Rituraj Pal

Src-dependent impairment of autophagy by oxidative stress in a mouse model of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a fatal degenerative muscle disease resulting from mutations in the dystrophin gene. Increased oxidative stress and altered Ca2+ homeostasis are hallmarks of dystrophic muscle. While impaired autophagy has recently been implicated in the disease process, the mechanisms underlying the impairment have not been elucidated. Here we show that nicotinamide adenine dinucleotide phosphatase (Nox2)-induced oxidative stress impairs both autophagy and lysosome formation in mdx mice. Persistent activation of Src kinase leads to activation of the autophagy repressor mammalian target of rapamycin (mTOR) via PI3K/Akt phosphorylation. Inhibition of Nox2 or Src kinase reduces oxidative stress and partially rescues the defective autophagy and lysosome biogenesis. Genetic down regulation of Nox2 activity in the mdx mouse decreases ROS production, abrogates defective autophagy and rescues histological abnormalities and contractile impairment. Our data highlight mechanisms underlying the pathogenesis of DMD and identify NADPH oxidase and Src kinase as potential therapeutic targets.

MTORC1-independent TFEB activation via Akt inhibition promotes cellular clearance in neurodegenerative storage diseases

Neurodegenerative diseases characterized by aberrant accumulation of undigested cellular components represent unmet medical conditions for which the identification of actionable targets is urgently needed. Here we identify a pharmacologically actionable pathway that controls cellular clearance via Akt modulation of transcription factor EB (TFEB), a master regulator of lysosomal pathways. We show that Akt phosphorylates TFEB at Ser467 and represses TFEB nuclear translocation independently of mechanistic target of rapamycin complex 1 (mTORC1), a known TFEB inhibitor. The autophagy enhancer trehalose activates TFEB by diminishing Akt activity. Administration of trehalose to a mouse model of Batten disease, a prototypical neurodegenerative disease presenting with intralysosomal storage, enhances clearance of proteolipid aggregates, reduces neuropathology and prolongs survival of diseased mice. Pharmacological inhibition of Akt promotes cellular clearance in cells from patients with a variety of lysosomal diseases, thus suggesting broad applicability of this approach. These findings open new perspectives for the clinical translation of TFEB-mediated enhancement of cellular clearance in neurodegenerative storage diseases.

Real-Time Imaging of NADPH Oxidase Activity in Living Cells Using a Novel Fluorescent Protein Reporter

Production of reactive oxygen species (ROS) has been implicated in the pathology of many conditions, including cardiovascular, inflammatory and degenerative diseases, aging, muscular dystrophy, and muscle fatigue. NADPH oxidases (Nox) have recently gained attention as an important source of ROS involved in redox signaling. However, our knowledge of the source of ROS has been limited by the relatively impoverished array of tools available to study them and the limitations of all imaging probes to provide meaningful spatial resolution. By linking redox-sensitive GFP (roGFP) to the Nox organizer protein, p47phox, we have developed a redox sensitive protein to specifically assess Nox activity (p47-roGFP). Stimulation of murine macrophages with endotoxin resulted in rapid, reversible oxidation of p47-roGFP. In murine skeletal muscle, both passive stretch and repetitive electrical stimulation resulted in oxidation of p47-roGFP. The oxidation of p47-roGFP in both macrophages and skeletal muscle was blocked by a Nox specific peptide inhibitor. Furthermore, expression of p47-roGFP in p47phox deficient cells restored Nox activity. As Nox has been linked to pathological redox signaling, our newly developed Nox biosensor will allow for the direct assessment of Nox activity and the development of therapeutic Nox inhibitors.

Rotenone induces neurotoxicity through Rac1-dependent activation of NADPH oxidase in SHSY-5Y cells

Neurodegenerative diseases are attributed to impairment of the ubiquitin–proteasome system (UPS). Oxidative stress has been considered a contributing factor in the pathology of impaired UPS by promoting protein misfolding and subsequent protein aggregate formation. Increasing evidence suggests that NADPH oxidase is a likely source of excessive oxidative stress in neurodegenerative disorders. However, the mechanism of activation and its role in impaired UPS is not understood. We show that activation of NADPH oxidase in a neuroblastoma cell line (SHSY‐5Y) resulted in increased oxidative and nitrosative stress, elevated cytosolic calcium, ER‐stress, impaired UPS, and apoptosis. Rac1 inhibition mitigated the oxidative/nitrosative stress, prevented calcium‐dependent ER‐stress, and partially rescued UPS function. These findings demonstrate that Rac1 and NADPH oxidase play an important role in rotenone neurotoxicity.

Redox regulation of autophagy in skeletal muscle.

Autophagy is a cellular degradative pathway that involves the delivery of cytoplasmic components, including proteins and organelles, to the lysosome for degradation. Autophagy is implicated in the maintenance of skeletal muscle; increased autophagy leads to muscle atrophy while decreased autophagy leads to degeneration and weakness. A growing body of work suggests that reactive oxygen species (ROS) are important cellular signal transducers controlling autophagy. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and mitochondria are major sources of ROS generation in skeletal muscle that are likely regulating autophagy through different signaling cascades based on localization of the ROS signals. This review aims to provide insight into the redox control of autophagy in skeletal muscle. Understanding the mechanisms by which ROS regulate autophagy will provide novel therapeutic targets for skeletal muscle diseases.

NADPH oxidase promotes Parkinsonian phenotypes by impairing autophagic flux in an mTORC1-independent fashion in a cellular model of Parkinson's disease.

Oxidative stress and aberrant accumulation of misfolded proteins in the cytosol are key pathological features associated with Parkinson’s disease (PD). NADPH oxidase (Nox2) is upregulated in the pathogenesis of PD; however, the underlying mechanism(s) of Nox2-mediated oxidative stress in PD pathogenesis are still unknown. Using a rotenone-inducible cellular model of PD, we observed that a short exposure to rotenone (0.5 μM) resulted in impaired autophagic flux through activation of a Nox2 dependent Src/PI3K/Akt axis, with a consequent disruption of a Beclin1-VPS34 interaction that was independent of mTORC1 activity. Sustained exposure to rotenone at a higher dose (10 μM) decreased mTORC1 activity; however, autophagic flux was still impaired due to dysregulation of lysosomal activity with subsequent induction of the apoptotic machinery. Cumulatively, our results highlight a complex pathogenic mechanism for PD where short- and long-term oxidative stress alters different signaling pathways, ultimately resulting in anomalous autophagic activity and disease phenotype. Inhibition of Nox2-dependent oxidative stress attenuated the impaired autophagy and cell death, highlighting the importance and therapeutic potential of these pathways for treating patients with PD.

AKT modulates the autophagy-lysosome pathway via TFEB

The serine and threonine kinase AKT (also known as protein kinase B, PKB) integrates inputs from growth factors and metabolic effectors to control key multifunctional signaling hubs via direct phosphorylation of substrates that control cell growth, proliferation and survival. Among critical pro-survival mechanisms, autophagy is an evolutionarily conserved housekeeping pathway that functions to degrade and recycle macromolecules and organelles as part of basal cell metabolism as well as in response to specific stimuli. Autophagic degradation is based on the activity of the lysosome, a membrane-enclosed organelle that contains a wide array of hydrolytic enzymes with different macromolecule specificities. Our previous work has established that transcription factor EB (TFEB) acts as a master regulator of the autophagy-lysosome pathway (reviewed in ref. 3). Since the initial characterization of TFEB as a transcriptional modulator of the lysosomal system, cytoplasm-to-nucleus translocation of TFEB has been identified as the main mechanism underlying regulation of TFEB activity. While several proof-of-concept studies have shown that exogenous expression of TFEB can counteract appearance and progression of pathological changes in animal models of diseases with a prominent intracellular storage component, clinical translation has been limited by the lack of suitable entry points to modulate TFEB activity that are amenable to pharmacological manipulation with available drugs. Our recent work has uncovered AKT-mediated phosphorylation of TFEB as a mechanism by which AKT modulates the autophagy-lysosomal pathway. AKT phosphorylates TFEB on serine 467, a residue that is evolutionarily conserved in vertebrate species and that is also conserved in TFEB’s close homologs, TFE3 and MITF. A TFEB phosphomutant in which the AKT phosphoacceptor site was substituted by alanine to impede AKT-mediated phosphorylation (TFEB S467A) showed increased nuclear localization and stability as well as increased ability to activate TFEB downstream target genes, thus indicating that TFEB function is inhibited by AKT phosphorylation. Accordingly, both AKT knockdown and pharmacological inhibition of AKT promoted TFEB nuclear translocation; AKT inhibition also activated TFE3 and MITF, thus revealing conservation of this regulatory pathway (Fig. 1). Notably, AKT inhibition resulted in increased number of LC3-positive autophagic vesicles and increased expression of autophagic and lysosomal genes, thereby indicating global enhancement of the autophagy-lysosome pathway. Similar results were obtained with trehalose, a natural disaccharide that stimulates autophagy and exerts neuroprotection in various models of neurodegenerative proteinopathies. The mechanism of action of trehalose has been elusive for more than a decade; recent work has shown that trehalose inhibits the activity of glucose transporters at the plasma membrane, thus inducing a starvation-like state that promotes autophagy via the AMPK-ULK1 axis. We have shown that trehalose inhibits AKT activity in vitro and in vivo, thus resulting in TFEB nuclear translocation and activation of the autophagy-lysosome pathway. Importantly, expression of a constitutive active form of AKT prevented trehalose from inducing TFEB nuclear translocation, demonstrating that trehalose indeed activates TFEB via AKT inhibition. Activation of the autophagy-lysosome pathway obtained by pharmacological inhibition of AKT or trehalose administration resulted in enhanced clearance of aberrant proteolipid aggregates in cells from patients with neuronal ceroid lipofuscinosis, a genetically diverse subgroup of the lysosomal storage diseases. Significantly, oral administration of trehalose to a mouse model of juvenile neuronal ceroid lipofuscinosis (Batten disease) diminished AKT activity and promoted TFEB activity in the brain, again resulting in the activation of the autophagy-lysosome pathway and the subsequent clearance of ceroid lipopigments in neurons, which finally led to protection from neurodegeneration and elongation of the life span of affected mice. These findings provide a direct, pharmacologically actionable entry point to modulate TFEB activity and enhance the autophagy-lysosome pathway through increased TFEB function. The AKT-TFEB signaling pathway might be leveraged to devise treatments of degenerative diseases in which there is pathological storage of aberrant aggregates of proteins or other macromolecules, such as in Alzheimer disease, Parkinson disease, Huntington’s disease and lysosomal storage disorders. Clinical development of trehalose is currently focusing on the treatment of arterial aging (ClinicalTrial.gov ID:

Sphingomyelinase promotes oxidant production and skeletal muscle contractile dysfunction through activation of NADPH oxidase

Elevated concentrations of sphingomyelinase (SMase) have been detected in a variety of diseases. SMase has been shown to increase muscle derived oxidants and decrease skeletal muscle force; however, the sub-cellular site of oxidant production has not been elucidated. Using redox sensitive biosensors targeted to the mitochondria and NADPH oxidase (Nox2), we demonstrate that SMase increased Nox2-dependent ROS and had no effect on mitochondrial ROS in isolated FDB fibers. Pharmacological inhibition and genetic knockdown of Nox2 activity prevented SMase induced ROS production and provided protection against decreased force production in the diaphragm. In contrast, genetic overexpression of superoxide dismutase within the mitochondria did not prevent increased ROS production and offered no protection against decreased diaphragm function in response to SMase. Our study shows that SMase induced ROS production occurs in specific sub-cellular regions of skeletal muscle; however, the increased ROS does not completely account for the decrease in muscle function.

Inhibition of ERK1/2 Restores GSK3β Activity and Protein Synthesis Levels in a Model of Tuberous Sclerosis

Tuberous sclerosis (TS) is a multi-organ autosomal dominant disorder that is best characterized by neurodevelopmental deficits and the presence of benign tumors. TS pathology is caused by mutations in tuberous sclerosis complex (TSC) genes and is associated with insulin resistance, decreased glycogen synthase kinase 3β (GSK3β) activity, activation of the mammalian target of rapamycin complex 1 (mTORC1), and subsequent increase in protein synthesis. Here, we show that extracellular signal–regulated kinases (ERK1/2) respond to insulin stimulation and integrate insulin signaling to phosphorylate and thus inactivate GSK3β, resulting in increased protein synthesis that is independent of Akt/mTORC1 activity. Inhibition of ERK1/2 in Tsc2−/− cells—a model of TS—rescues GSK3β activity and protein synthesis levels, thus highlighting ERK1/2 as a potential therapeutic target for the treatment of TS.

EF24 prevents rotenone-induced estrogenic status alteration in breast cancer

Protein disulfide isomerase (PDI), an important endoplasmic reticulum‐resident oxidoreductase chaperone can bind to estrogens as well as intact with its receptor proteins [i.e. estrogen receptors (ER) α and β]. It has been postulated that PDI also acts as an intracellular 17β‐estradiol (E2)‐binding protein that transports and accumulates E2 in live cells. Drop in E2 level promotes dissociation of E2 from PDI and released in cytosol; the released E2 can augment estrogen receptor‐mediated transcriptional activity and mitogenic action in cultured cells by modulating the ERβ/ERα ratio. In this study, we observed rotenone‐induced damage to PDI leads to significant increase in ERβ/ERα ratio by down‐regulating ERα and up‐regulating ERβ. We demonstrated that nitrosative stress induced disruption of the cellular estrogenic status can be prevented through diphenyl difluoroketone (EF24, curcumin analog) intervention by protecting PDI from reactive oxygen species (ROS)‐induced damage. Together, our study suggests that both PDI and EF24 can play a vital role in maintaining cellular estrogenic homeostasis.