Functional activity of N-methyl-D-aspartate (NMDA) receptors requires both glutamate binding and the binding of an endogenous coagonist that has been presumed to be glycine, although D-serine is a more potent agonist. Localizations of D-serine and it biosynthetic enzyme serine racemase approximate the distribution of NMDA receptors more closely than glycine. We now show that selective degradation of D-serine with D-amino acid oxidase greatly attenuates NMDA receptor-mediated neurotransmission as assessed by using whole-cell patch-clamp recordings or indirectly by using biochemical assays of the sequelae of NMDA receptor-mediated calcium flux. The inhibitory effects of the enzyme are fully reversed by exogenously applied D-serine, which by itself did not potentiate NMDA receptormediated synaptic responses. Thus, D-serine is an endogenous modulator of the glycine site of NMDA receptors and fully occupies this site at some functional synapses. D-Amino acids play prominent roles in bacteria but have not been thought to occur in substantial quantity or to have any important function in vertebrates. Recently, techniques to distinguish isomers of amino acids in routine assays have led to the identification in some mammalian tissues of substantial amounts of at least two D-amino acids, D-serine and D-aspartate (1). Although D-aspartate is present in selected neuronal populations in the brain, it is concentrated mainly in glands, especially the epinephrine-containing cells of the adrenal medulla, the posterior pituitary, and the pineal gland (2-4).In contrast, D-serine occurs primarily in the brain, with highest concentrations in regions enriched in N-methyl-D-aspartate (NMDA) receptors (5-7). In these areas immunohistochemical studies have localized D-serine to protoplasmic astrocytes, which ensheathe nerve terminals especially in areas of the brain enriched in NMDA receptors (7). Stimulation of the kainate subtype of glutamate receptor releases D-serine from protoplasmic astrocytes (7).Because exogenous D-serine potentiates NMDA receptormediated neurotransmission (8-11) and D-[ 3 H]serine selectively binds to the glycine site (6), D-serine has been proposed as an endogenous ligand for the strychnine-insensitive glycine site of the NMDA receptor (6). Activation of NMDA receptors requires the presence of a coagonist, initially thought to be glycine (8,(12)(13)(14), and a glycine-selective recognition domain has been localized on NMDA receptors (15-17). However, D-serine is at least as potent as glycine as a coagonist at this site (8,10,14). In addition, immunohistochemical studies have revealed an overlapping distribution of D-serine and NMDA receptor immunoreactivity in forebrain (7). In the developing cerebellum, D-serine is localized to Bergmann glia that regulate granule cell migration during development via NMDA receptors (7). In contrast, glycine immunoreactivity is localized differently from that of NMDA receptors except in the brainstem, where it closely parallels the distribution of NMDA receptors (7). Extracell...

␥-Secretase facilitates the regulated intramembrane proteolysis of select type I membrane proteins that play diverse physiological roles in multiple cell types and tissue. In this study, we used biochemical approaches to examine the distribution of amyloid precursor protein (APP) and several additional ␥-secretase substrates in membrane microdomains. We report that APP C-terminal fragments (CTFs) and ␥-secretase reside in Lubrol WX detergent-insoluble membranes (DIM) of cultured cells and adult mouse brain. APP CTFs that accumulate in cells lacking ␥-secretase activity preferentially associate with DIM. Cholesterol depletion and magnetic immunoisolation studies indicate recruitment of APP CTFs into cholesterol-and sphingolipid-rich lipid rafts, and co-residence of APP CTFs, PS1, and syntaxin 6 in DIM patches derived from the trans-Golgi network. Photoaffinity cross-linking studies provided evidence for the preponderance of active ␥-secretase in lipid rafts of cultured cells and adult brain. Remarkably, unlike the case of APP, CTFs derived from Notch1, Jagged2, deleted in colorectal cancer (DCC), and N-cadherin remain largely detergent-soluble, indicative of their spatial segregation in non-raft domains. In embryonic brain, the majority of PS1 and nicastrin is present in Lubrol WXsoluble membranes, wherein the CTFs derived from APP, Notch1, DCC, and N-cadherin also reside. We suggest that ␥-secretase residence in non-raft membranes facilitates proteolysis of diverse substrates during embryonic development but that the translocation of ␥-secretase to lipid rafts in adults ensures processing of certain substrates, including APP CTFs, while limiting processing of other potential substrates. Sequential processing of amyloid precursor protein (APP)1 by ␤-and ␥-secretases releases the 39 -42-amino acid-long ␤-amyloid (A␤) peptides, which accumulate in the brains of aged individuals and patients with Alzheimer disease (AD) (1). The major ␤-secretase in neurons is an aspartyl protease termed BACE-1, which cleaves APP within the luminal domain, generating the N terminus of A␤ (2). The C terminus of A␤ is generated by intramembranous cleavage of APP C-terminal fragments (CTFs) by ␥-secretase, a multiprotein complex made of four essential components, presenilin (PS) 1 (or PS2), nicastrin, PEN-2, and APH-1 (3). Mutations in PSEN1 and PSEN2, encoding multipass membrane proteins PS1 and PS2, respectively, co-segregate with the majority of cases of autosomal dominant familial early-onset AD (4). Familial AD-linked PS1 and PS2 variants elevate the production of highly fibrillogenic A␤42 peptides (1). A role for PS1 in Notch function was first discovered in Caenorhabditis elegans screens and involves intramembranous cleavage of the Notch receptor, analogous to APP processing (5, 6). Nicastrin is a type I membrane protein independently identified as a novel component of the GLP-1/ Notch signaling pathway in C. elegans early embryos and in the biochemical characterization of proteins that interacted with PS1 (7,8). Multitransmembra...

BackgroundGenome-wide association studies have identified BIN1 within the second most significant susceptibility locus in late-onset Alzheimer’s disease (AD). BIN1 undergoes complex alternative splicing to generate multiple isoforms with diverse functions in multiple cellular processes including endocytosis and membrane remodeling. An increase in BIN1 expression in AD and an interaction between BIN1 and Tau have been reported. However, disparate descriptions of BIN1 expression and localization in the brain previously reported in the literature and the lack of clarity on brain BIN1 isoforms present formidable challenges to our understanding of how genetic variants in BIN1 increase the risk for AD.MethodsIn this study, we analyzed BIN1 mRNA and protein levels in human brain samples from individuals with or without AD. In addition, we characterized the BIN1 expression and isoform diversity in human and rodent tissue by immunohistochemistry and immunoblotting using a panel of BIN1 antibodies.ResultsHere, we report on BIN1 isoform diversity in the human brain and document alterations in the levels of select BIN1 isoforms in individuals with AD. In addition, we report striking BIN1 localization to white matter tracts in rodent and the human brain, and document that the large majority of BIN1 is expressed in mature oligodendrocytes whereas neuronal BIN1 represents a minor fraction. This predominant non-neuronal BIN1 localization contrasts with the strict neuronal expression and presynaptic localization of the BIN1 paralog, Amphiphysin 1. We also observe upregulation of BIN1 at the onset of postnatal myelination in the brain and during differentiation of cultured oligodendrocytes. Finally, we document that the loss of BIN1 significantly correlates with the extent of demyelination in multiple sclerosis lesions.ConclusionOur study provides new insights into the brain distribution and cellular expression of an important risk factor associated with late-onset AD. We propose that efforts to define how genetic variants in BIN1 elevate the risk for AD would behoove to consider BIN1 function in the context of its main expression in mature oligodendrocytes and the potential for a role of BIN1 in the membrane remodeling that accompanies the process of myelination.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0124-1) contains supplementary material, which is available to authorized users.

Aged memory-impaired (AI) and unimpaired (AU) 24–25-month-old Long- Evans rats were used to investigate the integrity of various cholinergic markers during normal aging and to establish if alterations can possibly relate to cognitive disabilities. AI and AU rats were classified on the basis of their performance in the Morris swim maze task. Choline acetyltransferase activity (ChAT) was not differentially altered in various cortical and hippocampal areas between these two groups. Similarly, quantitative receptor autoradiography did not reveal significant differences in 3H-pirenzepine/muscarinic M1 and 3H- hemicholinium-3/high-affinity choline uptake binding sites in AI versus AU rats. In contrast, 3H-AF-DX 384/putative muscarinic M2 binding was significantly increased in certain cortical and hippocampal areas of the age-impaired animals. These increments were correlated with decreased in vivo acetylcholine (ACh) release capacity in the AI rats. Most interestingly, the muscarinic M2 antagonist BIBN-99 reversed, in a dose-dependent manner, the impaired ACh release as well as the cognitive deficits observed in the AI group. Similarly, BIBN-99 reversed scopolamine-induced amnesia in young animals. The efficacy of BIBN-99 likely relates to its antagonistic properties on negative muscarinic M2 autoreceptors that are apparently increased in the AI animals, leading to altered ACh release. Taken together, these findings strengthen the role of ACh in learning and memory and may have implications for the treatment of degenerative disorders associated with impaired cholinergic functions, such as Alzheimer's disease.