X11 and X11-like proteins regulate the level of extrasynaptic glutamate receptors.

X11/Mint 1 and X11-like (X11L)/Mint 2 are neuronal adaptor protein to regulate trafficking and/or localization of various membrane proteins. By analyzing the localization of neuronal membrane proteins in X11-, X11L-, and X11/X11L doubly deficient mice with membrane fractionation procedures, we found that deficient of X11 and X11L decreased the level of glutamate receptors in non-PSD fraction. This finding suggests that X11 and X11L regulate the glutamate receptor micro-localization to the extrasynaptic region. In vitro coimmunoprecipitation studies of NMDA receptors lacking various cytoplasmic regions with X11 and X11L proteins harboring domain deletion suggest that extrasynaptic localization of NMDA receptor may be as a result of the multiple interactions of the receptor subunits with X11 and X11L regulated by protein phosphorylation, while that of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunits is not dependent on the binding with X11 and X11L proteins. Because the loss of X11 and X11L tends to impair the exocytosis, but not endocytosis, of glutamate receptors, NMDA receptors are likely to be supplied to the extrasynaptic plasma membrane with a way distinct from the mechanism regulating the localization of NMDA receptors into synaptic membrane region. Reduced localization of NMDA receptor into the extrasynaptic region increased slightly the phosphorylation level of cAMP responsible element binding protein in brain of X11/X11L doubly deficient mice compare to wild-type mice, suggesting a possible role of X11 and X11L in the regulation of signal transduction pathway through extrasynaptic glutamate receptors. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Facilitation of brain mitochondrial activity by 5-aminolevulinic acid in a mouse model of Alzheimer's disease.

The activities of mitochondrial enzymes, which are essential for neural function, decline with age and in age-related disease. In particular, the activity of cytochrome c oxidase (COX/complex IV) decreases in patients with Alzheimer's disease (AD). COX, a mitochondrial inner membrane protein complex that contains heme, plays an essential role in the electron transport chain that generates ATP. Heme synthesis begins with 5-aminolevulinic acid (5-ALA) in mitochondria. 5-ALA synthetase is the rate-limiting enzyme in heme synthesis, suggesting that supplementation with 5-ALA might help preserve mitochondrial activity in the aged brain. We administered a diet containing 5-ALA to triple-transgenic AD (3xTg-AD) model mice for 6 months, starting at 3 months of age. COX activity and protein expression, as well as mitochondrial membrane potential, were significantly higher in brains of 5-ALA-fed mice than in controls. Synaptotagmin protein levels were also significantly higher in 5-ALA-fed mice, suggesting improved preservation of synapses. Although brain Aß levels tended to decrease in 5-ALA-fed mice, we observed no other significant changes in other biochemical and pathological hallmarks of AD. Nevertheless, our study suggests that daily oral administration of 5-ALA could preserve mitochondrial enzyme activities in the brains of aged individuals, thereby contributing to the preservation of neural activity.

Expression and localization of X11 family proteins in neurons.

The X11/Mint family of proteins comprises X11/X11α/Mint1, X11L/X11ß/Mint2, and X11L2/X11γ/Mint3. Each of these molecules is an adaptor protein that contains a phosphotyrosine interaction/binding (PI/PTB) and two PDZ domains in its carboxy-terminal region. X11/Mint family members associate with a broad spectrum of membrane proteins, including Alzheimer's ß-amyloid precursor protein (APP), alcadeins, and low density lipoprotein receptor proteins, as well as various cytoplasmic proteins including Arf, kalirin-7, and Munc18. In particular, X11 and X11L are thought to play various roles in the regulation of neural functions in brain. Nevertheless, the protein levels and respective localization of individual family members remain controversial. We analyzed the protein levels of X11 and X11L in the corresponding single- and double-knockout mice. X11 and X11L did not exhibit obvious changes of their protein levels when the other was absent, especially in cerebrum in which they were widely co-expressed. In cerebellum, X11 and X11L localized in characteristic patterns in various types of neurons, and X11 protein level increased without an obvious ectopic localization in X11L-knockout mice. Interestingly, only X11L protein existed specifically in brain, whereas, contrary to the accepted view, X11 protein was detected at the highest levels in brain but was also strongly detected in pancreas, testis, and paranephros. Together, our results indicate that both X11 and X11L exert largely in brain neurons, but X11 may also function in peripheral tissues.