KF-1 ubiquitin ligase: anxiety suppressor model.

Anxiety disorders are the most popular psychiatric disease in any human societies irrespective of nation, culture, religion, economics or politics. Anxiety expression mediated by the amygdala may be suppressed by signals transmitted from the prefrontal cortex and hippocampus. KF-1 is an endoplasmic reticulum (ER)-based E3-ubiquitin (Ub) ligase with a RING-H2 finger motif at the C-terminus. The kf-1 gene expression is up-regulated in the frontal cortex and hippocampus in rats after anti-depressant treatments. The kf-1 null mice show no apparent abnormalities, but exhibit selectively pronounced anxiety-like behaviors or increased timidity-like responses. The kf-1 orthologous genes had been generated after the Poriferan emergence, and are found widely in all animals except insects, arachnids and threadworms such as Drosophila, Ixodes and Caenorhabditis, respectively. This suggests that the kf-1 gene may be relevant to some biological functions characteristic to animals. Based on these observations, the Anxiety Suppressor Model has been proposed, which assumes that KF-1 Ub ligase may suppress the amygdala-mediated anxiety by degrading some anxiety promoting protein(s), such as a neurotransmitter receptor, through the ER-associated degradation pathway in the frontal cortex and hippocampus. According to this model, the emotional sensitivity to environmental stresses may be regulated by the cellular protein level of KF-1 relative to that of the putative anxiety promoter. The kf-1 null mice should be useful in elucidating the molecular mechanisms of the anxiety regulation and for screening novel anxiolytic compounds, which may block the putative anxiety promoter.

KF-1 Ubiquitin Ligase: An Anxiety Suppressor.

Anxiety is an instinct that may have developed to promote adaptive survival by evading unnecessary danger. However, excessive anxiety is disruptive and can be a basic disorder of other psychiatric diseases such as depression. The KF-1, a ubiquitin ligase located on the endoplasmic reticulum (ER), may prevent excessive anxiety; kf-1(-/-) mice exhibit selectively elevated anxiety-like behavior against light or heights. It is surmised that KF-1 degrades some target proteins, responsible for promoting anxiety, through the ER-associated degradation pathway, similar to Parkin in Parkinson's disease (PD). Parkin, another ER-ubiquitin ligase, prevents the degeneration of dopaminergic neurons by degrading the target proteins responsible for PD. Molecular phylogenetic studies have revealed that the prototype of kf-1 appeared in the very early phase of animal evolution but was lost, unlike parkin, in the lineage leading up to Drosophila. Therefore, kf-1(-/-) mice may be a powerful tool for elucidating the molecular mechanisms involved in emotional regulation, and for screening novel anxiolytic/antidepressant compounds.

Mice lacking the kf-1 gene exhibit increased anxiety- but not despair-like behavior.

KF-1 was originally identified as a protein encoded by human gene with increased expression in the cerebral cortex of a patient with Alzheimer's disease. In mouse brain, kf-1 mRNA is detected predominantly in the hippocampus and cerebellum, and kf-1 gene expression is elevated also in the frontal cortex of rats after chronic antidepressant treatments. KF-1 mediates E2-dependent ubiquitination and may modulate cellular protein levels as an E3 ubiquitin ligase, though its target proteins are not yet identified. To elucidate the role of kf-1 in the central nervous system, we generated kf-1 knockout mice by gene targeting, using Cre-lox recombination. The resulting kf-1(-/-) mice were normal and healthy in appearance. Behavioral analyses revealed that kf-1(-/-) mice showed significantly increased anxiety-like behavior compared with kf-1(+/+) littermates in the light/dark transition and elevated plus maze tests; however, no significant differences were observed in exploratory locomotion using the open field test or in behavioral despair using the forced swim and tail suspension tests. These observations suggest that KF-1 suppresses selectively anxiety under physiological conditions probably through modulating protein levels of its unknown target(s). Interestingly, kf-1(-/-) mice exhibited significantly increased prepulse inhibition, which is usually reduced in human schizophrenic patients. Thus, the kf-1(-/-) mice provide a novel animal model for elucidating molecular mechanisms of psychiatric diseases such as anxiety/depression, and may be useful for screening novel anxiolytic/antidepressant compounds.

Bone mass increase specific to the female in a line of transgenic mice overexpressing human osteoblast stimulating factor-1.

We have reported that transgenic mice overexpressing human osteoblast stimulating factor-1 (osf1) under the control of the human osteocalcin promoter have a significantly higher bone mineral content and density than nontransgenic littermates. Consequently, bone mass loss due to estrogen deficiency was compensated for in ovariectomized female mice. Here, we show that in this transgenic line, the bone mass increase was evident in female, but not male, mice, as evaluated using the ash assay, double-emission X-ray analysis, and calcein double-labeling to determine the bone formation rate. To elucidate a possible influence on gene expression, we analyzed genomic structures of the inserted transgene and its flanking regions in mouse chromosomes. The results revealed that the transgene was integrated in the mouse repetitive sequences, 234-bp-long gamma-satellite repeats, as inverted multiple (5 + 8) copies. Twelve copies at most seemed to be functional, but no direct evidence supporting female-specific mRNA synthesis of the transgene was obtained.

A unifying model for functional difference and redundancy of presenilin-1 and -2 in cell apoptosis and differentiation.

Mutations in genes encoding the highly homologous proteins presenilin-1 and -2 (PS1 and PS2) are linked to the early onset of Alzheimer's disease (AD). Here, we report that polyclonal antibodies against Xenopus PSbeta (PS2), but not PSalpha (PS1), suppress the in vitro apoptotic activation of Xenopus egg extracts. To clarify the relationship between structural and functional differences in presenilins, we searched for presenilin homologues in various living sources, and found that presenilins were divided into three distinct groups, named alpha-, beta- and gamma-types, based on the size of the large hydrophilic loop (HL) regions as follows: HLalpha/HLbeta/HLgamma=4:3:6. No such size conservations were found in the N-terminal (NT) hydrophilic regions. Phylogenetic studies revealed that the presenilin genes were duplicated independently in different lineages of phyla/divisions, suggesting that there were functional requirements for and constraints on the generation and conservation of these HL sizes. On the basis of these findings, we propose a model postulating that both PS1 and PS2 can be differentiative or apoptotic when they are proteolytically processed within the HL regions or not, respectively, and PS1 may be more sensitive than PS2 to auto-proteolytic cleavage due to the larger size of the HL region of the former. Furthermore, the model assumes that C-terminal fragments (CTF) stabilized by phosphorylation may inhibit both the activities due to the dominant-negative effect. The model explains not only the functional redundancy but also apparently conflicting observations reported so far for PS1 and PS2.

Differential expression of presenilin-alpha and -beta (PSalpha and PSbeta) in Xenopus laevis: embryonic phosphorylation of PSalpha.

Mutations in genes encoding the highly homologous proteins, presenilin-1 and -2 (PS1 and PS2), are linked to the development of early-onset Alzheimer's disease. On the other hand, presenilins are known to play a critical role(s) in cell fate decisions during embryonic development in Caenorhabditis elegans. The messenger RNAs (mRNAs) of amphibian presenilin homologues PSalpha and PSbeta are most abundantly synthesized in the brain and the ovary, but are differentially degraded upon oocyte maturation and at the midblastula transition (MBT), respectively. In this study, we examined the spatiotemporal distribution of PSalpha and PSbeta proteins and their post-translational modification. The results were essentially consistent with the mRNA data and revealed moreover that PSalpha was present exclusively as processed molecules in the early embryos, while PSbeta was present mainly as unprocessed molecules (90%). Furthermore, the C-terminal fragment (CTF) of PSalpha was phosphorylated upon oocyte maturation and dephosphorylated at MBT, while no phosphorylation of the PSbeta CTF was detectable. Human PS1 CTF exogenously injected was also phosphorylated in Xenopus oocytes induced to mature in vitro by progesterone treatment. Two phosphorylation loci were mapped at Thr(320) and Ser(334) in the hydrophilic loop region of PSalpha. Our results suggest that PS1 and PS2 may play different roles under physiological conditions despite their high structural similarity.