Impaired Retrograde Transport Due to Lack of TBC1D5 Contributes to the Trafficking Defect of Lysosomal Cathepsins in Ischemic/Hypoxic Cardiomyocytes

Lysosomal dysfunction has been found in many pathological conditions, and methods to improve lysosomal function have been reported to be protective against infarcted hearts. However, the mechanisms underlying lysosomal dysfunction caused by ischemic injury are far less well-established. The retromer complex is implicated in the trafficking of cation-independent mannose 6-phosphate receptor (CI-MPR), which is an important protein tag for the proper transport of lysosomal contents and therefore is important for the maintenance of lysosomal function. In this study, we found that the function of retrograde transport in cardiomyocytes was impaired with ischemia/hypoxia (I/H) treatment, which resulted in a decrease in CI-MPR and an abnormal distribution of lysosomal cathepsins. I/H treatment caused a reduction in TBC1D5 and a blockade of the Rab7 membrane cycle, which impeded retromer binding to microtubules and motor proteins, resulting in an impairment of retrograde transport and a decrease in CI-MPR. We also established that TBC1D5 was an important regulator of the distribution of lysosomal cathepsins. Our findings shed light on the regulatory role of retromer in ischemic injury and uncover the regulatory mechanism of TBC1D5 over retromer.

Loss of TBK1 activity leads to TDP-43 proteinopathy through lysosomal dysfunction in human motor neurons

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron loss accompanied by cytoplasmic localization of TDP-43 proteins and their insoluble accumulations. Haploinsufficiency of TBK1 has been found to associate with or cause ALS. However, the cell-autonomous mechanisms by which reduced TBK1 activity contributes to human motor neuron pathology remain elusive. Here, we generated a human cellular model harboring loss-of-function mutations of TBK1 by gene editing and found that TBK1 deficiency was sufficient to cause TDP-43 pathology in human motor neurons. In addition to its functions in autophagy, we found that TBK1 interacted with endosomes and was required for normal endosomal maturation and subsequent lysosomal acidification. Surprisingly, TDP-43 pathology resulted more from the dysfunctional endo-lysosomal pathway than the previously recognized autophagy inhibition mechanism. Restoring TBK1 levels ameliorated lysosomal dysfunction and TDP-43 pathology and maintained normal motor neuron homeostasis. Notably, using patient-derived motor neurons, we found that haploinsufficiency of TBK1 sensitized neurons to lysosomal stress, and chemical regulators of endosomal maturation rescued the neurodegenerative process. Together, our results revealed the mechanism of TBK1 in maintaining TDP-43 and motor neuron homeostasis and suggested that modulating endosomal maturation was able to rescue neurodegenerative disease phenotypes caused by TBK1 deficiency.

Reactive Oxygen Species Mediate 6c-Induced Mitochondrial and Lysosomal Dysfunction, Autophagic Cell Death, and DNA Damage in Hepatocellular Carcinoma

Increasing the level of reactive oxygen species (ROS) in cancer cells has been suggested as a viable approach to cancer therapy. Our previous study has demonstrated that mitochondria-targeted flavone-naphthalimide-polyamine conjugate 6c elevates the level of ROS in cancer cells. However, the detailed role of ROS in 6c-treated cancer cells is not clearly stated. The biological effects and in-depth mechanisms of 6c in cancer cells need to be further investigated. In this study, we confirmed that mitochondria are the main source of 6c-induced ROS, as demonstrated by an increase in 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) and MitoSox fluorescence. Compound 6c-induced mitochondrial ROS caused mitochondrial dysfunction and lysosomal destabilization confirmed by absolute quantitation (iTRAQ)-based comparative proteomics. Compound 6c-induced metabolic pathway dysfunction and lysosomal destabilization was attenuated by N-acetyl-L-cysteine (NAC). iTRAQ-based comparative proteomics showed that ROS regulated the expression of 6c-mediated proteins, and treatment with 6c promoted the formation of autophagosomes depending on ROS. Compound 6c-induced DNA damage was characterized by comet assay, p53 phosphorylation, and γH2A.X, which was diminished by pretreatment with NAC. Compound 6c-induced cell death was partially reversed by 3-methyladenine (3-MA), bafilomycin (BAF) A1, and NAC, respectively. Taken together, the data obtained in our study highlighted the involvement of mitochondrial ROS in 6c-induced autophagic cell death, mitochondrial and lysosomal dysfunction, and DNA damage.

Callyspongiolide kills cells by inducing mitochondrial dysfunction via cellular iron depletion

The highly cytotoxic marine natural product callyspongiolide holds great promise as a warhead of antibody-drug conjugate in cancer therapeutics; however, the mechanism underlying its cytotoxicity remains unclear. To elucidate how callyspongiolide kills cells, we employed label-free target identification with thermal stability-shift-based fluorescence difference in two-dimensional (2-D) gel electrophoresis (TS-FITGE), which allowed observation of a unique phenomenon of protein-spot separation on 2-D gels upon treatment with callyspongiolide at increasing temperatures. During our exploration of what proteins were associated with this phenomenon as well as why it happens, we found that callyspongiolide induces mitochondrial/lysosomal dysfunction and autophagy inhibition. Moreover, molecular biology studies revealed that callyspongiolide causes lysosomal dysfunction, which induces cellular iron depletion and leads to mitochondrial dysfunction and subsequent cytotoxicity. Notably, these effects were rescued through iron supplementation. Although our approach was unable to reveal the direct protein targets of callyspongiolide, unique phenomena observed only by TS-FITGE provided critical insight into the mechanism of action of callyspongiolide and specifically its cytotoxic activity via induction of mitochondrial dysfunction through cellular iron depletion caused by lysosomal deacidification, which occurred independent of known programmed cell death pathways.