Risa Nonaka

Perlecan is required for FGF-2 signaling in the neural stem cell niche.

In the adult subventricular zone (neurogenic niche), neural stem cells double-positive for two markers of subsets of neural stem cells in the adult central nervous system, glial fibrillary acidic protein and CD133, lie in proximity to fractones and to blood vessel basement membranes, which contain the heparan sulfate proteoglycan perlecan. Here, we demonstrate that perlecan deficiency reduces the number of both GFAP/CD133-positive neural stem cells in the subventricular zone and new neurons integrating into the olfactory bulb. We also show that FGF-2 treatment induces the expression of cyclin D2 through the activation of the Akt and Erk1/2 pathways and promotes neurosphere formation in vitro. However, in the absence of perlecan, FGF-2 fails to promote neurosphere formation. These results suggest that perlecan is a component of the neurogenic niche that regulates FGF-2 signaling and acts by promoting neural stem cell self-renewal and neurogenesis.

Perlecan inhibits autophagy to maintain muscle homeostasis in mouse soleus muscle.

The autophagy-lysosome system is essential for muscle protein synthesis and degradation equilibrium, and its dysfunction has been linked to various muscle disorders. It has been reported that a diverse collection of extracellular matrix constituents, including decorin, collagen VI, laminin α2, endorepellin, and endostatin, can modulate autophagic signaling pathways. However, the association between autophagy and perlecan in muscle homeostasis remains unclear. The mechanical unloading of perlecan-deficient soleus muscles resulted in significantly decreased wet weights and cross-section fiber area compared with those of control mice. We found that perlecan deficiency in slow-twitch soleus muscles enhanced autophagic activity. This was accompanied by a decrease in autophagic substrates, such as p62, and an increase in LC3II levels. Furthermore, perlecan deficiency caused a reduction in the phosphorylation levels of p70S6k and Akt and increased the phosphorylation of AMPKα. Our findings suggested that perlecan inhibits the autophagic process through the activation of the mTORC1 pathway. This autophagic response may be a novel target for enhancing the efficacy of skeletal muscle atrophy treatment.

Laminin α1 Regulates Age-Related Mesangial Cell Proliferation and Mesangial Matrix Accumulation through the TGF-β Pathway

Laminin α1 (LAMA1), a subunit of the laminin-111 basement membrane component, has been implicated in various biological functions in vivo and in vitro. Although LAMA1 is present in kidney, its roles in the kidney are unknown because of early embryonic lethality. Herein, we used a viable conditional knockout mouse model with a deletion of Lama1 in the epiblast lineage (Lama1(CKO)) to study the role of LAMA1 in kidney development and function. Adult Lama1(CKO) mice developed focal glomerulosclerosis and proteinuria with age. In addition, mesangial cell proliferation was increased, and the mesangial matrix, which normally contains laminin-111, was greatly expanded. In vitro, mesangial cells from Lama1(CKO) mice exhibited significantly increased proliferation compared with those from controls. This increased proliferation was inhibited by the addition of exogenous LAMA1-containing laminin-111, but not by laminin-211 or laminin-511, suggesting a specific role for LAMA1 in regulating mesangial cell behavior. Moreover, the absence of LAMA1 increased transforming growth factor (TGF)-β1-induced Smad2 phosphorylation, and inhibitors of TGF-β1 receptor I kinase blocked Smad2 phosphorylation in both control and Lama1(CKO) mesangial cells, indicating that the increased Smad2 phosphorylation occurred in the absence of LAMA1 via the TGF-β1 receptor. These findings suggest that LAMA1 plays a critical role in kidney function and kidney aging by regulating the mesangial cell population and mesangial matrix deposition through TGF-β/Smad signaling.

Perlecan deficiency causes endothelial dysfunction by reducing the expression of endothelial nitric oxide synthase

Perlecan is a major heparan sulfate proteoglycan found in the subendothelial extracellular matrix of the vascular wall. The aim of this study was to investigate the role of perlecan in the regulation of vascular tone. A previously developed conditional perlecan‐deficient mouse model was used to measure changes in the isometric force of isolated aortic rings. The vessels were first precontracted with phenylephrine, and then treated with increasing concentrations of vasorelaxants. Endothelium‐dependent relaxation, elicited by acetylcholine, was significantly reduced in the perlecan‐deficient aortas, whereas endothelium‐independent relaxation caused by the exogenous nitric oxide donor sodium nitroprusside remained well preserved. The expression of the endothelial nitric oxide synthase (eNOS) gene, detected by real‐time polymerase chain reaction, was significantly decreased in the perlecan‐deficient aortas. The expression of eNOS protein detected using Western blotting was also significantly decreased in the perlecan‐deficient aortas. We examined the role of perlecan in eNOS gene expression by creating perlecan knockdown human aortic endothelial cells using small interfering RNA (siRNA) for perlecan. Perlecan gene expression was significantly reduced in the perlecan siRNA‐treated cells, resulting in a significant decrease in eNOS gene expression. Perlecan deficiency induced endothelial dysfunction, as indicated by a reduction in endothelium‐dependent relaxation due, at least partly, to a reduction in eNOS expression. These findings suggest that perlecan plays a role in the activation of eNOS gene expression during normal growth processes.

Perlecan is required for the chondrogenic differentiation of synovial mesenchymal cells through regulation of Sox9 gene expression.

We previously reported that perlecan, a heparan‐sulfate proteoglycan (Hspg2), expressed in the synovium at the cartilage‐synovial junction, is required for osteophyte formation in knee osteoarthritis. To examine the mechanism underlying this process, we examined the role of perlecan in the proliferation and differentiation of synovial mesenchymal cells (SMCs), using a recently established mouse synovial cell culture method. Primary SMCs isolated from Hspg2−/−‐Tg (Hspg2−/−;Col2a1‐Hspg2Tg/−) mice, in which the perlecan‐knockout was rescued from perinatal lethality, lack perlecan. The chondrogenic‐, osteogenic‐, and adipogenic‐potentials were examined in the Hspg2−/−‐Tg SMCs compared to the control SMCs prepared from wild‐type Hspg2+/+‐Tg (Hspg2+/+;Col2a1‐Hspg2Tg/−) littermates. In a culture condition permitting proliferation, both control and Hspg2−/−‐Tg SMCs showed similar rates of proliferation and expression of cell surface markers. However, in micromass cultures, the cartilage matrix production and Sox9 and Col2a1 mRNA levels were significantly reduced in Hspg2−/−‐Tg SMCs, compared with control SMCs. The reduced level of Sox9 mRNA was restored by the supplementation with exogenous perlecan protein. There was no difference in osteogenic differentiation between the control and Hspg2−/−‐Tg SMCs, as measured by the levels of Runx2 and Col1a1 mRNA. The adipogenic induction and PPARγ mRNA levels were significantly reduced in Hspg2−/−‐Tg SMCs compared to control SMCs. The reduction of PPARγ mRNA levels in Hspg2−/−‐Tg SMCs was restored by supplementation of perlecan. Perlecan is required for the chondrogenic and adipogenic differentiation from SMCs via its regulation of the Sox9 and PPARγ gene expression, but not for osteogenic differentiation via Runx2.

Identification of Peptides Derived from the C-terminal Domain of Fibulin-7 Active for Endothelial Cell Adhesion and Tube Formation Disruption.

Despite the research done on pathological angiogenesis, there is still a need for the development of new therapies against angiogenesis‐related diseases. Fibulin‐7 (Fbln7) is a member of the extracellular matrix fibulin protein family. The Fbln7 C‐terminal fragment, Fbln7‐C, binds to endothelial cells and inhibits their tube formation in culture. In this study, we screened 12 synthetic peptides, covering the fibulin‐globular domain of Fbln7‐C, to identify active sites for endothelial cell adhesion and in vitro antiangiogenic activity. Three peptides, fc10, fc11, and fc12, promoted Human Umbilical Vein Endothelial Cells (HUVECs) adhesion, and the morphology of HUVECs on fc10 was similar to that on Fbln7‐C. EDTA and the anti‐integrin β1 function‐blocking antibody inhibited HUVECs adhesion to both fc10 and fc12, and heparin inhibited HUVECs adhesion to both fc11 and fc12. fc10 and fc11 inhibited HUVECs tube formation. Our results suggest that three peptides from Fbln7‐C are biologically active for endothelial cell adhesion and disrupt the tube formation, suggesting a potential therapeutic use of these peptides for angiogenesis‐related diseases.

A C-terminal fragment of fibulin-7 interacts with endothelial cells and inhibits their tube formation in culture.

We have previously demonstrated that fibulin-7 (Fbln7) is expressed in teeth by pre-odontoblast and odontoblast cells, localized in the basement membrane and dentin matrices, and is an adhesion molecule for dental mesenchyme cells and odontoblasts. Fbln7 is also expressed in blood vessels by endothelial cells. In this report, we show that a recombinant C-terminal Fbln7 fragment (Fbln7-C) bound to Human Umbilical Vein Endothelial Cells (HUVECs) but did not promote cell spreading and actin stress fiber formation. Fbln7-C binding to HUVECs induced integrin clustering at cell adhesion sites with other focal adhesion molecules, and sustained activation of FAK, p130Cas, and Rac1. In addition, RhoA activation was inhibited, thereby preventing HUVEC spreading. As endothelial cell spreading is an important step for angiogenesis, we examined the effect of Fbln7-C on angiogenesis using in vitro assays for endothelial cell tube formation and vessel sprouting from aortic rings. We found that Fbln7-C inhibited the HUVEC tube formation and the vessel sprouting in aortic ring assays. Our findings suggest potential anti-angiogenic activity of the Fbln7 C-terminal region.

Heparan sulfate alterations in extracellular matrix structures and fibroblast growth factor-2 signaling impairment in the aged neurogenic niche

Adult neurogenesis in the subventricular zone of the lateral ventricle decreases with age. In the subventricular zone, the specialized extracellular matrix structures, known as fractones, contact neural stem cells and regulate neurogenesis. Fractones are composed of extracellular matrix components, such as heparan sulfate proteoglycans. We previously found that fractones capture and store fibroblast growth factor 2 (FGF‐2) via heparan sulfate binding, and may deliver FGF‐2 to neural stem cells in a timely manner. The heparan sulfate (HS) chains in the fractones of the aged subventricular zone are modified based on immunohistochemistry. However, how aging affects fractone composition and subsequent FGF‐2 signaling and neurogenesis remains unknown. The formation of the FGF‐fibroblast growth factor receptor‐HS complex is necessary to activate FGF‐2 signaling and induce the phosphorylation of extracellular signal‐regulated kinase (Erk1/2). In this study, we observed a reduction in HS 6‐O‐sulfation, which is critical for FGF‐2 signal transduction, and failure of the FGF‐2‐induced phosphorylation of Erk1/2 in the aged subventricular zone. In addition, we observed increased HS 6‐O‐endo‐sulfatase, an enzyme that may be responsible for the HS modifications in aged fractones. In conclusion, the data revealed that heparan sulfate 6‐O‐sulfation is reduced and FGF‐2‐dependent Erk1/2 signaling is impaired in the aged subventricular zone. HS modifications in fractones might play a role in the reduced neurogenic activity in aging brains.

Soluble epoxide hydrolase plays a key role in the pathogenesis of Parkinson’s disease

Significance Parkinson’s disease (PD) is a chronic and progressive movement disorder; however, the precise mechanisms of its etiology remain largely unknown. Soluble epoxide hydrolase (sEH) plays a key role in the inflammation associated with PD pathogenesis. The sEH inhibitor or deletion of the sEH gene protected against MPTP-induced neurotoxicity in mouse brain. Furthermore, expression of the sEH protein (or mRNA) was higher in the striatum of MPTP-treated mice, patients with dementia of Lewy bodies (DLB), and neurons from iPSCs of a PD patient with PARKIN mutations. Interestingly, treatment with sEH inhibitor protected against apoptosis in human PARK2 iPSC-derived dopaminergic neurons. Our findings indicate that sEH inhibitors or epoxy fatty acids mimics may be promising prophylactic or therapeutic drugs for PD. Parkinson’s disease (PD) is characterized as a chronic and progressive neurodegenerative disorder, and the deposition of specific protein aggregates of α-synuclein, termed Lewy bodies, is evident in multiple brain regions of PD patients. Although there are several available medications to treat PD symptoms, these medications do not prevent the progression of the disease. Soluble epoxide hydrolase (sEH) plays a key role in inflammation associated with the pathogenesis of PD. Here we found that MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced neurotoxicity in the mouse striatum was attenuated by subsequent repeated administration of TPPU, a potent sEH inhibitor. Furthermore, deletion of the sEH gene protected against MPTP-induced neurotoxicity, while overexpression of sEH in the striatum significantly enhanced MPTP-induced neurotoxicity. Moreover, the expression of the sEH protein in the striatum from MPTP-treated mice or postmortem brain samples from patients with dementia of Lewy bodies (DLB) was significantly higher compared with control groups. Interestingly, there was a positive correlation between sEH expression and phosphorylation of α-synuclein in the striatum. Oxylipin analysis showed decreased levels of 8,9-epoxy-5Z,11Z,14Z-eicosatrienoic acid in the striatum of MPTP-treated mice, suggesting increased activity of sEH in this region. Interestingly, the expression of sEH mRNA in human PARK2 iPSC-derived neurons was higher than that of healthy control. Treatment with TPPU protected against apoptosis in human PARK2 iPSC-derived dopaminergic neurons. These findings suggest that increased activity of sEH in the striatum plays a key role in the pathogenesis of neurodegenerative disorders such as PD and DLB. Therefore, sEH may represent a promising therapeutic target for α-synuclein–related neurodegenerative disorders.

NDP52 interacts with mitochondrial RNA poly(A) polymerase to promote mitophagy

Parkin‐mediated mitophagy is a quality control pathway that selectively removes damaged mitochondria via the autophagic machinery. Autophagic receptors, which interact with ubiquitin and Atg8 family proteins, contribute to the recognition of damaged mitochondria by autophagosomes. NDP52, an autophagy receptor, is required for autophagic engulfment of damaged mitochondria during mitochondrial uncoupler treatment. The N‐terminal SKICH domain and C‐terminal zinc finger motif of NDP52 are both required for its function in mitophagy. While the zinc finger motif contributes to poly‐ubiquitin binding, the function of the SKICH domain remains unclear. Here, we show that NDP52 interacts with mitochondrial RNA poly(A) polymerase (MTPAP) via the SKICH domain. During mitophagy, NDP52 invades depolarized mitochondria and interacts with MTPAP dependent on the proteasome but independent of ubiquitin binding. Loss of MTPAP reduces NDP52‐mediated mitophagy, and the NDP52–MTPAP complex attracts more LC3 than NDP52 alone. These results indicate that NDP52 and MTPAP form an autophagy receptor complex, which enhances autophagic elimination of damaged mitochondria.