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Loss of Munc18-1 long splice variant in GABAergic terminals is associated with cognitive decline and… Ramos-Miguel, Alfredo; Hercher, Christa; Beasley, Clare L; Barr, Alasdair M; Bayer, Thomas A; Falkai, Peter; Leurgans, Sue E; Schneider, Julie A; Bennett, David A; Honer, William G Dec 2, 2015

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RESEARCH ARTICLE Open AccessLoss of Munc18-1 long splice variant inGABAergic terminals is associated withcognitive decline and increased risk ofdementia in a community sampleAlfredo Ramos-Miguel1,2, Christa Hercher1,2, Clare L. Beasley1,2, Alasdair M. Barr1,3, Thomas A. Bayer4, Peter Falkai5,Sue E. Leurgans6, Julie A. Schneider6, David A. Bennett6 and William G. Honer1,2*AbstractBackground: Presynaptic terminals contribute to cognitive reserve, balancing the effects of age-related pathologies oncognitive function in the elderly. The presynaptic protein Munc18-1, alternatively spliced into long (M18L) or short (M18S)isoforms, is a critical modulator of neurotransmission. While subtle alterations in Munc18-1 have been shown to causesevere neuropsychiatric disorders with cognitive impairment, little information is known regarding the specific roles ofMunc18-1 splice variants. We first investigated functional and anatomical features evidencing the divergent roles of M18Land M18S, and then evaluated their contribution to the full range of age-related cognitive impairment in the dorsolateralprefrontal cortex of a large sample of participants from a community-based aging study, including subjects withno-(NCI, n = 90), or mild-(MCI, n = 86) cognitive impairment, or with clinical dementia (n = 132). Finally, we usedAPP23 mutant mice to study the association between M18L/S and the time-dependent accumulation of commonAlzheimer’s disease pathology.Results: Using isoform-specific antibodies, M18L was localized to the synaptosomal fraction, with a distribution matchinglipid raft microdomains. M18S was found widely across cytosolic and synaptosomal compartments. Immunocytochemicalstudies identified M18L in perisomatic, GABAergic terminals, while M18S was broadly distributed in GABAergic andglutamatergic terminals. Using regression models taking into account multiple age-related pathologies, age, educationand sex, global cognitive function was associated with the level of M18L (p = 0.006) but not M18S (p = 0.88). Mean M18Lin dementia cases was 51 % lower than in NCI cases (p < 0.001), and each unit of M18L was associated with a lowerlikelihood of dementia (odds ratio = 0.68, 95 % confidence interval = 0.50–0.90, p = 0.008). In contrast, M18S balancedacross clinical and pathologically diagnosed groups. M18L loss may not be caused by age-related amyloid pathology,since APP23 mice (12- and 22-months of age) had unchanged cortical levels of M18L/S compared with wild-type animals.Conclusions: M18L was localized to presynaptic inhibitory terminals, and was associated with cognitive function andprotection from dementia in an elderly, community-based cohort. Lower M18L in inhibitory presynaptic terminals maybe an early, independent contributor to cognitive decline.Keywords: Syntaxin-binding protein, SNARE, Protein-protein interactions, VGAT, VGLUT1, Human postmortem brain,Aging study, Mild cognitive impairment, Synaptic pathology, Alzheimer’s disease* Correspondence: william.honer@ubc.ca1Child and Family Research Institute, 938 West 28th Avenue, Vancouver, BCV5Z 4H4, Canada2Department of Psychiatry, University of British Columbia, 2255 WesbrookMall, Vancouver, BC V6T 2A1, CanadaFull list of author information is available at the end of the article© 2015 Ramos-Miguel et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Ramos-Miguel et al. Molecular Neurodegeneration  (2015) 10:65 DOI 10.1186/s13024-015-0061-4BackgroundCommon age-related neuropathologies, includingAlzheimer’s disease pathology (i.e. neuritic plaquesand neurofibrillary tangles), cerebral infarcts, andLewy bodies, accumulate in the human brain acrossthe lifetime. The susceptibility of cognitive functionto decline as growing amounts of neuropathologydevelop in the forebrain varies greatly between indi-viduals, raising the concept of cognitive reserve [1–3].Large community-based studies have been designed,at least in part, to investigate the biological substratesof cognitive reserve [4–7]. The initial model that peoplewith mentally enriched lives may exhibit greater resistanceagainst brain-damaging diseases [2] is not yet supportedwith clear neurochemical correlates. Synaptic pathology iscommon in aging, being first reported as a deficit ofpresynaptic markers [8–11]. In some studies, synapticpathology showed stronger association with cognitivedecline than other common age-related pathologies [12],suggesting that intact, functional synapses may contributeto cognitive reserve.In this context, we previously reported that bettercognitive performance, a reduced odds of dementia,and slower rate of cognitive decline over multiple yearsprior to death were associated with greater densities ofspecific presynaptic proteins, and enhanced presynapticprotein-protein interactions (PPIs) [13, 14]. Interest-ingly, these PPIs involved the associations betweensyntaxin-1, synaptosomal-associated protein-25 (SNAP-25), and vesicle-associated membrane protein (VAMP,or synaptobrevin), which together assemble the machineryfacilitating vesicle trafficking and neurotransmitter release,known as the soluble N-ethylmaleimide-sensitive factorattachment protein receptor (SNARE) [15]. However, otherfrequent targets of synaptic pathology, such as synapto-physin, were not associated with cognitive function orAlzheimer’s disease pathology in the same community-based cohort study, which included subjects with thefull range of cognition from normality, to mild cogni-tive impairment, to dementia. Of note, many of thestudies related to synaptic pathology were based onsubpopulations of hospital- or clinic-based patientsdisplaying moderate-to-severe degrees of dementia, notnecessarily representative of the spectrum of cognitivedecline in aging [16]. Previous analyses in mixed post-mortem brain series found biphasic alterations of brainpresynaptic markers (including synaptophysin) acrossage-related cognitive decline and/or Alzheimer’s diseaseprogression, with upregulated densities at intermediatestages and reduced levels in more severe cases [17–19].Together, these observations suggest that an initialfailure of presynaptic terminals to maintain synapticfunction (reflected by SNARE disruption) might becompensated by upregulating the expression of certainpresynaptic genes; further accumulation of neuropathol-ogies may result in the observed synaptic degeneration atrelatively more severe stages. The mechanisms underlyinginitial SNARE and presynaptic dysfunction could bedirectly linked to age-related cognitive impairment andprogression to dementia. Consistent with these observa-tions, murine models with impaired SNARE PPIs undergorelated behavioral and physiological neurodegenerativesyndromes [20, 21].Although the biochemical and physiological nature ofneurosecretion is well established [22–24], the associationbetween SNARE dysregulation and the pathophysiology ofaging-related diseases is unclear. Among the regulatorspotentially involved in SNARE dysfunction, mammal unc-18-1 (Munc18-1 or n-Sec1; herein M18) is a strong candi-date. First identified by random mutagenesis screening foruncoordinated phenotypes in the worm Caenorhabditiselegans, M18 caught the attention of many research teamswhen described as a ‘syntaxin-binding protein’ (coining theM18 coding gene as STXBP1) twenty years later [25–27].While still controversial, M18 is believed to have multipleactivities during SNARE-regulated exocytosis, which mayinclude trafficking and chaperoning syntaxin-1, transitionfrom cis-to-trans SNARE conformations, and/or dockingthe presynaptic vesicle [23, 28, 29]. The observation thatSTXBP1 null mice lack neocortical synaptic activity [30]highlights the irreplaceable role of M18 for neurosecretion.Interestingly, M18 knockout mice also exhibit massiveneuronal apoptosis and widespread neurodegeneration, andconsequently do not survive after birth [30].In mammals, the STXBP1 gene is alternatively splicedto yield either a long (M18L) or a short (M18S) variant[31, 32], also called isoforms a and b, respectively. Pro-cessing of the final exon in the STXBP1 primary tran-script may include (M18L) or skip (M18S) a sequence of110 bp containing a stop codon [32], resulting in twodifferent C-terminal amino acid sequences for M18L/S(see Fig. 1a). To our knowledge, specific regulatorymechanisms of STXBP1 gene splicing have not beendescribed. Although intensive research focuses on M18activities in regulated exocytosis, the potentially diver-gent roles of the M18 variants are relatively neglected,assuming no substantial differences between them [32].However, recent observations indicate that M18 variantsmay not have overlapping functions. Transgenic miceexpressing a fluorophore-tagged, functional M18S weregenerated using a replacement strategy that erased theM18L splice variant [33, 34]. These animals show severephysical and behavioral deficits, and die prematurely afew weeks after birth. Conversely, mice overexpressingM18L in both glutamatergic and GABAergic neuronsdisplay aspects of a schizophrenia-like phenotype [35],compatible with alterations observed in human postmor-tem studies [36–38]. At a cellular level, transfection ofRamos-Miguel et al. Molecular Neurodegeneration  (2015) 10:65 Page 2 of 18Fig. 1 (See legend on next page.)Ramos-Miguel et al. Molecular Neurodegeneration  (2015) 10:65 Page 3 of 18either M18L or M18S to excitatory neuronal cell culturesfrom the hippocampus of M18 knockouts succeeded to asimilar extent in rescuing basal synaptic activity [39].However, M18S appeared to support high frequencystimulation more efficiently than the long variant, suggest-ing different roles in short-term plasticity. Altogether,these findings indicate that the functions of M18 splicevariants may not be interchangeable, and the multiplicityof activities attributed to M18 may represent a compositeof M18L + S.It has been reported that de novo mutations affectingSTXBP1 gene can cause Ohtahara syndrome in humans[40–45], a devastating neurological disease characterizedby early onset of epileptic seizures and a profound intel-lectual disability. Most STXBP1 mutations associated withOhtahara syndrome impair M18–syntaxin-1 interaction(and thereby proper SNARE assembly), compromisingcellular availability of functional M18 (haploinsufficiency)[41, 43]. Conversely, other mutations are predicted toconfer aberrant alternative splicing of M18 RNA [44]. Inaddition, some STXBP1 de novo mutations were associ-ated with similar mental retardation without epilepticseizures [42].In the context of aging, two previous studies addressedalterations in M18 protein levels in Alzheimer’s diseasepostmortem brain. In the first, M18 immunodensity waslower in two cortical areas in n = 32 Alzheimer’s diseasecases, although after synaptophysin normalization, theauthors concluded that productive synapses in Alzheimer’sdisease brains might be enriched in M18 [46]. A laterproteomic study [47] found increased M18 levels in cellmembrane extractions from cortical samples of n = 5Alzheimer’s disease cases. These two studies did notexamine the correlation of M18 with cognitive out-comes, or with the likelihood of dementia; nor werepotential differences in M18 splice variants assessed. Inaddition, the small sample sizes limited the possibilityof investigating possible effects of stage of illness, orpotential confounders.Altogether, clinical and preclinical data demonstratedthat M18 is essential for neurosecretion, subtle alter-ations have a dramatic impact on normal synaptic func-tion and cognition in humans and rodents, and couldrepresent an early sign of synaptic pathology and cogni-tive impairment. For the present study we hypothesizedthat cortical downregulation of M18 long and/or short(M18L/S) splice variants could contribute to poorercognitive performance and increased likelihood ofdementia in old age. Due to the scarce data on specificfunctions of M18 isoforms, we first performed func-tional and anatomical characterization experiments thatevidenced the complementary, rather than overlappingroles of M18L and M18S. Using tissue samples fromthe community-based Memory and Aging Project (MAP)[7], cortical expression of M18 variants was quantified in alarge sample of MAP participants with and without clinicaldementia, representing the range of cognitive impairmentin an elderly, aging population. The potential effects ofAlzheimer’s disease pathology on M18 levels were furthermodeled using APP23 transgenic mice, which overexpressa pathogenic variant of the human amyloid precursorprotein (APP) [48].ResultsBiochemical and anatomical evidence indicates differentroles of M18 splice variantsWe first used immunoprecipitation (IP) followed byeither sodium dodecyl sulfate (SDS) or blue native (BN)polyacrylamide gel electrophoresis (PAGE) to investigatefunctional binding affinities of M18 isoforms for theSNARE complex. As expected from their target immuno-genic sequences (Fig. 1a), and also from previous data[36], variant-specific antibodies selectively immunopreci-pitated M18L or M18S without cross-reacting, confirmedby immunoblot analyses (Fig. 1b). Surprisingly, anti-M18SIP products were 7–12 fold enriched in SNARE proteins(i.e. syntaxin-1, SNAP-25 and VAMP), compared to thoseof anti-M18L (Fig. 1b). This was not due to a lower affinity(See figure on previous page.)Fig. 1 Biochemical characterization of M18 splice variants. a Alignment of M18L/S C-terminal amino acid (aa) sequences. Mismatching residuesare in red. Amino acids highlighted (in yellow) represent the immunogenic sequences used for production of variant-specific antibodies. OmittedM18L/S N-terminal sequences are 100 % identical. b–c Immunoprecipitation (IP) of M18L/S with variant specific antibodies (and anti-mouse IgGas a negative control) using human brain homogenates. NCI control subjects (n = 3) were tested with similar results. IP products, along with inputsamples and negative controls (IgG), were resolved by (b) standard SDS- or (c) BN-PAGE, followed by either silver staining (Silver) or immunoblotting (IB)with specific antibodies against M18 variants, syntaxin-1 (STX1), SNAP-25 (S25), or VAMP. c M18S antibody recognized two bands at ~150 and ~70 kDa,putatively corresponding to a SNARE-M18 heterotetramer (pointed with a red arrowhead) and the monomeric form, respectively. d, f Schematic illustrationdepicting the sequential extraction of (d) synaptosomes and (f) lipid rafts from human cortical homogenates. g, relative centrifugal forces; IF, interface; P,pellet; S, supernatant. e Equivolumetric amounts of fractions obtained in (d) were resolved by SDS-PAGE and immunoblotted using antibodies targetingM18L/S, STX1, S25, VAMP, and markers for synaptic vesicles (synaptophysin [SYP]), nuclei (FosB), nuclei + cytosol (NeuN), and myelin fragments (myelin basicprotein [MBP]). Note that MBP strongly labels P1, as heavy myelin fragments precipitate along with cell debris. g Equivolumetric amounts of fractionsobtained in (f) were resolved by SDS-PAGE and immunoblotted using the antibodies above. α-Synuclein (α-syn) antibody was additionally used, showingsimilar distribution across fractions as previously reported [66]. e, g IF1 and S4 fractions were framed in a red, dashed box to highlight synaptosome andlipid raft-enriched proteins, respectively. b–g Masses (in kDa) of prestained markers are indicated on the left side of immunoblotsRamos-Miguel et al. Molecular Neurodegeneration  (2015) 10:65 Page 4 of 18of the antibody for M18L, since comparable amounts ofboth isoforms were observed in silver stained gels (Fig. 1b).Rather, M18L displayed (under current assay conditions) apoorer ability to bind syntaxin-1. Accordingly, M18Sformed a stable ~150-kDa complex with SNARE proteins,which was barely detected in M18L co-IP products, as re-vealed by BN-PAGE (Fig. 1c).Substantial differences in the subcellular localization ofM18 variants were also observed (Fig. 1d–g). While M18Lwas apparently restricted to the synaptosomal fraction(IF1), M18S showed the expected wide distribution acrosscytosolic (S2) and synaptosomal compartments (Fig. 1e).Furthermore, M18L was almost exclusively found withinthe Triton X-100 (TritonX)-insoluble, SDS-soluble proteinfraction (S4), matching lipid raft microdomains, whileM18S was present in water- and detergent-soluble subcel-lular compartments (Fig. 1g).Immunohistochemical analyses of rat brain revealed adifferential distribution of M18 variants, particularly inthe hippocampus (Fig. 2a). M18L was highly abundantin the pyramidal cell layer of the Ammon's horn (CA)regions, as well as in the dentate gyrus granule cell layer.In marked contrast, M18S showed greatest immunostain-ing in pyramidal cell-flanking strata (with particularlystrong labeling of the mossy fibers), but was barely presentwithin the pyramidal layer. In cortical regions, M18L accu-mulated in neuronal perisomatic areas, whereas M18Sshowed the punctate staining of neuropil typically observedfor presynaptic proteins (Fig. 2a). Despite their apparentlydistinct patterns of distribution (best characterized in CA3),confocal imaging showed some degree of overlap betweensplice variants in areas such as the granule cell layer(Fig. 2b). Of note, colocalization of M18 isoforms also over-lapped with syntaxin-1. Similar results were observed inhuman hippocampus (Additional file 1: Figure S1A). InNeuN co-stained sections, preferential perisomatic accu-mulation of M18L was highlighted, and additional evidenceof the cytosolic localization of M18S (but not M18L orsyntaxin-1) obtained (Fig. 2c–d).Triple co-immunolabeling with antibodies against M18L,vesicle GABA (VGAT) and glutamate (VGLUT1) trans-porters showed a high degree of M18L localization withinthe inhibitory (VGAT-positive) synapses, with little (orpossibly no) presence at excitatory (VGLUT1-positive)terminals, in both cortex and hippocampus (Fig. 2e–g). Incontrast, overlap of M18S was greater with VGLUT1 thanwith VGAT (Fig. 3h–j), although this could be explained bythe relative abundance of glutamatergic over GABAergicterminals. This difference was most remarkable in CA3,where a complete segregation of M18L and M18Swith respectively, VGAT or VGLUT1 positive termi-nals was observed (Fig. 2f, i). In human hippocam-pus, similar distributions were seen (Additional file1: Figure S1B–C).Characteristics of MAP participants and potentialconfoundsDemographic, cognitive and pathological variables of MAPparticipants are summarized in Table 1. No correlation wasdetected between M18 splice variants and potentialconfounders, including age at death, postmortem interval,years of education and brain weight. Similarly, M18L/Scortical immunodensity did not vary across sex, race, orApoE genotype. Furthermore, tobacco and/or alcohol usersdid not present different M18L/S levels from those withoutthese histories.Similar numbers of clinically diagnosed MAP partici-pants with no (NCI) or mild (MCI) cognitive impairment,and dementia were observed (Table 1). As expected, com-mon Alzheimer’s disease pathology was largely abundantin dementia cases, compared to NCI and MCI participants(Fig. 3a), although some degree of variability was presentin all clinically diagnosed groups, as documented previ-ously in larger epidemiologic studies [5, 7]. Similarly,clinical diagnoses showed substantial, but not complete,overlap with CERAD and Braak rating scales (Fig. 3b).Association of cortical M18L with cognitive function anddementiaWe investigated possible alterations in the amounts ofM18 splice variants, and their potential associationwith cognitive function, in samples from the dorsolat-eral prefrontal cortex (DLPFC) of MAP participants(n = 308). Initial inspection of quantitative immuno-blotting datasets (normalized by β-actin content) revealedlarge variability in M18L immunodensities among MAPparticipants (interquartile range = 7–185 %), compared tothat of M18S (86–122 %) or β-actin (85–117 %). Theimmunodensities of the M18 variants showed a non-linearassociation (Additional file 1: Figure S2A, Spearmanrho = 0.53), best fitted by a semi-log curve (r = 0.56,p < 0.001). Due to the large number of values proximateto zero (see Additional file 1: Figure S2), and consequentskewness (skew = 1.712), the distribution of M18L valuesdid not pass the Kolmogorov-Smirnov normality test. Toconstruct linear models, we log-transformed M18L values,and then standardized (by subtracting the mean anddividing by the standard deviation) both variant datasets.Data transformations symmetrized the distribution ofM18L values and rendered linear associations between thevariants (r = 0.51, p < 0.001).We initially conducted regression models (controlling fordemographics) to address basic associations between M18variants, Alzheimer’s disease pathology and global cognitivefunction. Lower cortical immunodensity of M18L, but notM18S, was associated with the severity of Alzheimer’sdisease pathology, and lower cognitive scores (Fig. 4a).Importantly, for those participants above the 90th percent-ile of M18L the effect of higher levels of Alzheimer’s diseaseRamos-Miguel et al. Molecular Neurodegeneration  (2015) 10:65 Page 5 of 18pathology on cognition was not statistically significant,although the number of cases with high pathology waslimited, preventing definitive conclusions (Fig. 4b). In con-trast, in subjects with M18L score below the 10th percent-ile, cognitive function was highly sensitive to Alzheimer’sdisease pathology. While these observations suggested aninteraction between Alzheimer’s disease pathology andM18L cortical levels, the inclusion of a statistical interactionterm in the full sample analysis did not render significance.Decay curves for pathology-cognition associations weresimilar across M18S-ranked MAP participants (Fig. 4b).Based on prior work showing that select presynapticproteins were related to cognition in a relatively independ-ent manner [13], we next generated linear regressionmodels to evaluate the impact of M18L on cognitive func-tion, accounting for age-related neuropathology. In theseanalyses, each of the common age-related neuropathologieswas associated to a variable degree with global and/orFig. 2 Immunohistochemical characterization of M18 splice variants in rat brain. (a) Photomicrographs showing M18L (upper panels) and M18S (bottompanels) immunostaining with variant specific antibodies at various magnifications. Hippocampus and CA3 are magnified captions from the framed areasat immediate left-side images. (b–j) Confocal images and analyses from triple co-immunolabeled sections with the antibodies indicated at the top-leftcorners. Colors were arbitrarily assigned to maximize overlap visualization. (e–j) Rat brain sections were co-immunolabeled with antibodies against vesicularGABA (VGAT) and glutamate (VGLUT1) transporters, along with (e–f) anti-M18L or (h–i) anti-M18S, and representative images from (e and h) cortical and(f and i) hippocampal CA3 are shown. In every 6-panel composite: left and middle panels correspond to single-labeled or merged-channel images; rightpanels are ImageJ-generated bitmaps resulting from colocalization analyses, in which pixels mirror the intensity of colocalization (in white) between VGAT(top) or VGLUT1 (bottom) and the corresponding M18 splice variant. (g and j) Quantitative colocalization analyses of (G) M18L or (J) M18S witheach of the vesicular transporters (VGAT and VGLUT1) Bars represent mean ± standard error of n = 4 rats. *p < 0.05, **p < 0.01, and ***p < 0.001 (pairedt-test). Abbreviations: DG, dentate gyrus; hil, hilus; Hipp, hippocampus sg; stratum granulosum (i.e. granule cell layer); sm/l, stratum moleculare/lacunosum;so, stratum oriens; sp, stratum pyramidale (i.e. pyralmidal cell layer); sr, stratum radiatum (in CA3 also contains stratum lucidum. Scale bars: a, 200 (CA3) or20 (cortex) μm; b, 30 μm; c, 50 (left) or 10 (right) μm; d, 30 (left) or 10 (right) μm; e and h, 10 μm; f and i, 25 μmRamos-Miguel et al. Molecular Neurodegeneration  (2015) 10:65 Page 6 of 18domain-specific cognitive functions (Table 2, Model 1).Alzheimer’s disease pathology, cerebral macroinfarcts, hip-pocampal sclerosis, arteriolosclerosis, and atherosclerosiswere all associated with poorer cognitive abilities. Lewybodies and microinfarcts were not, in this subset ofMAP participants. Overall, greater M18L corticalimmunodensity was associated with better cognitiveabilities (Model 2). Cortical amounts of M18L ex-plained an additional 1.7 % of the variance in globalcognition, relatively independent of any neuropathol-ogy. Of note, the frontal cortical M18L contributionwas significant for episodic, semantic and workingmemory, but not for perceptual speed or visuospatialskills. Variations in M18S immunodensities were notrelated to global cognition or any of its domains(Model 3); when including both M18L and M18S(Model 4) only the M18L effect remained significant.In a final model (not shown), we analyzed the possibleinteraction between Alzheimer’s disease pathology andM18L cortical density by adding a statistical inter-action term to the model. No significant effect was de-tected for this interaction term.Grouping MAP participants by clinical diagnosisrevealed that dementia cases had lower M18L corticalimmunodensity (−51 %, p < 0.001) than those with nodementia (Fig. 4c–d). Differences were also noted whensubjects were graded using either NIA/Reagan or Braak,with lower M18L levels (−77 % and−64 %, respectively)in participants with high probability of Alzheimer’sdisease (i.e. subjects falling into NIA/Reagan group 1and/or Braak stage V–VI) (Additional file 1: Figure S2B).Minimal differences for M18S were observed across clinicaldiagnoses or postmortem rankings. In further multiplelogistic regression analyses we aimed to evaluate the signifi-cance of M18L cortical downregulation to the likelihood ofclinical dementia (Table 3). As expected, each unit ofAlzheimer’s disease pathology, cerebrovascular disease(excluding microinfarcts), Lewy bodies and hippocampalsclerosis increased the odds of dementia (Table 3, Model 1).Each unit of M18L, however, was associated with lowerodds of dementia, without altering the effects of neuropa-thologies (Model 2). The probability of dementia was notrelated to M18S cortical levels (Model 3), which in turn didnot modify M18L effects (Model 4).Fig. 3 Burden of Alzheimer’s disease pathology in MAP participants and relation to clinical diagnoses. a Composite Alzheimer’s disease (AD) pathologyvalues were estimated for each participant and plotted by clinical diagnosis criteria into no- (NCI, n = 90), or mild-cognitive impairment (MCI, n = 86), ordementia (DEM, n = 132). Whiskers represent 10th and 90th percentiles and boxes enclose interquartile ranges crossed by the median of Alzheimer’sdisease pathology scores within groups. As expected, Kruskal-Wallis test detected differences on the accumulated Alzheimer’s disease pathology acrossclinical diagnoses (KW-statistic = 57.0, p < 0.001; Mean rank differences: NCI vs MCI = 30.7; NCI vs DEM = 88.7; MCI vs DEM = 58.0). ns, notsignificant, ***p < 0.001, Kruskal-Wallis followed by Dunn’s multiple comparison test. b Bubble plot illustrating the distribution of MAP participants acrossclinically diagnosed (NCI/MCI/DEM) and neuropathologically graded [by NIA/Reagan, CERAD (Consortium to Establish a Registry for Alzheimer’s Disease),and Braak scales] groupsRamos-Miguel et al. Molecular Neurodegeneration  (2015) 10:65 Page 7 of 18APP23 mice do not show alterations in M18L/S corticalamountsWe used APP23 transgenic mice to examine the associ-ation between M18L and a common Alzheimer’s diseasepathology (i.e. amyloid plaques). These animals developan Alzheimer’s disease-like syndrome via expression of amutant APP that causes abnormal, age-dependent extra-synaptic amyloid-β accumulation [48]. M18L/S corticalimmunodensities were compared between adult (12-month old) and aged (22-month old) APP23 mice, andage-matched wild type (WT) littermates. Although overtsynaptic damage is reported in APP23 mice [48], corticallevels of M18L/S were not significantly different fromthose in WT controls, at either age (Fig. 5). However,M18L was slightly reduced in aged rats regardless ofgenotype (−13 to −16 %, p > 0.05), while APP23 micetended to display lower M18S cortical immunodensitiesregardless of age (−21 to−22 %, p > 0.05). These marginaleffects were considerably smaller than the M18L loss(−64 %) observed in the DLPFC of MAP participantsgraded according to Braak’s V–VI stages.DiscussionIn the present study, variations in frontal corticalM18 levels were associated with cognitive functionand the likelihood of dementia in the elderly, beyondthe effects of common age-related neuropathologies.Specific loss of M18L (but not M18S), which wasfound preferentially at GABAergic presynapticTable 1 Demographic, cognitive and pathological characteristicsa of MAP participants included in the present studyVariable All participants(n = 308)NCI(n = 90)MCI(n = 86)Dementia(n = 132)DemographicFemale, no. (%) 194 (63 %) 64 (71 %) 48 (56 %) 82 (62 %)Age at death, years 88.8 ± 6.0 86.8 ± 6.3 88.1 ± 6.4 90.7 ± 5.0Education, years 14.4 ± 2.8 14.0 ± 2.4 14.7 ± 2.5 14.5 ± 3.2Race, no. W:AA:NA 300:7:1 86:4:0 84:1:1 130:2:0APOE ε4 allele, no. (%) 84 (27 %) 15 (17 %) 22 (26 %) 47 (36 %)PMI, hours 7.2 ± 4.8 6.9 ± 4.4 8.1 ± 5.5 6.7 ± 4.6Cognitive function proximate to deathGlobal cognition score −0.85 ± 1.09 0.16 ± 0.41 −0.43 ± 0.45 −1.83 ± 0.88Episodic memory −0.84 ± 1.19 0.34 ± 0.48 −0.55 ± 0.70 −1.84 ± 0.90Semantic memory −0.70 ± 1.23 0.12 ± 0.56 −0.19 ± 0.53 −1.61 ± 1.31Working memory −0.66 ± 1.14 0.16 ± 0.77 −0.29 ± 0.79 −1.50 ± 1.01Perceptual speed −1.13 ± 1.13 −0.28 ± 0.85 −0.70 ± 0.88 −2.02 ± 0.77Visuospatial ability −0.63 ± 1.23 0.12 ± 0.62 −0.16 ± 0.78 −1.49 ± 1.29MMSE 21.3 ± 8.8 28.1 ± 1.8 25.7 ± 3.8 13.6 ± 8.2PathologicalNIA/Reagan scale, no. in 1:2:3:4b 49:135:120:4 4:25:59:2 4:44:36:2 41:66:25:0CERAD scale, no. in 1:2:3:4c 95:99:40:74 13:21:19:37 19:31:13:23 63:47:8:14Braak stage, no. in 0:I:II:III: 4:20:33:91: 2:14:16:33: 2:2:14:28: 0:4:3:30:IV:V:VI 88: 67:5 21:4:0 30:10:0 37:53:5Global AD pathology 0.69 ± 0.62 0.38 ± 0.39 0.60 ± 0.57 0.97 ± 0.66Macroinfarcts, no. (%) 108 (35 %) 20 (22 %) 29 (34 %) 59 (45 %)Microinfarcts, no. (%) 71 (23 %) 20 (22 %) 14 (16 %) 37 (28 %)Lewy body disease, no. (%) 27 (9 %) 2 (2 %) 4 (5 %) 21 (16 %)Hippocampal sclerosis, no. (%) 26 (8 %) 1 (1 %) 5 (6 %) 20 (15 %)Abbreviations: AA Afro-American, AD, Alzheimer’s disease, BL baseline, CERAD Consortium to establish a registry for AD, MCI mild cognitive impairment, MMSE minimental state examination, NA Native-American, NCI no cognitive impairment, NIA National Institute on Aging, no number of subjects, PMI postmortem interval, SDstandard deviation, W WhiteaValues are mean ± SD unless noted otherwisebNIA/Reagan scale: high (1), intermediate (2), low (3), or no (4) likelihood of AD, according to neuritic plaques and tanglescCERAD scale: definite (1), possible (2), probable (3), or no (4) AD, according to neuritic plaquesRamos-Miguel et al. Molecular Neurodegeneration  (2015) 10:65 Page 8 of 18Fig. 4 (See legend on next page.)Ramos-Miguel et al. Molecular Neurodegeneration  (2015) 10:65 Page 9 of 18terminals, may initiate synaptic dysfunction and con-sequent synaptic pathology.We first characterized biochemical, cellular andanatomical aspects of M18 splice variants. Compared toM18S, M18L showed minimal binding affinity forsyntaxin-1 (and hence for the SNARE complex) in twofunctional assays, co-IP and BN-PAGE. A previous studyshowed similar, but less pronounced, variant-specificbinding affinities to syntaxin-1 [32]. Furthermore, in ayeast two-hybrid setting, depletion of any segment of the(See figure on previous page.)Fig. 4 Associations of M18 splice variants with Alzheimer’s disease pathology, global cognition and clinical dementia. a Contour plots illustrating MAPparticipants’ (n = 308) global cognition (z-score) as a function of M18L or M18S DLPFC immunodensities, and Alzheimer’s disease (AD) pathology. OnlyAD pathology ×M18L (r =−0.251, p < 0.001), AD pathology × global cognition (r =−0.545, p < 0.001), and M18L × global cognition (r = 0.373, p < 0.001)were significantly correlated. b Regression analyses predicting global cognition as a function of AD pathology in groups of MAP participants having M18Lor M18S immunodensities above 90th (green), between 10th and 90th (blue), or below 10th percentile (red). Lines represent best-fit curvesand 95 % confident intervals. For M18L, significant correlations between AD pathology and global cognitive function were observed for participants amongthe lowest (r =−0.735, p < 0.001, n = 31) and intermediate (r =−0.512, p < 0.001, n = 243), but not the highest (r =−0.399, p = 0.074, n = 31), M18L groups. ForM18S, statistically significant correlations were seen in all three M18S ranked groups: low (r =−0.460, p = 0.036, n = 31), intermediate (r =−0.559, p < 0.001,n = 245) and high (r =−0.509, p = 0.021, n = 29). cWhiskers represent 10th and 90th percentiles of M18L or M18S values, with boxed interquartile rangescrossed by the median, in participants with no- (NCI, n = 90), or mild-cognitive impairment (MCI, n = 86), or with dementia (DEM, n = 132). One-way ANOVAdetected a significant effect only for M18L (F(2305) = 8.77, p < 0.001). *p < 0.05 and ***p < 0.001, ANOVA followed by Bonferroni’s test. d Representativeimmunoblots of M18L/S and β-actin, with various participants and standard (std) samples. Masses are indicated in kDaTable 2 Linear regression modelsa predicting global or single-domain cognitive functionModel no.and termsGlobal cognition Episodic memory Semantic memory Working memory Perceptual speed Visuospatial ability(n = 302) (n = 302) (n = 296) (n = 301) (n = 293) (n = 289)r2 or βb p-value r2 or β p-value r2 or β p-value r2 or β p-value r2 or β p-value r2 or β p-valueModel 1 0.328 0.394 0.208 0.174 0.189 0.121AD pathology −0.872 <0.001 −1.091 <0.001 −0.713 <0.001 −0.634 <0.001 −0.477 <0.001 −0.362 0.014Macroinfarcts −0.283 0.016 −0.253 0.039 −0.366 0.011 −0.470 0.001 −0.052 0.681 −0.065 0.657Lewy bodies −0.184 0.193 −0.111 0.452 −0.027 0.875 −0.102 0.533 −0.027 0.857 −0.112 0.518Hipp sclerosis −0.660 0.001 −1.034 <0.001 −0.662 0.005 −0.143 0.529 −0.687 0.001 −0.315 0.198Model 2 0.345 0.406 0.226 0.193 0.193 0.123AD pathology −0.793 <0.001 −1.018 <0.001 −0.628 <0.001 −0.549 <0.001 −0.434 0.001 −0.323 0.032Macroinfarcts −0.269 0.021 −0.240 0.048 −0.347 0.014 −0.455 0.001 −0.043 0.731 −0.058 0.693Lewy bodies −0.141 0.314 −0.071 0.630 0.021 0.900 −0.055 0.735 −0.004 0.981 −0.088 0.611Hipp sclerosis −0.658 0.001 −1.032 <0.001 −0.665 0.005 −0.141 0.530 −0.686 0.001 −0.317 0.196M18Lc 0.0010 0.003 0.0009 0.009 0.0012 0.006 0.0011 0.006 0.0005 0.141 0.0005 0.226Model 3 0.328 0.392 0.209 0.177 0.188 0.120AD pathology −0.865 <0.001 −1.090 <0.001 −0.707 <0.001 −0.624 <0.001 −0.470 <0.001 −0.357 0.016Macroinfarcts −0.290 0.014 −0.255 0.038 −0.372 0.010 −0.483 <0.001 −0.057 0.650 −0.071 0.629Lewy bodies −0.175 0.217 −0.108 0.464 −0.011 0.947 −0.086 0.598 −0.018 0.907 −0.100 0.566Hipp sclerosis −0.670 0.001 −1.037 <0.001 −0.676 0.005 −0.160 0.480 −0.696 0.001 −0.325 0.184M18Sc 0.0016 0.331 0.0004 0.794 0.0024 0.247 0.0027 0.150 0.0015 0.389 0.0018 0.383Model 4 0.343 0.405 0.224 0.191 0.190 0.120AD pathology −0.793 0.001 −1.014 <0.001 −0.629 <0.001 −0.551 <0.001 −0.436 0.001 −0.626 0.021Macroinfarcts −0.267 0.022 −0.232 0.057 −0.348 0.014 −0.460 0.001 −0.047 0.714 −0.062 0.673Lewy bodies −0.141 0.313 −0.074 0.614 0.022 0.896 −0.053 0.745 −0.002 0.990 −0.085 0.625Hipp sclerosis −0.657 0.001 −1.023 <0.001 −0.667 0.005 −0.146 0.515 −0.690 0.001 −0.321 0.189M18Lc 0.0010 0.006 0.0011 0.006 0.0011 0.013 0.0010 0.017 0.0005 0.217 0.0004 0.343M18Sc −0.0003 0.877 −0.0015 0.419 0.0003 0.890 0.0009 0.669 0.0006 0.741 0.0010 0.658Abbreviations: AD Alzheimer’s disease Hipp, hippocampal; M18L/S, Munc18-1 long/short splice variantaAll models included terms (not shown) for age, sex, education, microinfarcts, arteriolosclerosis, and atherosclerosisbValues are whole model adjusted r-squared, or individual β-coefficients for each termcStandardized M18L/S values (z-scores) were usedRamos-Miguel et al. Molecular Neurodegeneration  (2015) 10:65 Page 10 of 18M18S (M18L was not reported) amino acid sequence(including the 25 C-terminal residues) completelyabolished M18-syntaxin-1 association [49]. It is possiblethat the present assay conditions (e.g. absence of Ca2+)may induce a SNARE conformation that favors M18Srelative to M18L binding in vitro. Since binding tosyntaxin-1 is the major mechanism by which M18 exertsits functions [23, 28, 29], future studies should addressthis apparent difference in M18 variants in more detail.Given its globular, hydrophilic nature, M18 attachment tocell membranes is thought to occur through syntaxin-1interaction [50]. Despite the lower affinity for syntaxin-1,M18L seemed confined to the highly hydrophobic, lipidraft-enriched fraction. While the role of these cholesterol-enriched microdomains in regulated exocytosis is unclear,some studies proposed that specific pools of target SNAREproteins may concentrate at lipid rafts, which in turn coulddiscriminate between different kinds of neurotransmitter-containing vesicles [51]. Of note, the M18L C-terminalsequence does not display specific motifs for palmitoylation(cysteine enriched sequences), myristoylation (typicallyoccurring at a N-terminal glycine), or prenylation (typicallyin a C-terminal cysteine), major post-translational modifica-tions determining membrane localization for globularproteins. Possible explanations for M18L localization atlipid rafts could include a preferential M18L–syntaxin-1interaction in the biochemical environment of these mem-brane microdomains, or alternatively, M18L may displaygreater affinity for another raft-attached protein. Munc18-interacting proteins (Mint1/2) may be key partners in thisprocess, as they bear two membrane-attaching PDZdomains in addition to a M18-binding sequence [52].Remarkably, Mint1/2 can regulate M18-syntaxin-1 inter-action and were found also altered in Alzheimer's diseasedpostmortem brains [46]. Additionally, the presence of aputative phosphoserine site for Ca2+-calmodulin kinase II(CaMKII) activity in the C-terminal tail of M18L, but notM18S, may indicate a differential regulatory mechanism forthe M18 variants [39]. This CaMKII activity was presumedto be responsible for the enhanced depression of synchron-ous vesicle release in M18 null pyramidal neurons rescuedwith M18L, compared to those carrying M18S only.Immunohistochemical assays were consistent with tissuefractionation experiments. In contrast to the wide distribu-tion of M18S across subcellular compartments, M18L wasrestricted to nerve endings and enriched in perisomaticareas. The M18S subcellular and histological distributionwas similar to that previously reported for M18 [32, 53],and no cell-type specificity was presumed. The distributionof M18L was consistent with the localization expected forGABAergic presynaptic proteins [54]. Indeed, M18L closelymatched VGAT distribution in all brain areas analyzed,especially in the hippocampus, where it was abundantlyexpressed in the perisomatic area of pyramidal cells. Inter-estingly, a previous in situ hybridization showed greatestexpression of M18L in cells within the granule and pyram-idal layers of mouse hippocampus [35]. Combined, theseexperiments indicate that M18L protein is mainly presentin the presynaptic terminals of GABAergic interneuronswithin these hippocampal layers, although we cannotexclude some residual M18L expression in glutamatergic orother neuromodulatory terminals. This finding suggests aqualitative difference between the neurosecretory mecha-nisms of excitatory and inhibitory neurons. Several epilepticsyndromes have been associated with firing abnormalitiesof dentate gyrus granule cells, likely due to an inappropriateinhibitory tone [55, 56]. This model is consistent withthe mutations in M18 related to Ohtahara syndrome[41, 45], a disease accompanied by seizures and severeintellectual disability.We found marked reductions of M18L, but not M18S,in DLPFC of MAP participants displaying clinicalTable 3 Logistic regression modelsa predicting likelihood of clinical dementia per unit of termModel terms Model 1 Model 2 Model 3 Model 4Oddsratio95 % CI p-value Oddsratio95 % CI p-value Oddsratio95 % CI p-value Oddsratio95 % CI p-valueAD pathology 3.15 1.80–5.73 <0.001 2.84 1.60–5.22 <0.001 3.16 1.81–5.75 <0.001 2.83 1.59–5.21 <0.001Macroinfarcts 2.16 1.20–3.94 0.010 2.06 1.14–3.79 0.017 2.20 1.22–4.02 0.009 2.05 1.13–3.78 0.018Microinfarcts 0.73 0.39–1.36 0.326 0.77 0.41–1.43 0.412 0.75 0.40–1.40 0.370 0.77 0.41–1.43 0.409Arteriolosclerosis 1.30 0.95–1.78 0.098 1.34 0.98–1.84 0.068 1.32 0.97–1.82 0.078 1.33 0.98–1.84 0.071Atherosclerosis 1.36 1.02–1.82 0.035 1.36 1.02–1.84 0.038 1.34 1.00–1.80 0.046 1.36 1.02–1.84 0.038Lewy bodies 4.92 1.79–15.40 0.002 4.63 1.67–14.70 0.003 4.93 1.79–15.51 0.002 4.62 1.66–14.66 0.003Hipp sclerosis 4.39 1.72–12.16 0.002 4.41 1.70–12.39 0.002 4.59 1.78–12.80 0.001 4.38 1.68–12.34 0.002M18Lb 0.68 0.50–0.90 0.008 0.67 0.47–0.93 0.018M18Sb 0.84 0.64–1.10 0.208 1.03 0.75–1.43 0.871Abbreviations: AD Alzheimer’s disease, CI confidence interval Hipp, hippocampal, M18L/S Munc18-1 long/short splice variantaAll models were controlled for age, sex and educationbStandardized M18L/S values (z-scores) were usedRamos-Miguel et al. Molecular Neurodegeneration  (2015) 10:65 Page 11 of 18dementia and/or a large burden of Alzheimer’s diseasepathology. Pairwise correlations between M18L levels,the burden of Alzheimer’s disease pathology, and cogni-tive function scores were all significant. Accordingly,participants having high M18L cortical density displayedgreater cognitive resistance against the accumulation ofAlzheimer’s disease pathology, which may directly im-plicate M18L in the molecular mechanisms of cogni-tive reserve. Statistical modeling indicated that loss ofM18L contributed moderately to a higher likelihood ofdementia and to cognitive impairment. Additionally,including synaptophysin as a covariate did not alterthe results, suggesting that loss of M18L is an earlyevent in synaptic pathology. The importance of main-taining appropriate splicing balance between M18L/Sisoforms is probably best represented by the dramaticendophenotypes observed in murine strains eitherlacking or overexpressing the M18L variant [34, 35].While specific regulatory splicing mechanisms of theSTXBP1 transcript are unclear, other splicing deficitshave been associated with aging related neuropathol-ogies and dementia [57].The predominant expression of M18L in GABAergicterminals suggests that synaptic deficits may be initi-ated in inhibitory, rather than excitatory synapses. Thiscould be related to the surprising relative upregulationof glutamatergic terminals observed in MCI [58]. Indeed,there is compelling evidence involving GABAergic systemin early stages of dementia [59, 60]. By contrast, previousobservations in transgenic mouse models suggested thatcholinergic and glutamatergic terminals may collapse firstas a result of APP-mediated synaptic damage [61]. In thepresent study, APP23 mice did not display reduced levelsof M18L/S, which agrees with previous findings [46].Thus, cortical loss of M18L may be an independent eventfrom amyloid-β accumulation, and perhaps other com-mon age-related neuropathologies, and could represent adistinct contribution to cognitive impairment, possiblythrough erosion of cognitive reserve.ConclusionsThe present study identified the M18L isoform as a rela-tively specific component of the GABAergic presynapticmachinery. In contrast to the alternatively spliced M18S,which is highly abundant and ubiquitously expressed ininhibitory and excitatory neurons, M18L appears restrictedto lipid raft microdomains within the presynaptic compart-ment, and may have a function independent from its inter-action with syntaxin-1. Importantly, M18L, but not M18S,is associated with cognitive function in the elderly, and maycontribute to the mechanisms of cognitive reserve that pro-tect against dementia related to age-associated pathologies.MethodsParticipants, cognitive evaluations and neuropathologicalassessmentsMAP recruits volunteers without known dementia,living in Chicago (IL, USA) [7]. Since 1997, this studyenrolled over 1750 community-dwelling participants.At enrollment, all participants signed an informedconsent and an Anatomic Gift Act for organ donation upondeath. All protocols were approved by the InstitutionalReview Board of Rush University Medical Center. Theoverall follow-up rate is 95 % and the autopsy rate exceeds80 %. Samples obtained in a consecutive series of autopsiesFig. 5 M18 splice variants are not altered in APP23 mice. aImmunodensities of M18L/S splice variants were quantified byWestern blotting in brain homogenates (frontal cortex) fromadult (12-month-old; T12) and aged (22-month-old; T22) wildtype (WT) and APP23 transgenic mice. Columns are immunodensitymean values ± standard error (normalized by β-actin) of n = 6 miceper group, and represented in percentage to control (T12–WT) animals.Two-way ANOVA only detected a significant effect for genotype(but not age) on M18S (F(1,20) = 4.51, p = 0.0462), and a borderlineage (but not genotype) effect on M18L (F(1,20) = 3.44, p = 0.0784), withoutspecific between-group differences in the following Dunnett’s post hoctests. b Representative immunoblots of M18L, M18S and β-actin, withone sample per group. Masses (in kDa) of proximal prestained markersare indicated on the left side of immunoblotsRamos-Miguel et al. Molecular Neurodegeneration  (2015) 10:65 Page 12 of 18of n = 308 participants were included in the present study.A summary of demographic, cognitive and pathologicalcharacteristics is listed in Table 1.Methodological approaches to systematic cognitive,clinical and neuropathological evaluations were extensivelyreported [7, 62]. Annual cognitive evaluations comprised abattery of 21 standard tests, 19 of which are used tosummarize one of the following domains: episodic memory,semantic memory, working memory, perceptual speed orvisuospatial ability. For the present work, last valid cogni-tive tests were used in all analyses. The 19 tests could alsobe combined into a single variable, based on an averagez-score to summarize global cognitive function nearestto death [7, 63]. The final clinical diagnoses of dementiafollowed the National Institute of Neurological and Com-municative Disorders and Stroke and the Alzheimer’sDisease and Related Disorders Association criteria [64], andwere made by a board-certified neurologist blind to allpathological data.Neuropathological examinations documented Alzheimer’sdisease-related pathology (neuritic and diffuse plaques, andneurofibrillary tangles), vascular diseases (macroscopic andmicroscopic infarcts, arteriolosclerosis and atheroscler-osis), Lewy bodies, and hippocampal sclerosis [7, 62]. Aboard-certified neuropathologist made all diagnosesblind to all clinical data. A composite measure of globalAlzheimer’s disease pathology was created using a stan-dardized average of neuritic and diffuse plaques andneurofibrillary tangles [62]. The burden of Alzheimer’sdisease pathology was also categorized using Braak,CERAD and NIA/Reagan scales.AnimalsApproval from the UBC’s Animal Care Committee wasobtained prior to experiments involving laboratoryanimals. Adult Sprague–Dawley rats were supplied byCharles-River (Montreal, QC, Canada). Novartis Pharma(Basel, Switzerland) provided APP23 transgenic mice, over-expressing a variant of human APP carrying the ‘Swedishdouble mutation’ KM670/671NL [48]. Pentobarbital-anaesthetized adult rats, as well as 12- or 22-month oldAPP23 mice and wild-type (WT) littermates, were killedby decapitation. Hemispheres were separated, and usedfor electrophoretic or immunohistochemical assays.Purification of synaptosomesCortical synaptosomes were obtained following standardprocedures (see cartoon in Fig. 1d), similar to thoseoriginally developed by Grey and Whittaker in the1960’s [65]. All sucrose solutions described below wereHEPES-buffered (4 mM, pH 7.4), supplemented with1 % of a protease inhibitor cocktail (Sigma, St. Louis,MO, USA), and pre-chilled at 4 °C. Approximately 1 gof human inferior temporal cortex was homogenized in10 ml of 0.32 M sucrose buffer, using a motorized 20-mlPotter-Elvehjem tissue grinder, with a clearance of 0.13–0.18 mm between the Teflon pestle and the glass chamber.The following centrifugal separations were all per-formed at 4 °C in an Avanti J-30I high performancecentrifuge (Beckman Coulter, Fullerton, CA, USA),equipped with a JA-30.50 fixed angle rotor. Tissuedebris, along with cell nuclei, were removed at 1000 ×g for 1 min (P1). Supernatants (S1) were centrifuged at17,000 × g for 15 min, and the resulting pellets (P2)washed in 0.32 M sucrose and re-centrifuged. P2 fractionswere resuspended in 0.32 M sucrose and layered onto asucrose discontinuous gradient, with 1.2 (bottom), 0.8 M(middle), and 0.32 M (topping) layers. After a 12-hcentrifugation at 96,000 × g, the interfaces between 0.8and 1.2 M (IF1, containing the crude synaptosomalfraction), and 0.32–0.8 M (IF2, mainly myelin-coatedfragments) were collected. IF1 was again overlaid on0.32 M sucrose buffer and centrifuged at 30,000 × gfor 20 min. The resulting pellet, containing purifiedsynaptosomes, was resuspended in 1 ml ofhomogenization buffer supplemented with 0.5 %TritonX. In all steps, aliquots of each fraction wereseparated for further SDS-PAGE and immunoblottinganalyses.Extraction of lipid raftsPurification of lipid raft-enriched fraction was based onthe TritonX-insoluble, SDS-soluble property of thesemembrane microdomains. The present method, initiallydesigned for separating cytosolic from insoluble, raft-associated α-synuclein [66], offers a simple and quickassay to yield a fraction (S4) of TritonX-insoluble pro-teins highly enriched in lipid rafts through sequentialcentrifugation steps [67]. Figure 1f in the main textillustrates the procedure. Briefly, ~150 mg of corticaltissue were homogenized in 1.5 mL of ice-cold 10 mMTris-buffer, pH 6.8, containing 1 % of a protease inhibitorcocktail (Sigma). Total homogenate (TH) was centrifugedat 31,000 × g for 1 h at 4 °C. Supernatant was collected (S1),and the pellet (P1) was resuspended in 1 ml of the sameice-cold Tris-buffered solution supplemented with 0.5 %TritonX. To achieve a homogenous solution, P1 was gentlysonicated (F60 Sonic Dismembrator, Fisher Scientific, Wal-tham, MA, USA). The same centrifugation step was per-formed and the supernatant (S2) was kept. The pellet (P2)was again resuspended and sonicated in Tris-buffer con-taining 2 % TritonX, and the centrifugation step wasrepeated. The subsequent supernatant (S3) was separatedand the pellet (P3) homogenized as above in ice-coldTris-buffer containing 0.5 % SDS. P3 was incubated at15 °C with rotation for 10 min before a final centrifuga-tion at 31,000 × g and at 12 °C was performed for 1 h.The supernatant (S4), enriched in lipid-raft proteins,Ramos-Miguel et al. Molecular Neurodegeneration  (2015) 10:65 Page 13 of 18was collected, and the final pellet (P4) resuspended asabove. All fractions were stored at−80 °C until resolvedby SDS-PAGE and immunoblotting.AntibodiesA list of primary antibodies used appears in Additionalfile 2: Table S1. Commercial antibodies selectivelytargeting M18L and M18S variants were raised againsttheir respective C-terminal amino acidic sequences(Fig. 1a), which share 100 % homology across humanand rodent orthologues. The isoform-selectivity ofthese antibodies was previously reported [36], andfurther characterized by co-immunoprecipitation.Production and characterization of mouse monoclonalantibodies against syntaxin-1, SNAP-25, VAMP andsynaptophysin was described elsewhere [68, 69]. Peroxid-ase- and Alexa-Fluor 488/555/647-conjugated secondaryantibodies were from Jackson ImmunoResearch Laborator-ies (West Grove, PA, USA) or Molecular Probes (Eugene,OR, USA), respectively.ImmunoprecipitationTarget proteins were immunoprecipitated using protein G-coated magnetic Dynabeads (Life Technologies, Carlsbad,CA, USA), as reported [38]. In each reaction, 50 μg ofbeads were incubated in phosphate-buffered saline (PBS),supplemented with 0.1 % TritonX, and containing 0.33 μgof anti-mouse IgG (negative control), or antibodies againsteither M18L or M18S splice variants. Nonspecific bindingsites were blocked in the same buffer supplemented with3 % bovine serum albumin (BSA). In parallel, humancortical samples were ground homogenized in PBS contain-ing 1 % TritonX, and 1 % of protease inhibitors (Sigma),and solubilized for 1 h at 4 °C. Prior to IP reactions, solubi-lized brain proteins were pre-cleared with antibody-freebeads. Antibody-conjugated magnetic beads were com-bined with excess (2 mg) of pre-cleared brain proteins, andincubated overnight at 4 °C. After washing, IP productswere eluted in 25 μl of 20 mM tricine, pH 2.7 to preservenative protein structures and interactions.Quantitative immunoblottingGrey matter samples from the middle-frontal gyrus(Brodmann’s area 46/9) of the DLPFC were obtained atautopsies of MAP participants, following a standard atlas[70]. The DLPFC was selected for its central role incomplex cognitive tasks and contribution to age-relatedcognitive decline [71]. Samples (40–80 mg) were ground-homogenized using a Teflon pestle in ice-cold PBS,pH 7.4, and stored at−80 °C until use [13]. Prior to immu-noassays, protein concentrations were determined by DCassay (Bio-Rad, Hercules, CA, USA), and samples adjustedto equal concentrations with homogenization buffer.Quantification and characterization of M18L/S vari-ants and β-actin in MAP cortical samples was achievedby SDS-PAGE, using 10 % or 12 % minigels (Bio-Rad),followed by immunoblotting, as previously reported [38].Quantification of other presynaptic proteins was previ-ously reported [13]. Total brain homogenates (and alsosubcellular fractions or IP products) were combinedwith equal volumes of 2× loading buffer (100 mM Tris,pH 6.8, 4 % SDS, 0.2 % bromophenol blue, 20 % glycerol,200 mM β-mercaptoethanol). All samples were boiledfor 5 min prior to electrophoretical separations. Prelim-inary analyses determined that 10-μg protein aliquots oftotal brain homogenates from DLPFC were optimal fordensitometric quantifications [36]. The standard sample(pool of n = 132 MAP participants) was spaced-loaded intriplicate in all gels to control for correct loading andprotein transfer to the membranes (see Fig. 4d), and alsoto account for between-gel variability. For quality con-trol, immunoblots were rejected when the coefficient ofvariation (CV) of in-gel standard samples exceeded10 %, although typical CV < 5 % values were obtained.For the analysis of brain fractions, equivolumetricamounts were loaded onto the SDS-gels, while 5 μl of IPproducts were found optimal. All brain samples wereresolved in 10–12 % polyacrylamide minigels (Bio-Rad,Hercules, CA, USA). After electrophoresis, proteinswere transferred to polyvinylidene difluoride (PVDF)membranes, and subsequently blocked (1 h), and incu-bated with primary (overnight, 4 °C; see Additional file2: Table S1) and secondary (1 h; 1:5000) antibodies, inTBS containing 5 % milk and 0.1 % Tween-20. Chemilu-minescence was enhanced with commercial reagents(Perkin Elmer, Waltham, MA, USA), and images weredigitized using a LAS-3000 Image Reader (Fujifilm,Tokyo, Japan).In quantitative studies, membrane stripping and reprob-ing with anti-β-actin antibody was done for housekeepingand data normalization purposes. Densitometric analyses ofthe immunoblots were done with ImageGauge software,version 4.22 (Fujifilm). Every gel was run with 14 brainsamples (including 11 MAP participants randomly selected,and the triplicate standard sample), and a molecular weightladder (Bio-Rad). Gels containing the same subset ofsamples were assessed at least twice in different days. Forquality control, a minimal between-gel Pearson’s correlationcoefficient of r = 0.80 was required for M18L/S and β-actinimmunoreactivity values. Gels not meeting this criterionwere discarded and repeated. For each sample, immunore-activity of M18L/S (in arbitrary optical density units) wasfirst divided by that of β-actin within the same gel, and thencalculated as a percentage of in-gel standards. Mean valuebetween the two different, valid gels was used as a final es-timate. This procedures were reported to reduce the ex-perimental variability in studies that used immunoblottingRamos-Miguel et al. Molecular Neurodegeneration  (2015) 10:65 Page 14 of 18as a quantitative technique for large cohorts of samples[36, 38].BN-PAGEOriginally developed to study mitochondrial mem-brane complexes [72], separation of solubilized brainproteins by BN-PAGE was recently shown suitable toidentify and quantify presynaptic complexes in humanpostmortem tissue [38]. Brain samples were combinedwith equal volumes of ice-cold 100 mM Bis-tris,pH 7.0, solubilization buffer, containing 50 mM NaCl,2 mM EDTA, 4 mM 6-aminohexanoic acid, 1 %TritonX, and 1 % protease inhibitor cocktail, and incu-bated for 1 h at 4 °C with gentle rotation. After centri-fugation (16,000 × g, 30 min, 4 °C), supernatants or IPproducts were combined with equal volumes of a loadingbuffer (0.5 % TritonX, 0.25 % Coomassie brilliant blue G-250, 10 % glycerol). Samples were loaded in 4–16 % gradi-ent NativePAGE precast gels (Novex, Carlsbad, CA, USA).Electrophoresis was run under constant voltage (150 V), at4 °C, with pre-chilled anode (50 mM Bis-tris, pH 7.0) andcathode (15 mM Bis-tris, 50 mM tricine, pH 7.0, 0.02 %Coomassie dye) buffers. Before transferring proteins andcomplexes to PVDF membranes, BN-gels were incubatedan ice-cold 12 mM Tris, 96 mM glycine buffer, pH 8.3,containing 0.1 % SDS. To remove excess of Coomassie dye,membranes were rinsed in 100 % methanol, followed byimmediate rehydration in TBS for 15 min. Followingimmunoblotting procedures were as described for SDS-PAGE. Molecular markers (NativeMARK, Novex, range20–1240 kDa) were loaded in all gels to estimate proteincomplex sizes.Immunohistochemistry and immunofluorescenceForty-μm floating coronal sections (−3.30 to−4.20 rela-tive to bregma) were obtained from paraformaldehyde-fixed rat brain hemispheres [73]. Other floating coronalsections were from the mid-hippocampus of selectedcases with or without dementia [74]. Retrieval of theM18L epitope required 20-min incubation at 95 °C in20 mM Tris buffer (pH 9.0), containing 1 mM EDTAand 0.05 % Tween-20, possibly because of the majorlocalization in TritonX-insoluble subcellular compart-ments (see Results). Other antigen retrievals were donein 20 mM citrate buffer (pH 6.0) at 80 °C for 15 min. Forprocedures involving 3,3′–diaminobenzidine staining, weused commercially available kits (Vector Labs, Burlingame,CA, USA), with incubations as reported [75]. Images wereacquired with an Olympus BX61 microscope (Olympus,Tokyo, Japan). Immunofluorescence assays were performedas described [74, 76]. A series of orthogonal images wereobtained using a LSM 5 Pascal confocal microscope (Zeiss;Jena, Germany). Co-staining of M18L/S isoforms withVGAT and VGLUT1 was assessed to determinepresence in inhibitory or excitatory presynaptic termi-nals, respectively. Appropriate negative controls wereincluded in all experiments.Statistical analysesIn quantitative immunoblotting assays, M18L/S immu-nodensities were first normalized to corresponding β-actin values, and calculated as a percentage of in-gelstandards [36, 38]. Linear models were constructed withlog-transformed (M18L) and/or standardized (M18L andM18S) values (z-scores). Data transformations did notalter the observed differences between groups.Multivariate analyses were performed to survey puta-tive associations between M18 isoforms and cognitive,pathological, neurochemical and/or putative confound-ing variables. An additional exploratory analysis wasperformed by ranking participants according to theirM18L/S cortical immunodensities and comparingpathology-cognition decay curves across subjects withhigh (above the 90th percentile) and low (below 10thpercentile) M18L/S values. Given the potential effect ofcortical M18L levels on cognitive performance, wegenerated multiple linear regression models with cogni-tive measures as outcomes and pathological and neuro-chemical variables as predictors. Additionally, using thesame predictors, we built logistic regression modelswith clinical diagnosis of dementia as the outcome.Linear and logistic models were controlled for sex, ageand education. Differences in M18 immunodensitiesbetween clinically diagnosed or pathologically gradedgroups were assessed by ANOVA followed by Bonferroni’spost hoc test for standardized data, or Kruskal-Wallisfollowed by Dunn’s post hoc tests for non-transformedvalues. Experiments involving APP23 mice were analyzedwith two-way ANOVA, with genotype and age as inde-pendent factors, followed by Dunnet’s post hoc test.For colocalization analyses of confocal imaging, weused an ImageJ 2.0 (NIH, Bethesda, MA, USA) built-inmethod [76, 77]. Comparisons of M18L/S colocalizationsacross inhibitory versus excitatory compartments wereperformed with paired t-tests.All tests were two-tailed, and p-values < 0.05 wereconsidered significant. Datasets were analyzed and plot-ted with JMP 10.0.2 (SAS Institute, Cary, NC, USA),and/or GraphPad Prism 6.0 (GraphPad Software, LaJolla, CA, USA).Additional filesAdditional file 1: Figure S1. Immunohistochemical characterization ofMunc18-1 splice variants in human dentate gyrus reveals similar cellularand subcellular distributions of M18L and M18S than those in rat brain.Confocal images show a preferential localization of M18L to inhibitorypresynaptic terminals, as its immunofluorescence fully overlaps with thatRamos-Miguel et al. Molecular Neurodegeneration  (2015) 10:65 Page 15 of 18of VGAT, but not VGLUT1. M18S shows ubiquitous distribution. Figure S2.M18L, but not M18S, immunodensity is reduced in the DLPFC of MAPparticipants with clinical dementia, compared to those with no or mildcognitive impairment, as well as in those presenting high burden ofAlzheimer’s disease pathology, using either NIA/Reagan or Braak scales.(PDF 986 kb)Additional file 2: Table S1. List of both commercially available andlocally produced primary antibodies used in the present study.References cited in Table S1 are listed below the table. (PDF 107 kb)AbbreviationsANOVA: Analysis of variance; ANCOVA: Analysis of covariance; APP: Amyloidprecursor protein; BN-PAGE: Blue native-PAGE; BSA: Bovine serum albumin;CA: Ammon’s horn; CERAD: Consortium to Establish a Registry forAlzheimer’s Disease; CV: Coefficient of variation; DC: Detergent compatible;DG: Dentate gyrus; DLPFC: Dorsolateral prefrontal cortex; ECL: Enhancedchemiluminescence; EDTA: Ethylenediaminetetraacetic acid; ELISA: Enzyme-linked immunoadsorbant assay; GABA: Gamma-aminobutyric acid;GCL: Granule cell layer; IP: Immunoprecipitation; M18: Munc18-1; M18L/S: M18 long/short splice variant; MAP: Memory and aging project;MCI: Mild cognitive impairment; NCI: No cognitive impairment;PAGE: Polyacrylamide gel electrophoresis; PBS: Phosphate-buffered saline;PMI: Postmortem interval; PPIs: Protein-protein interactions; PVDF: Polyvinylidenedifluoride; SDS: Sodium dodecyl sulfate; SNAP-25: Synaptosome-associated proteinof 25 kDa; SNARE: soluble N-ethylmaleimide-sensitive factor attachment proteinreceptor; STXBP1: Syntaxin-binding protein-1; TBS: TRIS-buffered saline;TritonX: Triton X-100; VAMP: Vesicle-associated membrane protein; VGAT: VesicleGABA transporter; VGLUT1: Vesicle glutamate transporter-1; WT: Wild-type.Competing interestsWGH has received consulting fees or sat on paid advisory boards for: In Silico,Lundbeck/Otsuka, Eli Lilly, and Roche. AMB is on the advisory board or receivedconsulting fees from Roche Canada, and received educational grant supportfrom BMS Canada. The Organizations cited above had no role in (and thereforedid not influence) the design of the present study, the interpretation of results,and/or preparation of the manuscript. All other authors have no financialinterest on the reported data and declare that no competing interests exist.Authors’ contributionsWGH, DAB, AR-M and AMB, designed the study. AR-M performed allcharacterization and quantification experiments of M18L/S splicevariants in human and rat brain tissues. CH and CLB participated inthe immunohistochemical and immunofluorescence assays in rat and humanbrain sections. TAB and PF contributed to the APP23 mice study. DAB and JASconceived the Memory and Aging Project, performed all clinical and pathologicalexams, and procured human tissue samples. SEL compiled all participants’demographic, clinical and pathological data and, with the collaboration of AR-Mand WGH, run all statistical analyses. AR-M, WGH and DAB wrote the first draft ofthe manuscript. All authors critically contributed to the discussion of the resultsand approved the final version of the manuscript.Authors’ informationAR-M is a Post-Doctoral Fellow of the BC Schizophrenia Society Foundationand the Michael Smith Foundation for Health Research.AcknowledgementsWe wish to express our gratitude to all participants in MAP, and to the staffin Rush Alzheimer's Disease Center. We also thank Hong-Ying Li and JennyYang for their skillful technical assistance. The study was supported by theCanadian Institutes of Health Research (MT-14037, MOP-81112), the MichaelSmith Foundation for Health Research (Grant #5401), and the Jack Bell Chairin Schizophrenia. The Memory and Aging Project is a collaborative, multidis-ciplinary and translational research project subsidized by the National Insti-tute on Aging (Grants R01AG17917, R01AG42210).Author details1Child and Family Research Institute, 938 West 28th Avenue, Vancouver, BCV5Z 4H4, Canada. 2Department of Psychiatry, University of British Columbia,2255 Wesbrook Mall, Vancouver, BC V6T 2A1, Canada. 3Department ofAnesthesiology, Pharmacology and Therapeutics, University of BritishColumbia, 2176 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada.4Department of Psychiatry, University Medicine Goettingen,von-Siebold-Strasse 5, D-37075 Goettingen, Germany. 5Department ofPsychiatry and Psychotherapy, Ludwig-Maximilians-University Munich,Nussbaumstrasse 7, D-80336 Munich, Germany. 6Rush Alzheimer’s DiseaseCenter, Rush University Medical Center, 600S. Paulina StreetIL 60612 Chicago,USA.Received: 28 July 2015 Accepted: 24 November 2015References1. Satz P. 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