PLEKHM2 Loss of Function Impairs the Activity of iPSC-Derived Neurons via Regulation of Autophagic Flux

Pleckstrin Homology And RUN Domain Containing M2 (PLEKHM2) [delAG] mutation causes dilated cardiomyopathy with left ventricular non-compaction (DCM-LVNC), resulting in a premature death of PLEKHM2[delAG] individuals due to heart failure. PLEKHM2 is a factor involved in autophagy, a master regulator of cellular homeostasis, decomposing pathogens, proteins and other cellular components. Autophagy is mainly carried out by the lysosome, containing degradation enzymes, and by the autophagosome, which engulfs substances marked for decomposition. PLEKHM2 promotes lysosomal movement toward the cell periphery. Autophagic dysregulation is associated with neurodegenerative diseases’ pathogenesis. Thus, modulation of autophagy holds considerable potential as a therapeutic target for such disorders. We hypothesized that PLEKHM2 is involved in neuronal development and function, and that mutated PLEKHM2 (PLEKHM2[delAG]) neurons will present impaired functions. Here, we studied PLEKHM2-related abnormalities in induced pluripotent stem cell (iPSC)-derived motor neurons (iMNs) as a neuronal model. PLEKHM2[delAG] iMN cultures had healthy control-like differentiation potential but exhibited reduced autophagic activity. Electrophysiological measurements revealed that PLEKHM2[delAG] iMN cultures displayed delayed functional maturation and more frequent and unsynchronized activity. This was associated with increased size and a more perinuclear lysosome cellular distribution. Thus, our results suggest that PLEKHM2 is involved in the functional development of neurons through the regulation of autophagic flux

Functional defects in hiPSCs-derived cardiomyocytes from patients with a PLEKHM2-mutation associated with dilated cardiomyopathy and left ventricular non-compaction

Abstract Dilated cardiomyopathy (DCM) is a primary myocardial disease, leading to heart failure and excessive risk of sudden cardiac death with rather poorly understood pathophysiology. In 2015, Parvari's group identified a recessive mutation in the autophagy regulator, PLEKHM2 gene, in a family with severe recessive DCM and left ventricular non-compaction (LVNC). Fibroblasts isolated from these patients exhibited abnormal subcellular distribution of endosomes, Golgi apparatus, lysosomes and had impaired autophagy flux. To better understand the effect of mutated PLEKHM2 on cardiac tissue, we generated and characterized induced pluripotent stem cells-derived cardiomyocytes (iPSC-CMs) from two patients and a healthy control from the same family. The patient iPSC-CMs showed low expression levels of genes encoding for contractile functional proteins (α and β-myosin heavy chains and 2v and 2a-myosin light chains), structural proteins integral to heart contraction (Troponin C, T and I) and proteins participating in Ca2+ pumping action (SERCA2 and Calsequestrin 2) compared to their levels in control iPSC-derived CMs. Furthermore, the sarcomeres of the patient iPSC-CMs were less oriented and aligned compared to control cells and generated slowly beating foci with lower intracellular calcium amplitude and abnormal calcium transient kinetics, measured by IonOptix system and MuscleMotion software. Autophagy in patient’s iPSC-CMs was impaired as determined from a decrease in the accumulation of autophagosomes in response to chloroquine and rapamycin treatment, compared to control iPSC-CMs. Impairment in autophagy together with the deficiency in the expression of NKX2.5, MHC, MLC, Troponins and CASQ2 genes, which are related to contraction-relaxation coupling and intracellular Ca2+ signaling, may contribute to the defective function of the patient CMs and possibly affect cell maturation and cardiac failure with time

NKp44-Derived Peptide Binds Proliferating Cell Nuclear Antigen and Mediates Tumor Cell Death

Proliferating cell nuclear antigen (PCNA) is considered as a hub protein and is a key regulator of DNA replication, repair, cell cycle control, and apoptosis. PCNA is overexpressed in many cancer types, and PCNA overexpression is correlated with cancer virulence. Membrane-associated PCNA is a ligand for the NKp44 (NCR2) innate immune receptor. The purpose of this study was to characterize the PCNA-binding site within NKp44. We have identified NKp44-derived linear peptide (pep8), which can specifically interact with PCNA and partly block the NKp44–PCNA interaction. We then tested whether NKp44-derived pep8 (NKp44-pep8) fused to cell-penetrating peptides (CPPs) can be employed for targeting the intracellular PCNA for the purpose of anticancer therapy. Treatment of tumor cells with NKp44-pep8, fused to R11-NLS cell-penetrating peptide (R11-NLS-pep8), reduced cell viability and promoted cell death, in various murine and human cancer cell lines. Administration of R11-NLS-pep8 to tumor-bearing mice suppressed tumor growth in the 4T1 breast cancer and the B16 melanoma in vivo models. We therefore identified the NKp44 binding site to PCNA and further developed an NKp44-peptide-based agent that can inhibit tumor growth through interfering with the function of intracellular PCNA in the tumor cell