a bidirectional feedback system between integrin-mediated extracellular cues and autophagy-dependent intracellular trafficking that jointly orchestrates axon growth and regeneration.

Autophagy is a degradative pathway that is critical in neurons to maintain their homeostasis and to direct nervous system development. Neurons are large, highly polarized cells with distinct compartments that perform discrete functions. How an individual neuron can differentially mobilize autophagy in the axon, dendrite, and cell body is unknown. Here we interrogate the role of autophagy in the neurodevelopment of a single neuron in Caenorhabditis elegans to identify how the spatial compartmentalization of neuronal autophagy ultimately restricts neurite outgrowth in multiple neuronal compartments. However, while canonical autophagy restricts dendrite outgrowth, noncanonical forms of autophagy appear to restrain neurite outgrowth in the axon and soma. Through mutant analysis of the autophagy pathway, we identify that WIPI2-independent autophagy modulates ectopic neurite formation in the soma and that ATG9-independent autophagy regulates axon arborization. Further, we find that unrelated lipid scramblases can compensate for the loss of ATG9 in axon arborization. Our data indicate that neurons marshal both canonical and non-canonical autophagy to spatially control development of separate compartments.

Lipid droplets (LDs) are unique organelles, surrounded by a phospholipid monolayer. They are present in most eukaryotic cells including the unicellular model organism S. cerevisiae. LDs store neutral lipids which serve as precursors for amphipathic membrane lipids and as an energy reserve. Loss of LDs in S. cerevisiae results in multiple cellular defects impairing lipid homeostasis and the biogenesis and function of other organelles. Here, we find that the expression levels of many proteins in isolated mitochondrial fractions are altered in cells that cannot synthesize neutral lipids and therefore lack LDs. In addition, among several downregulated proteins, we identified the previously uncharacterized Ylr001c (which we name Vlf1 for Vacuolar Lipophagy Factor 1). We show that Vlf1 is glycosylated and, in contrast to some previous reports, is actually localized to the vacuole. Furthermore, we demonstrate that changes in Vlf1 expression alter growth sensitivity to rapamycin, and detected a physical interaction of Vlf1 with Atg15, a lipase involved in autophagy. Additionally, we observe higher levels of autophagy/lipophagy in the absence of Vlf1 and a reduction upon overexpression of the protein. Taken together, the effects on lipohagy by Vlf1 makes it, according to our knowledge, the first vacuolar lipophagy regulator identified in S. cerevisiae.

Zebrafish can regenerate the heart by proliferation of cardiomyocytes. While the innate immune response and wound re-vascularization are pre-requisites for cardiomyocyte regeneration, little is known about signals linking early injury responses with the initiations of regenerative programs in cardiomyocytes. Here we show that mTOR (mechanistic target of rapamycin) signaling is rapidly activated in response to heart injury in many cell types of the heart including endothelial cells, cardiomyocytes and macrophages, but surprisingly not in neutrophils. We find that mTORC2 regulates macrophage recruitment to the wound, while mTORC1 is required for wound debris clearance by macrophages. In addition, mTOR signaling is required for wound re-vascularization. Interestingly, it also appears to directly regulate cardiomyocyte dedifferentiation and proliferation, making mTOR signaling a central hub for regenerative responses. Anabolic mTOR signaling acts as potent inhibitor of catabolic autophagy in many systems. Yet, we observed upregulation of autophagy within border zone cardiomyocytes where mTOR signaling is active. We show that mTOR signaling limits, but does not block autophagy, and that autophagic flux is regulated by both inhibitory mTOR signaling and stimulatory JNK and MEK pathways. Our results indicate that a fine-balanced anabolic and catabolic injury response is essential for zebrafish heart regeneration. Furthermore, they reveal interesting differences in the regulation of mTOR signaling and autophagy between the regenerative zebrafish heart and non-regenerative mammalian hearts.

Mutations in the LRRK2 gene are the most common genetic cause of both familial and sporadic Parkinsons disease (PD). LRRK2 belongs to the leucine-rich repeat kinase (LRRK) family. Two members of the LRRK family exist in humans (LRRK1 and LRRK2). Although there is strong structural similarity between the two proteins, they have attracted very different levels of attention by the scientific community owing to the strong association between LRRK2 and PD. In contrast, the role of LRRK1 is relatively unexplored. LRRK2 is also known to regulate endolysosomal function, but its precise role in this process remains incompletely understood. Our study investigated the interaction between LRRK1 and LRRK2 under different cellular conditions, uncovering their role in modulating the endolysosomal system. We found that LRRK1 and LRRK2 interact and modulate each others activity, and that this interaction is reduced under starvation conditions. We also found that LRRK1 and LRRK2 have contrasting effects on lysosomal size, impacting on lysosomal exocytosis. Together, our findings suggest that LRRK2 regulates endolysosomal homeostasis, at least in part, by modulating LRRK1. Our findings offer new insight into the molecular mechanisms associated with lysosomal function and, ultimately, we anticipate this knowledge will help us better understand the molecular crosstalk between LRRK kinases and their contribution to PD pathogenesis. Graphical abstractStarvation reduces the interaction between LRRK2 and LRRK1 due to a conformational change in LRRK2. Under normal conditions, LRRK2/LRRK1 interaction enhances LRRK1 activity, leading to increased phosphorylation of Rab7. Disruption of the Rab7 cycle impairs lysosomal homeostasis, leading to lysosomal accumulation and an increase in lysosomal diameter. This enlargement negatively impacts lysosomal exocytosis. Created with BioRender.com. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC="FIGDIR/small/731951v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@13ce58forg.highwire.dtl.DTLVardef@1034543org.highwire.dtl.DTLVardef@1b84096org.highwire.dtl.DTLVardef@198598f_HPS_FORMAT_FIGEXP M_FIG C_FIG

HSPA1A is a stress-inducible molecular chaperone that localizes to the plasma membrane (PM) of heat-shocked and cancer cells, where its membrane-associated form contributes to therapeutic resistance, membrane stabilization, and immune modulation. Because HSPA1A lacks a signal peptide, it does not follow the classical secretory pathway; instead, it reaches the PM through unconventional routes whose vesicular intermediates and intracellular lipid requirements remain largely undefined. Here, we show that following heat shock, HSPA1A undergoes coordinated redistribution across the endo-lysosomal network. It transiently associates with PI(3)P-enriched early endosomes, progresses through Rab4A- and Rab4B-positive recycling endosomes, and accumulates in LAMP1-positive lysosomes, while avoiding degradative and slow-recycling routes. Pharmacological inhibition of the ER-Golgi pathway did not affect HSPA1As PM localization, while disruption of endosomal maturation and lysosomal function resulted in significant reductions. Heat shock drives a progressive increase in lysosomal BMP immunoreactivity, and pharmacological BMP accumulation increased PM-HSPA1A, whereas intracellular antibody-mediated BMP blockade reduced it, identifying BMP-enriched lysosomes as regulatory hubs that govern HSPA1A PM competence. Using a rapamycin-inducible compartment-specific phosphatase system, we further demonstrate that PI(4)P is required not only at the PM for final docking but within early endosomes, late endosomes, and lysosomes, establishing a distributed PI(4)P requirement across the endosomal network. Together, these findings define a lipid-gated vesicular trafficking mechanism for HSPA1A PM localization and identify lysosomal BMP and endosomal PI(4)P as additional regulatory layers relevant to cancer cells in which constitutive lipid remodeling may sustain membrane-associated HSPA1A and its pro-survival functions.

Intracellular parasites cause over 226 million disability-adjusted life years (DALY) globally each year. Current treatment methods fall short of desirable impact due to adverse effects and growing resistance, shifting focus to parasitic effectors for future therapeutic development. Unfortunately, the divergent nature of parasitic proteins has impeded in silico discovery of - and functional inference for - parasitic effectors. Here, we present SecretTarget, a pipeline designed to identify host-interacting effector candidates through secondary localization features. By truncating signal peptides from predicted extracellular proteins ({Delta}SP), we unmask potential underlying localization features that dictate subcellular trafficking within the host. Applying our pipeline to a set of Toxoplasma gondii secreted effectors with known localizations and interactions, we propose a novel host ER-parasite interaction critical for parasite survival, recapitulate published localizations, and provide meaningful biological insights aligning with recent host-parasite interaction discoveries.

Mitophagy is the process of selective autophagic clearance of damaged mitochondria and is closely implicated in neurodegenerative disease. PTEN-induced kinase 1 (PINK1) and a RBR E3 ubiquitin-protein ligase (Parkin) constitute a positive feedback loop in mitophagy initiation. It is known that reactive oxygen species (ROS) modulate mitophagy, while the exact regulatory mechanism remains largely elusive. Here, we found that exogenously applied ROS effectively block mitophagy induced by acute mitochondrial damage agents, which could be reversed by antioxidants. Mechanistically, ROS activate poly(ADP-ribose) polymerase 1 (PARP1), and suppression of PARP1 eliminates the inhibitory effect of ROS on mitophagy. Notably, PARP1 directly interacts with PINK1 and mediates its PARylation at residue E417, thereby negatively regulating PINK1 function. Collectively, our study identifies PARylation as a new form of post-translational modification of PINK1 and reveals a novel mechanism underlying the regulatory role of ROS in mitophagy by PARP1 activation and PARylation of PINK1. In briefGao et al. demonstrate that exogenous ROS inhibit mitophagy. Mechanistically, ROS activate PARP1, which mediates PARylation of PINK1, a central regulator of mitophagy, leading to its functional impairment. This study reveals a novel regulatory mechanism of ROS on mitophagy through PARP1 activation and identifies PARylation as a novel form of post-translational modification of PINK1. HighlightsO_LIROS block PINK1-Parkin-mediated mitophagy. C_LIO_LIROS activate PARP1. C_LIO_LIPARP1 suppression eliminates the inhibitory effect of ROS on mitophagy. C_LIO_LIPARylation of PINK1 by PARP1 impairs its activity and mitophagy. C_LI

The SARS-CoV-2 envelope (E) protein is a virulence factor that remodels host endomembranes, but mechanisms remain incompletely understood. We recently demonstrated that E protein interacts with and inhibits the sarco/endoplasmic reticulum Ca2-ATPase (SERCA), disrupting ER calcium homeostasis. Here, we investigated how this perturbation affects autophagy-associated membrane organization. E protein expression induced lipidated LC3 accumulation and enlarged p62-positive structures, consistent with dysregulated autophagic turnover. Although E protein partially colocalized with LC3 and p62, enlarged p62-positive structures were also observed in cells retaining the reticular ER distribution of E protein, indicating that their formation does not require association with E protein or ER reorganization. E protein also increased the association of p62-positive structures with lysosomes without altering lysosome abundance. Pharmacological SERCA activation attenuated E protein-induced remodeling of autophagy-associated structures, demonstrating that SERCA inhibition contributes to these alterations. Together, our findings establish SERCA-dependent ER calcium homeostasis as a host pathway linking E protein expression to remodeling of autophagy-associated membrane compartments, providing a mechanistic framework for how the SARS-CoV-2 E protein promotes ER membrane remodeling associated with coronavirus replication.

Aquaporins at the plant plasmalemma are divided into two highly conserved subclasses, PLASMA MEMBRANE INTINSIC PROTEINs 1 (PIP1) and PIP2. Arabidopsis thaliana encodes five PIP1 and eight PIP2 isoforms. Individual loss-of-function mutants had been employed for functional analyses. Here, we observe that the pip2;1 pip2;2 pip2;4 pip2;6 pip2;7 quintuple mutant defective of major PIP2 isoforms concomitantly leads to a strongly reduced PIP1 protein level. Lower order mutants pip2;1 pip2;2 and pip2;1 pip2;2 pip2;7 still harbor only 60% and 20% residual PIP1, respectively. This repression is established post-translationally, since neither PIP1s steady-state transcripts nor polysome-associated PIP1 mRNAs are suppressed by pip2;1 pip2;2 pip2;7. Thus, the two major pathways operating in eukaryotes for removal of aberrant proteins, ubiquitin proteasome system (UPS)-dependent ER-associated degradation (ERAD) and autophagy/vacuole-linked degradation, were assessed. Introgression of atg7 blocking autophagy-mediated degradation does not affect the PIP1 protein level of pip2;1 pip2;2 pip2;7. In contrast, introgression of ERAD loss-of-function mutations hrd1A hrd1B and dln1 into pip2;1 pip2;2 pip2;7 partially stabilizes its PIP1 protein level. PIP1 accumulates intracellularly upon pharmacological inhibition of proteasomal degradation by MG132. Nevertheless, the lack of a full PIP1 recovery by these means suggests the flexible operation of parallel ERAD components or unknown pathways. In conclusion, the essential dependence of PIP1 expression on PIP2 isoforms intrinsically interconnects the two PIP subclades at the protein level and will thereby affect their mutual functions. Significance statementPlasma membrane intrinsic proteins constituting the most homogenous plant aquaporin family are nonetheless split into two highly conserved subfamilies, PIP1 and PIP2. The loss of major Arabidopsis PIP2 isoforms does not lead to compensation by PIP1 members, but rather to PIP1s concomitant, post-translational repression. This dependence of PIP1 isoforms inevitably ties the two PIP subfamilies and their function.

Type 2 Diabetes mellitus (T2DM) is a metabolic syndrome characterized by hyperglycemia and various complications. Current drugs are limited by side effects and resistance, necessitating novel targets and therapies. Previous studies have shown that MK-83, a synthetic agonist of transient receptor potential mucolipin 1 (TRPML1/ MCOLN1), a lysosomal Ca2+ channel, activates adenosine 5'-monophosphate-activated protein kinase (AMPK), a key therapeutic target in diabetes. However, whether targeting TRPML1 can treat T2DM remains unclear. In this study, we found that transgenic overexpression or pharmacological activation of TRPML1 finely controls AMPK phosphorylation via a Ca2+-CaMKK{beta}-dependent mechanism. This activation promotes glucose transporter 4 (GLUT4) translocation and dramatically increases intracellular glucose uptake. Conversely, genetic inactivation or pharmacologically inhibition of TRPML1 blocks both AMPK activation and glucose uptake. More importantly, pharmacological activation of TRPML1 in vivo dramatically alleviates hyperglycemia in db/db mice (a T2DM model), as evidenced by random blood glucose levels, fasting blood glucose levels and HbA1c levels. Furthermore, other hallmark features of db/db mice-including impaired oral glucose tolerance, reduced insulin tolerance, and elevated ALT and AST levels- were all ameliorated upon TRPML1 activation. Hence, targeting lysosomal TRPML1 channel represents a promising therapeutic strategy for T2DM, with highly specific TRPML1 agonists as potential anti-diabetic agents.

Mitochondrial diseases are a heterogeneous group of genetic disorders caused by impaired oxidative phosphorylation (OxPhos). When skeletal muscle is predominantly affected, they are defined as primary mitochondrial myopathies. Although the genetic causes of mitochondrial myopathies and the resulting bioenergetic impairments are well established, the metabolic drivers behind progressive muscle dysfunction remain poorly defined. This gap in knowledge may contribute to the lack of effective treatments for these disorders. OxPhos defective muscle initiates a metabolic response coordinated by systemic signals which invokes the mobilization of fatty acids from white adipose tissue despite muscle inability to fully oxidize fatty acids due to OxPhos impairment. Here, we show that in human patients with mitochondrial disease and mice with OxPhos defective muscle, increased fatty acid mobilization from white adipose tissue leads to ectopic lipid accumulation and lipotoxicity in skeletal muscle. We find an increase in very long chain ceramides which are mechanistically linked to chronic ER stress, phosphorylation of eIF2, and activation of ATF4 signaling. We propose that Ph-eIF2-mediated attenuation of global protein synthesis and ATF4-initiated atrophy pathways contribute to muscle wasting and weakness. Importantly, inhibition of de novo synthesis of ceramides with myriocin reduces ER stress and improves muscle proteostasis, body weight, and motor functions in a conditional COX10 KO mouse model of mitochondrial myopathy. Together, these findings highlight altered lipid metabolism as a contributor of mitochondrial myopathy pathogenesis and identify lipid-mediated stress pathways that can be targeted therapeutically.