Overexpression of CNP in chondrocytes rescues achondroplasia through a MAPK-dependent pathway

Achondroplasia is the most common genetic form of human dwarfism, for which there is presently no effective therapy. C-type natriuretic peptide (CNP) is a newly identified molecule that regulates endochondral bone growth through GC-B, a subtype of particulate guanylyl cyclase. Here we show that targeted overexpression of CNP in chondrocytes counteracts dwarfism in a mouse model of achondroplasia with activated fibroblast growth factor receptor 3 (FGFR-3) in the cartilage. CNP prevented the shortening of achondroplastic bones by correcting the decreased extracellular matrix synthesis in the growth plate through inhibition of the MAPK pathway of FGF signaling. CNP had no effect on the STAT-1 pathway of FGF signaling that mediates the decreased proliferation and the delayed differentiation of achondroplastic chondrocytes. These results demonstrate that activation of the CNP-GC-B system in endochondral bone formation constitutes a new therapeutic strategy for human achondroplasia.     

Phytosulfokine stimulates somatic embryogenesis in Cryptomeria japonica

Phytosulfokine (PSK), which has been identified as a plant growth factor, had a dramatic stimulatory effect on the formation of somatic embryos of sugi (Cryptomeria japonica) in the presence of polyethylene glycol. The resultant somatic embryos germinated with synchronous sprouting of cotyledons, hypocotyls and roots, and most of the seedlings grew normally. A cDNA clone for the precursor to the PSK peptide of C. japonica was identified in an expressed sequence tags database. Our results support the existence of a PSK signaling pathway in C. japonica.     

Insights into links between familial and sporadic Parkinson's disease: physical relationship between UCH-L1 variants and chaperone-mediated autophagy

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is expressed abundantly in neurons and has been reported to be a major target of oxidative/carbonyl damage associated with sporadic Parkinson's disease (PD). The I93M mutation in UCH-L1 is also associated with familial PD. We recently reported that UCH-L1 physically interacts with LAMP-2A, the lysosomal receptor for chaperone-mediated autophagy (CMA), and Hsc70 and Hsp90, both of which can function as components of the CMA pathway. We found that the levels of these interactions were aberrantly increased by the I93M mutation, and that expression of I93M UCH-L1 in cells induced the CMA inhibition-associated increase in the amount of alpha-synuclein, a risk factor for PD. The interactions of UCH-L1 with LAMP-2A, Hsc70 and Hsp90 were also abnormally enhanced by carbonyl modification of UCH-L1. We propose that aberrant interactions of UCH-L1 variants with CMA machinery, at least partly, underlie the pathogenesis of I93M UCH-L1-associated PD, and possibly of sporadic PD. Our findings may provide novel insights into the links between familial and sporadic PD.     

Cell-penetrating peptide-conjugated lipid nanoparticles for siRNA delivery

Lipid nanoparticles (LNP) modified with cell-penetrating peptides (CPP) were prepared for the delivery of small interfering RNA (siRNA) into cells. Lipid derivatives of CPP derived from protamine were newly synthesized and used to prepare CPP-decorated LNP (CPP-LNP). Encapsulation of siRNA into CPP-LNP improved the stability of the siRNA in serum. Fluorescence-labeled siRNA formulated in CPP-LNP was efficiently internalized into B16F10 murine melanoma cells in a time-dependent manner, although that in LNP without CPP was hardly internalized into these cells. In cells transfected with siRNA in CPP-LNP, most of the siRNA was distributed in the cytoplasm of these cells and did not localize in the lysosomes. Analysis of the endocytotic pathway indicated that CPP-LNP were mainly internalized via macropinocytosis and heparan sulfate-mediated endocytosis. CPP-LNP encapsulating siRNA effectively induced RNA interference-mediated silencing of reporter genes in B16F10 cells expressing luciferase and in HT1080 human fibrosarcoma cells expressing enhanced green fluorescent protein. These data suggest that modification of LNP with the protamine-derived CPP was effective to facilitate internalization of siRNA in the cytoplasm and thereby to enhance gene silencing.     

Microtubule destruction induces tau liberation and its subsequent phosphorylation

Neurofibrillary tangle-bearing neurons, a pathological hallmark of Alzheimer’s disease, are mostly devoid of normal microtubule (MT) structure and instead have paired helical filaments that are composed of abnormal hyperphosphorylated tau. However, a causal relationship between tau phosphorylation and MT disruption has not been clarified. To examine whether MT disruption induces tau phosphorylation, stathmin, an MT-disrupting protein, was co-expressed with tau in COS-7 cells. Stathmin expression induced apparent MT catastrophe and tau hyperphosphorylation at Thr-181, Ser-202, Thr-205, and Thr-231 sites. In contrast, c-Jun N-terminal kinase activation, or phosphatase inhibition, led to significant tau phosphorylation without affecting MT structure. These findings suggest that MT disruption induces subsequent tau phosphorylation.