Deletion of Lrp4 increases the incidence of microphthalmia.

Microphthalmia is a malformation that reduces the size of the ocular globe. The etiologies of this anomaly are various, but the genetic background appears to have a predominant influence on its development through mutations of genes controlling ocular developmental processes. LRP4 is a type I single transmembrane protein that is essential for the formation of neuromuscular junctions. We created and experimented on homozygous Lrp4-deficient mice and found the microphthalmia phenotype in their eyes. The loss of Lrp4 resulted in an elevated incidence of microphthalmia and affected the mRNA expression of the members of bone morphogenetic protein, fibroblast growth factor, Sonic hedgehog, and WNT signaling pathways and of several pathogenic genes for microphthalmia. Moreover, the loss of Lrp4 enhanced the incidence of aberrant retinal folds, which appeared pleated and corrugated in the eyeball.

MafB protein stability is regulated by the JNK and ubiquitin-proteasome pathways.

MafB is a basic leucine zipper transcription factor that plays important roles in development and differentiation processes. During osteoclastogenesis, its expression is downregulated at the transcriptional level via the JNK and p38 MAP kinase pathways. In the present study, we demonstrated that MafB protein stability is regulated by JNK and identified a phosphorylation site, Thr62. The expression of a constitutively active form of JNK (a fusion protein MKK7alpha1-JNK1beta1) promoted the degradation of MafB in COS7 cells, and a T62A substitution significantly reduced the instability of MafB. The introduction of a fourfold (T58A/T62A/S70A/S74A) substitution in an acidic transcription-activating domain almost protected the instability resulting from the activation of JNK. Furthermore, treatment with proteasome inhibitors increased the MafB level, and a high-molecular-weight smear, characteristic of polyubiquitination, was observed in lysates from cells in which MafB, ubiquitin, and MKK7alpha1-JNK1beta1 were co-expressed. These results suggest that phosphorylation of MafB by JNK confers susceptibility to proteasomal degradation.

RNA interference silencing of DRAL affects processing of amyloid precursor protein.

In a previous study, we reported that Alzheimer's disease-associated presenilin-2 interacts with a LIM-domain protein, namely, DRAL/FHL2/SLIM3. In this study, we investigated whether DRAL modifies the metabolism of the amyloid precursor protein (APP). We used small interfering RNA (siRNA) to knockdown DRAL in COS7 and HEK293 cells that stably overexpress APP695. We found that the knockdown was accompanied by a decrease in the amount of secreted alpha-secretase-cleaved APP and the membrane-bound C-terminal fragment C83 and an increase in the amount of secreted beta-amyloid peptide (Abeta) from the cells. We also found that in addition to a disintegrin and metalloprotease (ADAM)-17, DRAL binds to ADAM-10. Thus, DRAL may be involved in the processing of APP through the alpha-secretase pathway.

A novel beta-site amyloid precursor protein cleaving enzyme (BACE) isoform regulated by nonsense-mediated mRNA decay and proteasome-dependent degradation.

Proteolytic cleavage of amyloid beta-peptide (Abeta) from amyloid precursor protein (APP) is a key event in the pathogenesis of Alzheimer's disease. Beta-site amyloid precursor protein cleaving enzyme (BACE) cleaves the APP at the N-terminus of Abeta. We investigated whether particular stress conditions modify the expression and activity of BACE, and found that treatment of human neuroblastoma cells with protein synthesis inhibitors induced expression of a novel splice variant of BACE. This unusual transcript, I-127, is produced by usage of an internal splicing donor site in exon 3. The splicing event leads to a premature termination codon, as well as elimination of one of two conserved aspartic protease active sites, a transmembrane domain, and a C-terminal cytoplasmic tail from BACE. Low levels of this mRNA were found in the human brain. When expressed in cells, I-127 had no effect on Abeta secretion and was retained in the endoplasmic reticulum without propeptide removal. It was also unstable with a turnover t(1/2) of approximately 2h; normal BACE had a turnover t(1/2) of approximately 8h. Finally, I-127 was degraded in a proteasome-dependent manner. Thus, I-127 is regulated by both nonsense-mediated mRNA decay (NMD) and proteasome-dependent degradation.

Polyhydramnios in Lrp4 knockout mice with bilateral kidney agenesis: Defects in the pathways of amniotic fluid clearance.

Amniotic fluid volume during mid-to-late gestation depends mainly on the urine excretion from the foetal kidneys and partly on the fluid secretion from the foetal lungs during foetal breathing-like movements. Urine is necessary for foetal breathing-like movements, which is critical for foetal lung development. Bilateral renal agenesis and/or obstruction of the urinary tract lead to oligohydramnios, which causes infant death within a short period after birth due to pulmonary hypoplasia. Lrp4, which functions as an agrin receptor, is essential for the formation of neuromuscular junctions. Herein, we report novel phenotypes of Lrp4 knockout (Lrp4(-/-)) mice. Most Lrp4(-/-) foetuses showed unilateral or bilateral kidney agenesis, and Lrp4 knockout resulted in polyhydramnios. The loss of Lrp4 compromised foetal swallowing and breathing-like movements and downregulated the expression of aquaporin-9 in the foetal membrane and aquaporin-1 in the placenta, which possibly affected the amniotic fluid clearance. These results suggest that amniotic fluid removal was compromised in Lrp4(-/-) foetuses, resulting in polyhydramnios despite the impairment of urine production. Our findings indicate that amniotic fluid removal plays an essential role in regulating the amniotic fluid volume.

Association between tau polymorphism and male early-onset Alzheimer's disease.

Neurofibrillary tangles, containing hyperphosphorylated microtubule-associated protein tau, are one of the major pathological hallmarks of Alzheimer's disease. To investigate a possible association between tau genotypes and the risk of Alzheimer's disease, we screened for polymorphisms in the tau gene and found a novel polymorphism IVS11 + 90G --> A. A case-control study (874 patients and 678 controls) showed a significant association between possession of the A allele and male Alzheimer's disease with early-onset (age of onset before 65, odds ratio = 2.65; 95% confidence interval 1.30-5.42), suggesting that age and gender modify the risk effect. However, we failed to replicate the reported association between the Saitohin gene located in the tau intron 9 and Japanese Alzheimer's disease.

Characterization of an amyloid precursor protein-binding protein Fe65L2 and its novel isoforms lacking phosphotyrosine-interaction domains.

Human Fe65L2 is a member of the Fe65 protein family, which interacts with amyloid precursor protein (APP). Fe65L2 contains an N-terminal WW (Trp-Trp) domain followed by two phosphotyrosine-interaction domains, and consists of 486 amino acids. In the present study, we cloned and characterized two novel isoforms of Fe65L2, designated I-214 and I-245, which are produced by alternative splicing of the RNA. The splicing events disrupt the ability to bind with APP and low-density-lipoprotein-receptor-related protein. Fe65L2 was highly expressed in the brain, whereas I-214 and I-245 were expressed in various tissues. In HEK-293 cells, Fe65L2 was expressed in the nucleus and cytosol, whereas I-245 and I-214 were localized exclusively to the nucleus. The ratio of I-214 to Fe65L2 mRNA was increased by apoptotic stimuli. Although the overexpression of either Fe65L2 or I-214 did not significantly affect the half-life and maturation of APP, or the secretion of secreted APP, the secretion of beta-amyloid peptide (Abeta)40 and Abeta42 was increased by overexpression of Fe65L2, but not by that of I-214. These results suggest that Fe65L2 affects Abeta production and a possible regulation of Fe65L2 function by alternative splicing.