PFKP Activation Ameliorates Foot Process Fusion in Podocytes in Diabetic Kidney Disease

BackgroundGlycolysis dysfunction is an important pathogenesis of podocyte injury in diabetic kidney disease (DKD). Foot process fusion of podocytes and increased albuminuria are markers of early DKD. Moreover, cytoskeletal remodeling has been found to be involved in the foot process fusion of podocytes. However, the connections between cytoskeletal remodeling and alterations of glycolysis in podocytes in DKD have not been clarified.MethodsmRNA sequencing of glomeruli obtained from db/db and db/m mice with albuminuria was performed to analyze the expression profiling of genes in glucose metabolism. Expressions of phosphofructokinase platelet type (PFKP) in the glomeruli of DKD patients were detected. Clotrimazole (CTZ) was used to explore the renal effects of PFKP inhibition in diabetic mice. Using Pfkp siRNA or recombinant plasmid to manipulate PFKP expression, the effects of PFKP on high glucose (HG) induced podocyte damage were assessed in vitro. The levels of fructose-1,6-bisphosphate (FBP) were measured. Targeted metabolomics was performed to observe the alterations of the metabolites in glucose metabolism after HG stimulation. Furthermore, aldolase type b (Aldob) siRNA or recombinant plasmid were applied to evaluate the influence of FBP level alteration on podocytes. FBP was directly added to podocyte culture media. Db/db mice were treated with FBP to investigate its effects on their kidney.ResultsmRNA sequencing showed that glycolysis enzyme genes were altered, characterized by upregulation of upstream genes (Hk1, and Pfkp) and down-regulation of downstream genes of glycolysis (Pkm, and Ldha). Moreover, the expression of PFKP was increased in glomeruli of DKD patients. The CTZ group presented more severe renal damage. In vitro, the Pfkp siRNA group and ALDOB overexpression group showed much more induced cytoskeletal remodeling in podocytes, while overexpression of PFKP and suppression of ALDOB in vitro rescued podocytes from cytoskeletal remodeling through regulation of FBP levels and inhibition of the RhoA/ROCK1 pathway. Furthermore, targeted metabolomics showed FBP level was significantly increased in HG group compared with the control group. Exogenous FBP addition reduced podocyte cytoskeletal remodeling and renal damage of db/db mice.ConclusionsThese findings provide evidence that PFKP may be a potential target for podocyte injury in DN and provide a rationale for applying podocyte glycolysis enhancing agents in patients with DKD.

Biological Implications of a Stroke Therapy Based in Neuroglobin Hyaluronate Nanoparticles. Neuroprotective Role and Molecular Bases

Exogenous neuroprotective protein neuroglobin (Ngb) cannot cross the blood–brain barrier. To overcome this difficulty, we synthesized hyaluronate nanoparticles (NPs), able to deliver Ngb into the brain in an animal model of stroke (MCAO). These NPs effectively reached neurons, and were microscopically identified after 24 h of reperfusion. Compared to MCAO non-treated animals, those treated with Ngb-NPs showed survival rates up to 50% higher, and better neurological scores. Tissue damage improved with the treatment, but no changes in the infarct volume or in the oxidative/nitrosative values were detected. A proteomics approach (p-value < 0.02; fold change = 0.05) in the infarcted areas showed a total of 219 proteins that significantly changed their expression after stroke and treatment with Ngb-NPs. Of special interest, are proteins such as FBXO7 and NTRK2, which were downexpressed in stroke, but overexpressed after treatment with Ngb-NPs; and ATX2L, which was overexpressed only under the effect of Ngb. Interestingly, the proteins affected by the treatment with Ngb were involved in mitochondrial function and cell death, endocytosis, protein metabolism, cytoskeletal remodeling, or synaptic function, and in regenerative processes, such as dendritogenesis, neuritogenesis, or sinaptogenesis. Consequently, our pharmaceutical preparation may open new therapeutic scopes for stroke and possibly for other neurodegenerative pathologies.

MYH10 governs adipocyte function and adipogenesis through its interaction with GLUT4

Adipocyte differentiation is dependent on cytoskeletal remodeling processes that determine and maintain cellular shape and function. In turn, cytoskeletal proteins contribute to the filament-based network responsible for controlling adipocyte’s shape and promoting the intracellular trafficking of key cellular components. Currently, our understanding of these mechanisms remains incomplete. In this study, we identified the non-muscle myosin 10 (MYH10) as an important regulator of adipogenesis and adipocyte function through its interaction with the insulin dependent, Glucose transporter 4 (GLUT4). MYH10 depletion in preadipocytes resulted in impaired adipogenesis, with knockdown cells exhibiting disrupted morphology and reduced molecular adipogenic signals. MYH10 was shown to be in complex with GLUT4 in adipocytes, an interaction regulated by insulin induction. The missing adipogenic capacity of MYH10-KD cells was restored when they uptook GLUT4 vesicles up from neighbor wild-type cells in a co-culture system. Our results provide the first demonstration that MYH10 interacts with GLUT4 in cells and adipose tissue through the insulin pathway. The signaling cascade is regulated by the protein kinase C ζ (PKCζ), which interacts with MYH10 to modify the localization and interaction of both GLUT4 and MYH10 in adipocytes as PKCζ inhibition resulted in reduced GLUT4 and MYH10 translocation and interactions. Overall, our study establishes MYH10 as an essential regulator of GLUT4 translocation, affecting both adipogenesis and adipocyte function, highlighting its importance in future cytoskeleton-based studies in adipocytes.

Differential mobility and self-association of Arc/Arg3.1 in the cytoplasm and nucleus of living cells

ABSTRACTArc, also known as Arg3.1, is an activity-dependent immediate-early gene product that plays essential roles in memory consolidation. A pool of Arc is located in the postsynaptic cytoplasm, where it promotes AMPA receptor endocytosis and cytoskeletal remodeling. However, Arc is also found in the nucleus, a major portion being associated with promyelocytic leukemia nuclear bodies (PML-NBs). Nuclear Arc has been implicated in epigenetic control of gene transcription associated with learning and memory. In this study, we use a battery of fluorescence nanoimaging approaches to characterize the behavior of Arc in living cells. Our results indicate that in the cytoplasm, Arc exists predominantly as monomers and dimers associated with slowly diffusing particles. In contrast, nuclear Arc is almost exclusively monomeric and displays a higher diffusivity than cytoplasmic Arc. We further show that Arc moves freely and rapidly between PML-NBs and the nucleoplasm, and that its movement within PML-NBs is relatively unobstructed.

Smoothelin-like Protein 1 Regulates the Thyroid Hor-Mone-Induced Homeostasis and Remodeling of C2C12 Cells via the Modulation of Myosin Phosphatase

The pathological elevation of the active thyroid hormone (T3) level results in the manifestation of hyperthyroidism, which is associated with alterations in the differentiation and contractile function of skeletal muscle (SKM). Myosin phosphatase (MP) is a major cellular regulator that hydrolyzes the phosphoserine of phosphorylated myosin II light chain. MP consists of an MYPT1/2 regulatory and a protein phosphatase 1 catalytic subunit. Smoothelin-like protein 1 (SMTNL1) is known to inhibit MP by directly binding to MP as well as by suppressing the expression of MYPT1 at the transcriptional level. Supraphysiological vs. physiological concentration of T3 were applied on C2C12 myoblasts and differentiated myotubes in combination with the overexpression of SMTNL1 to assess the role and regulation of MP under these conditions. In non-differentiated myoblasts, MP included MYPT1 in the holoenzyme complex and its expression and activity was regulated by SMTNL1, affecting the phosphorylation level of MLC20 assessed using semi-quantitative Western blot analysis. SMTNL1 negatively influenced the migration and cytoskeletal remodeling of myoblasts measured by high content screening. In contrast, in myotubes, the expression of MYPT2 but not MYPT1 increased in a T3-dependent and SMTNL1-independent manner. T3 treatment combined with SMTNL1 overexpression impeded the activity of MP. In addition, MP interacted with Na+/K+-ATPase and dephosphorylated its inhibitory phosphorylation sites, identifying this protein as a novel MP substrate. These findings may help us gain a better understanding of myopathy, muscle weakness and the disorder of muscle regeneration in hyperthyroid patients.

First person – Dipanjana Ghosh

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Dipanjana Ghosh is first author on ‘ PLP2 drives collective cell migration via ZO-1-mediated cytoskeletal remodeling at the leading edge in human colorectal cancer cells’, published in JCS. Dipanjana is an independent DST Woman Scientist-A researcher at the Indian Institute of Science Education and Research, Bhopal, India, investigating cancer cell metastasis at the proteome-wide level.