Inhibition of DUSP6 Activates Autophagy and Rescues the Retinal Pigment Epithelium in Sodium Iodate-Induced Retinal Degeneration Models In Vivo and In Vitro

Autophagy plays a protective role in the retinal pigment epithelium (RPE) by eliminating damaged organelles in response to reactive oxygen species (ROS). Dual-specificity protein phosphatase 6 (DUSP6), which belongs to the DUSP subfamily, works as a negative-feedback regulator of the extracellular signal-regulated kinase (ERK) pathway. However, the complex interplay between DUSP6 and autophagy induced by ROS in RPE is yet to be investigated. To investigate the relationship between DUSP6 and autophagy, we exposed the ARPE-19 cell line and C57BL/6N mice to sodium iodate (NaIO3) as an oxidative stress inducer. Our data showed that the inhibition of DUSP6 activity promotes autophagy flux through the ERK pathway via the upregulation of immunoblotting expression in ARPE-19 cells. Live imaging showed a significant increase in autophagic flux activities, which suggested the restoration autophagy after treatment with the DUSP6 inhibitor. Furthermore, the mouse RPE layer exhibited an irregular structure and abnormal deposits following NaIO3 injection. The retina layer was recovered after being treated with DUSP6 inhibitor; this suggests that DUSP6 inhibitor can rescue retinal damage by restoring the mouse retina’s autophagy flux. This study suggests that the upregulation of DUSP6 can cause autophagy flux malfunctions in the RPE. The DUSP6 inhibitor can restore autophagy induction, which may serve as a potential therapeutic approach for retinal degeneration disease.

Protectin DX Attenuates Lumbar Radicular Pain of Non-compressive Disc Herniation by Autophagy Flux Stimulation via Adenosine Monophosphate-Activated Protein Kinase Signaling

Background: Neuroinflammation plays a crucial role in initiating and sustaining lumbar radicular pain (LRP). Protectin DX (PDX) has been experimentally verified to possess pro-resolving properties and anti-inflammatory effects. This study aimed to observe the analgesic effects of PDX and its potential mechanisms in LRP rats with non-compressive lumbar disc herniation (NCLDH).Method: Only male rats were selected to avoid gender-related interferences. Rat models of NCLDH were established, and rats were randomly divided into four groups: the sham group, the vehicle group, the PDX (10 ng PDX) group, and the PDX (100 ng PDX) group. Changes in the mechanical withdrawal threshold and thermal withdrawal latency were observed for 7 days. The mRNAs of pro-inflammatory and anti-inflammatory mediators were evaluated via real-time polymerase chain reaction, whereas western blot and immunohistochemistry were separately conducted to assess the expression levels of autophagy-related proteins and adenosine monophosphate-activated protein kinase (AMPK) signaling.Results: Intrathecal delivery of PDX reduced interleukin (IL)-6 and IL-1β mRNA levels and facilitated mRNA transcription of transforming growth factor-β1, with attenuation of mechanical and thermal hyperalgesia in LRP rat models. With the application of nucleus pulposus to the dorsal root ganglion, autophagy flux and AMPK signaling were severely disrupted in the spinal dorsal horns, and intrathecal treatment with PDX could dose-dependently restore the dysfunction of autophagy flux and AMPK signaling.Conclusion: These data suggest that PDX possesses pro-resolving properties and exerts potent analgesic effects in LRP by affecting autophagy flux via AMPK signaling.

In search of autophagy biomarkers in breast cancer: Receptor status and drug agnostic transcriptional changes during autophagy flux in cell lines

Autophagy drives drug resistance and drug-induced cancer cell cytotoxicity. Targeting the autophagy process could greatly improve chemotherapy outcomes. The discovery of specific inhibitors or activators has been hindered by challenges with reliably measuring autophagy levels in a clinical setting. We investigated drug-induced autophagy in breast cancer cell lines with differing ER/PR/Her2 receptor status by exposing them to known but divergent autophagy inducers each with a unique molecular target, tamoxifen, trastuzumab, bortezomib or rapamycin. Differential gene expression analysis from total RNA extracted during the earliest sign of autophagy flux showed both cell- and drug-specific changes. We analyzed the list of differentially expressed genes to find a common, cell- and drug-agnostic autophagy signature. Twelve mRNAs were significantly modulated by all the drugs and 11 were orthogonally verified with Q-RT-PCR (Klhl24, Hbp1, Crebrf, Ypel2, Fbxo32, Gdf15, Cdc25a, Ddit4, Psat1, Cd22, Ypel3). The drug agnostic mRNA signature was similarly induced by a mitochondrially targeted agent, MitoQ. In-silico analysis on the KM-plotter cancer database showed that the levels of these mRNAs are detectable in human samples and associated with breast cancer prognosis outcomes of Relapse-Free Survival in all patients (RSF), Overall Survival in all patients (OS), and Relapse-Free Survival in ER+ Patients (RSF ER+). High levels of Klhl24, Hbp1, Crebrf, Ypel2, CD22 and Ypel3 were correlated with better outcomes, whereas lower levels of Gdf15, Cdc25a, Ddit4 and Psat1 were associated with better prognosis in breast cancer patients. This gene signature uncovers candidate autophagy biomarkers that could be tested during preclinical and clinical studies to monitor the autophagy process.

Quantitative and temporal measurement of dynamic autophagy rates

Autophagy is a multistep degradative process that is essential for maintaining cellular homeostasis. Systematically quantifying flux through this pathway is critical for gaining fundamental insights and effectively modulating this process that is dysregulated during many diseases. Established methods to quantify flux use steady state measurements, which provide limited information about the perturbation and the cellular response. We present a theoretical and experimental framework to measure autophagic steps in the form of rates under non-steady state conditions. We use this approach to measure temporal responses to rapamycin and wortmannin treatments, two commonly used autophagy modulators. We quantified changes in autophagy rates in as little as 10 minutes, which can establish direct mechanisms for autophagy perturbation before feedback begins. We identified concentration-dependent effects of rapamycin on the initial and temporal progression of autophagy rates. We also found variable recovery time from wortmannin’s inhibition of autophagy, which is further accelerated by rapamycin. In summary, this new approach enables the quantification of autophagy flux with high sensitivity and temporal resolution and facilitates a comprehensive understanding of this process.

β-arrestin1 promotes tauopathy by transducing GPCR signaling, disrupting microtubules and autophagy

G protein–coupled receptors (GPCRs) have been shown to play integral roles in Alzheimer’s disease pathogenesis. However, it is unclear how diverse GPCRs similarly affect Aβ and tau pathogenesis. GPCRs share a common mechanism of action via the β-arrestin scaffolding signaling complexes, which not only serve to desensitize GPCRs by internalization, but also mediate multiple downstream signaling events. As signaling via the GPCRs, β2-adrenergic receptor (β2AR), and metabotropic glutamate receptor 2 (mGluR2) promotes hyperphosphorylation of tau, we hypothesized that β-arrestin1 represents a point of convergence for such pathogenic activities. Here, we report that β-arrestins are not only essential for β2AR and mGluR2-mediated increase in pathogenic tau but also show that β-arrestin1 levels are increased in brains of Frontotemporal lobar degeneration (FTLD-tau) patients. Increased β-arrestin1 in turn drives the accumulation of pathogenic tau, whereas reduced ARRB1 alleviates tauopathy and rescues impaired synaptic plasticity and cognitive impairments in PS19 mice. Biochemical and cellular studies show that β-arrestin1 drives tauopathy by destabilizing microtubules and impeding p62/SQSTM1 autophagy flux by interfering with p62 body formation, which promotes pathogenic tau accumulation.

Melatonin-mediated Calcineurin Inactivation Attenuates Prion Protein-induced Nf-Kb- Driven Pro-Inflammation Response

Background: Prion diseases are a group of prevalent and rapidly progressive neurodegenerative disorders that lead to chronic inflammation and neuronal cell death. Calcineurin and autophagy mediate prion-induced neurodegeneration, suggesting that inhibition of calcineurin and autophagy could be a target for therapy. Melatonin has been reported to exert neuroprotective effects against calcium-dependent neuronal cell death.Methods: Real-time quantitative PCR was used to detect mRNA levels of proinflammatory cytokines. Western blot was used to analysis p-nfkb, p-bcl10, calcineurin, prpc and autophagy flux pathway. Immunocytochemistry was used to analysis p-nfkb and calcineurin. Ca2+ levels were measured by fluo-4 using confocal microscope. Calcineurin activity was used to detect with calcineurin cellular activity assay kit. Transmission electron microscopy (TEM) was used to detect autophagy flux.Results: In the present study, we investigated whether melatonin attenuates prion peptide-mediated neuroinflammation and reduces calcineurin. We found that melatonin treatment inhibits prion protein-induced apoptosis. Melatonin inhibited calcium up-regulation and protected the cells against prion peptide‑induced neuron cell death by calcineurin inactivation. Furthermore, melatonin increased p62 protein levels and decrease LC3-II protein levels indicating autophagic flux inhibition and melatonin inhibited prion protein-induced neurotoxicity through autophagy flux inhibition.Conclusions: Taken together, our results illuminate that melatonin attenuated prion protein-induced neurinflammation through calcineurin inactivation and autophagic flux reduction, and also suggest that melatonin may provide effective strategy for therapy against neurodegenerative diseases, including prion diseases.

Fluoxetine Potentiates Phagocytosis and Autophagy in Microglia

Fluoxetine is a classic antidepressant drug, and its immunomodulatory effects have recently been reported in many disease models. In addition, it has strong antineuroinflammatory effects in stroke and neurodegenerative animal models. However, the effect of fluoxetine on microglia phagocytosis and its molecular mechanisms have not yet been studied. In this study, we investigated whether fluoxetine has a regulatory effect on microglial function. Microglia cell lines and primary mouse microglia were treated with fluoxetine, and the production of inflammatory cytokines and neurotrophic factors and the phagocytosis of amyloid β were measured. Fluoxetine significantly attenuated the production of lipopolysaccharide-induced proinflammatory cytokines and oxidative stress in microglia. Fluoxetine also significantly potentiated microglia phagocytosis and autophagy. In addition, autophagy flux inhibitors attenuated fluoxetine-induced phagocytosis. In conclusion, fluoxetine induces autophagy and potentiates phagocytosis in microglia, which can be a novel molecular mechanism of the neuroinflammatory and neuroprotective effects of fluoxetine.