Detecting Monoamine Oxidase A and B Proteins: A Western Blotting Protocol and Some Practical Considerations.

The expression of the two isoforms of monoamine oxidase (MAO A and MAO B) is often inferred from proxy measures such as mRNA transcript levels or catalytic activity. Yet the literature is clear that the proportionality of protein, mRNA, and activity does not guarantee that any of these measures can be used as a proxy for any of the others. Here we provide a protocol for the detection of MAO proteins in cell lysates that can be adapted readily to tissue preparations. Given that MAOs influence many physiological and pathological processes, we feel it is essential to include measures of protein expression when exploring genetic regulation or catalytic properties of these important enzymes.

An (Immuno) Fluorescence Protocol for Monitoring Monoamine Oxidase A/B Protein Distribution Within the Cell.

The influence of a protein is not determined exclusively by its level of expression, but also by its localization within the cell. The literature often refers to the enzyme monoamine oxidase (MAO) as a mitochondrial enzyme, yet there is evidence that mitochondria-independent pools of MAO exist. These pools of MAO could exert distinct influences across physiological as well as pathological phenotypes. Fluorescence microscopy is a powerful tool for spatially resolving target proteins in cell and tissue preparations. This can rely on an antibody-based probe that targets the endogenous protein, e.g., immunofluorescence. In the event that antibodies might not be readily available or if one is interested in characterizing a variant of the wild-type protein, then a recombinant protein with a fluorescent fusion "tag" is preferred. We now describe a protocol for the detection of endogenous MAO using indirect immunofluorescence and a version of the protocol with minor modification for detecting (green) fluorescent protein-tagged MAOs. One observation we can highlight using these easily adaptable approaches is that MAO A and MAO B do not follow similar patterns of distribution throughout the cell, suggesting potential expression of MAO A and MAO B on distinct pools of mitochondria. Furthermore, distinct subcellular compartmentalization is suggested by the fact that a pool of MAO A, but not MAO B, is associated with certain lysosomal compartments. However, directed and quantitative studies will be required before any definitive statement can be made on these intriguing possibilities.

The Aß(1-38) peptide is a negative regulator of the Aß(1-42) peptide implicated in Alzheimer disease progression.

The pool of ß-Amyloid (Aß) length variants detected in preclinical and clinical Alzheimer disease (AD) samples suggests a diversity of roles for Aß peptides. We examined how a naturally occurring variant, e.g. Aß(1-38), interacts with the AD-related variant, Aß(1-42), and the predominant physiological variant, Aß(1-40). Atomic force microscopy, Thioflavin T fluorescence, circular dichroism, dynamic light scattering, and surface plasmon resonance reveal that Aß(1-38) interacts differently with Aß(1-40) and Aß(1-42) and, in general, Aß(1-38) interferes with the conversion of Aß(1-42) to a ß-sheet-rich aggregate. Functionally, Aß(1-38) reverses the negative impact of Aß(1-42) on long-term potentiation in acute hippocampal slices and on membrane conductance in primary neurons, and mitigates an Aß(1-42) phenotype in Caenorhabditis elegans. Aß(1-38) also reverses any loss of MTT conversion induced by Aß(1-40) and Aß(1-42) in HT-22 hippocampal neurons and APOE ε4-positive human fibroblasts, although the combination of Aß(1-38) and Aß(1-42) inhibits MTT conversion in APOE ε4-negative fibroblasts. A greater ratio of soluble Aß(1-42)/Aß(1-38) [and Aß(1-42)/Aß(1-40)] in autopsied brain extracts correlates with an earlier age-at-death in males (but not females) with a diagnosis of AD. These results suggest that Aß(1-38) is capable of physically counteracting, potentially in a sex-dependent manner, the neuropathological effects of the AD-relevant Aß(1-42).

Age- and sex-dependent profiles of APP fragments and key secretases align with changes in despair-like behavior and cognition in young APPSwe/Ind mice.

Biological sex exerts distinct influences on brain levels of the ß-amyloid (Aß) peptide in both clinical depression and Alzheimer disease (AD), yet studies in animal models focus primarily on males. We examined behavioral 'despair'/depression (using the tail-suspension test) and memory (using the novel object recognition task) in J20 (hAPPSwe/Ind) mice. Three month-old male (but not female) J20 mice exhibited less despair-like behavior, but more evidence of cognitive deficits. In young J20 mice, only soluble Aß peptides -primarily Aß(1-40)- were detected. There was no evidence of an effect on despair-like behavior in the six month-old J20 mice, although cognitive deficits were now evident in both sexes, and coincided with a greater proportion of the neurotoxic Aß(1-42) species (in soluble as well as insoluble fractions). This age-dependent shift in Aß peptide profile coincided with reduced expression of glycosylated species of ADAM-10 (α-secretase) and BACE1 (ß-secretase), and an increased co-immunoprecipitation of presenilin-1 with nicastrin (components of the γ-secretase complex). Sex-dependent changes in depression-related monoaminergic, e.g. serotonin and dopamine (but not noradrenaline), systems were evident already in young J20 mice. It is critical to acknowledge that sex-dependent APP-related phenotypes might differentially influence modifiable depression-related monoaminergic signalling at some of the earliest pathological stages of clinical AD.

Glycosylation States of Pre- and Post-synaptic Markers of 5-HT Neurons Differ With Sex and 5-HTTLPR Genotype in Cortical Autopsy Samples.

The serotonin (5-hydroxytryptamine, 5-HT) transporter (5-HTT) gene-linked polymorphic region (5-HTTLPR) is thought to alter 5-HT signaling and contribute to behavioral and cognitive phenotypes in depression as well as Alzheimer disease (AD). We explored how well the short (S) and long (L) alleles of the 5-HTTLPR align with serotoninergic indices in 60 autopsied cortical samples from early-onset AD/EOAD and late-onset AD/LOAD donors, and age- and sex-matched controls. Stratifying data by either diagnosis-by-genotype or by sex-by-genotype revealed that the donor's 5-HTTLPR genotype, i.e., L/L, S/L, or S/S, did not affect 5-HTT mRNA or protein expression. However, the glycosylation of 5-HTT was significantly higher in control female (vs. male) samples and tended to decrease in female EOAD/LOAD samples, but remained unaltered in male LOAD samples. Glycosylated forms of the vesicular monoamine transporter (VMAT2) were lower in both male and female AD samples, while a sex-by-genotype stratification revealed a loss of VMAT2 glycosylation specifically in females with an L/L genotype. VMAT2 and 5-HTT glycosylation were correlated in male samples and inversely correlated in female samples in both stratification models. The S/S genotype aligned with lower levels of 5-HT turnover in females (but not males) and with an increased glycosylation of the post-synaptic 5-HT2C receptor. Interestingly, the changes in presynaptic glycosylation were evident primarily in female carriers of the APOE ε4 risk factor for AD. Our data do not support an association between 5-HTTLPR genotype and 5-HTT expression, but they do reveal a non-canonical association of 5-HTTLPR genotype with sex-dependent glycosylation changes in pre- and post-synaptic markers of serotoninergic neurons. These patterns of change suggest adaptive responses in 5-HT signaling and could certainly be contributing to the female prevalence in risk for either depression or AD.

Alzheimer Disease and Selected Risk Factors Disrupt a Co-regulation of Monoamine Oxidase-A/B in the Hippocampus, but Not in the Cortex.

Monoamine oxidase-A (MAO-A) and MAO-B have both been implicated in the pathology of Alzheimer disease (AD). We examined 60 autopsied control and AD donor brain samples to determine how well MAO function aligned with two major risk factors for AD, namely sex and APOE ε4 status. MAO-A activity was increased in AD cortical, but not hippocampal, samples. In contrast, MAO-B activity was increased in both regions (with a strong input from female donors) whether sample means were compared based on: (a) diagnosis alone; (b) diagnosis-by-APOE ε4 status (i.e., carriers vs. non-carriers of the ε4 allele); or (c) APOE ε4 status alone (i.e., ignoring 'diagnosis' as a variable). Sample means strictly based on the donor's sex did not reveal any difference in either MAO-A or MAO-B activity. Unexpectedly, we found that cortical MAO-A and MAO-B activities were highly correlated in both males and females (if focussing strictly on the donor's sex), while in the hippocampus, any correlation was lost in female samples. Stratifying for sex-by-APOE ε4 status revealed a strong correlation between cortical MAO-A and MAO-B activities in both non-carriers and carriers of the allele, but any correlation in hippocampal samples was lost in carriers of the allele. A diagnosis of AD disrupted the correlation between MAO-A and MAO-B activities in the hippocampus, but not the cortex. We observed a novel region-dependent co-regulation of MAO-A and MAO-B mRNAs (but not proteins), while a lack of correlation between MAO activities and the respective proteins corroborated previous reports. Overexpression of human APOE4 increased MAO activity (but not mRNA/protein) in C6 and in HT-22 cell cultures. We identified a novel co-regulation of MAO-A and MAO-B activities that is spared from any influence of risk factors for AD or AD itself in the cortex, but vulnerable to these same factors in the hippocampus. Sex- and region-dependent abilities to buffer influences on brain MAO activities could have significant bearing on ambiguous outcomes when monoaminergic systems are targeted in clinical populations.

Profiles of ß-Amyloid Peptides and Key Secretases in Brain Autopsy Samples Differ with Sex and APOE ε4 Status: Impact for Risk and Progression of Alzheimer Disease.

The APOE ε4 allele was originally reported to contribute to risk of Alzheimer's disease (AD) in women, yet male and female AD patient-derived data are routinely pooled. Histopathological hallmarks of AD include neurofibrillary tangles centered on hyperphosphorylated Tau and plaques composed of the ß-amyloid (Aß) peptide that is derived by sequential secretase-mediated cleavage of the Amyloid Protein Precursor (APP). We chose to examine profiles of Aß(1-40), Aß(1-42), and N-truncated (i.e., p3-related) fragments in the plaque-associated fraction of autopsied cortical and corresponding hippocampal samples from donors with a diagnosis of early-onset (EOAD) and late-onset (LOAD) AD. Levels of Aß(1-40), Aß(1-42), and the p3 fragment-enriched pool were increased in EOAD and LOAD samples, and correlated well within -but not between- regions. Counterintuitively, these increases were similar regardless of the AD donor's APOE ε4 status. Focusing on the donor's sex and APOE ε4 status as nominal variables (i.e., omitting diagnosis from the stratification) revealed that increases in Aß peptides were specific to female carriers of the ε4 allele and correlated with the proportional expression of BACE1/ß-secretase and ADAM10/α-secretase in the cortex and with nicastrin (γ-secretase) expression in the hippocampus. These data preliminarily support the possibility that AD follows distinct amyloidogenic processes in males and females, and that the APOE ε4 allele exerts a major influence on the disease process, particularly in women. This knowledge could significantly impact the (re)interpretation of unsuccessful outcomes of clinical interventions targeting either Aß peptides directly or the secretases implicated in APP processing.

RAGE mediates the inactivation of nAChRs in sympathetic neurons under high glucose conditions.

Autonomic dysfunction is a serious complication of diabetes and can lead to cardiovascular abnormalities and premature death. It was recently proposed that autonomic dysfunction is triggered by oxidation-mediated inactivation of neuronal nicotinic acetylcholine receptors (nAChRs), impairing synaptic transmission in sympathetic ganglia and resulting in autonomic failure. We investigated whether the receptor for advanced glycation end products (RAGE) and its role in the generation of reactive oxygen species (ROS) could be contributing to the events that initiate sympathetic malfunction under high glucose conditions. Using biochemical, live imaging and electrophysiological tools we demonstrated that exposure of sympathetic neurons to high glucose increases RAGE expression and oxidative markers, and that incubation with RAGE ligands (e.g. AGEs, S100 and HMGB1) mimics both ROS elevation and nAChR inactivation. In contrast, co-treatment with either antioxidants or an anti-RAGE IgG prevented the inactivation of nAChRs. Lastly, a role for RAGE in this context was corroborated by the lack of sensitivity of sympathetic neurons from RAGE knock-out mice to high glucose. These data define a pivotal role for RAGE in initiating the events associated with exposure of sympathetic neurons to high glucose, and strongly support RAGE signaling as a potential therapeutic target in the autonomic complications associated with diabetes.

The monoamine oxidase-A inhibitor clorgyline promotes a mesenchymal-to-epithelial transition in the MDA-MB-231 breast cancer cell line.

Monoamine oxidase-A (MAO-A) dysfunction has been historically associated with depression. Recently, depression as well as altered MAO-A expression have both been associated with a poor prognosis in cancers, although the mechanism involved remains ambiguous. For example, MAO-A mRNA is repressed across cancers, yet MAO-A protein and levels of serotonin, a substrate of MAO-A implicated in depression, are paradoxically increased in malignancies, including breast cancer. The effect of clorgyline (CLG), a selective inhibitor of MAO-A, on malignant behaviour, expression of transitional markers, and biochemical correlates was examined in two human breast carcinoma cell lines, i.e. the epithelial, oestrogen receptor (ER)-positive MCF-7 cell line and the post-EMT (mesenchymal), ER-negative MDA-MB-231 cell line. CLG exerted little effect on malignant behaviour in MCF-7 cells, but inhibited proliferation and anchorage-independent growth, and increased invasiveness and active migration of MDA-MB-231 cells. CLG induced the expression of the mesenchymal marker vimentin in MCF-7 cells, but not in MDA-MB-231 cells. In contrast, CLG induced the epithelial protein marker E-cadherin in both cell lines, with a more robust effect in MDA-MB-231 cells (where a nuclear E-cadherin signal was also detected). This effect appears to be independent of any canonical Snai1-mediated regulation of E-cadherin mRNA expression. CLG interfered with the ß-catenin/[phospho]GSK-3ß complex as well as the E-cadherin/ß-catenin complex in both cell lines cells, but, again, the effect was more robust in MDA-MB-231 cells. Parallel studies revealed a general lack of effect of CLG on the ER-negative, epithelial Au565 breast cancer cell line. Thus, any effect of CLG on metastatic behaviours appears to rely on the cell's EMT status rather than on the cell's ER status. These data suggest that inactivation of MAO-A triggers a mesenchymal-to-epithelial transition in MDA-MB-231 cells via a non-canonical mechanism. This potentially implicates an MAO-A-sensitive step in advanced breast cancer and should be borne in mind when considering pharmacological treatment options for co-morbid depression in breast cancer patients.

Aspartic acid substitutions in monoamine oxidase-A reveal both catalytic-dependent and -independent influences on cell viability and proliferation.

Post-translational influences could underlie the ambiguous roles of monoamine oxidase-A (MAO-A) in pathologies such as depression, cancer and Alzheimer disease. In support of this, we recently demonstrated that the Ca²âº-sensitive component of MAO-A catalytic activity is inhibited by a pro-survival p38 (MAPK)-dependent mechanism. We substituted three aspartic acid (D) residues in human MAO-A that reside in putative Ca²âº-binding motifs and overexpressed the individual proteins in the human HEK293 cell line. We assayed the overexpressed proteins for catalytic activity and for their influence on cell viability (using MTT conversion and trypan blue exclusion) and proliferation/DNA synthesis [using bromodeoxyuridine (BrdU) incorporation]. Innate MAO-A catalytic activity (and the capacity for generating hydrogen peroxide) was unaffected by the D61A substitution, but inhibited moderately or completely by the D248A and D328G substitutions, respectively. The Ca²âº-sensitive activities of wild-type and D248A MAO-A proteins were enhanced by treatment with the selective p38(MAPK) inhibitor, SB203580, but was completely abrogated by the D61A substitution. Monoamine oxidase-A(D61A) was toxic to cells and exerted no effect on cell proliferation, while MAO-A(D248A) was generally comparable to wild-type MAO-A. As expected, the catalytic-dead MAO-A(D328G) was not cytotoxic, but unexpectedly enhanced both MTT conversion and BrdU staining. Variant-dependent changes in Bax and Bcl-2/Bcl-XL protein expression were observed. A different pattern of effects in N2-a cells suggests cell line-dependent roles for MAO-A. A catalytic-dependent mechanism influences MAO-A-mediated cytotoxicity, whereas a catalytic-independent mechanism contributes to proliferation. Context-dependent inputs by either mechanism could underlie the ambiguous pathological contributions of MAO-A.

Monoamine oxidase-A physically interacts with presenilin-1(M146V) in the mouse cortex.

The concentration of presenilin-1 (PS-1) protein at the mitochondrial-associated aspect of the endoplasmic reticulum supports the potential for a mitochondrial influence of PS-1. Given that carriers of certain Alzheimer's disease (AD)-related PS-1 variants are predisposed to clinical depression and that depression has been historically associated with the mitochondrial enzyme, monoamine oxidase-A (MAO-A), we investigated cortical MAO-A function in the AD-related PS-1(M146V) knock-in mouse. The MAO-A system was clearly altered in the PS-1(M146V) mouse as revealed by (a) a mismatch between MAO-A protein expression and MAO-A activity; (b) changes in MAO-A-mediated monoaminergic neurotransmitter metabolism; (c) changes in non-cognitive behavior following treatment with the irreversible MAO-A inhibitor clorgyline; and (d) an increase in the potency of clorgyline in these same mice. We next investigated whether PS-1(M146V) could be influencing MAO-A directly. We observed (a) an enhanced MAO-A activity in necropsied PS-1(M146V) mouse cortical extracts incubated with DAPT (a PS-1 substrate-competitor); (b) the proximity of PS-1 with MAO-A and mitochondrial markers in cortical sections and in primary cortical neurons; (c) the co-segregation and co-immunoprecipitation of PS-1 and MAO-A within the mitochondrial fraction; and (d) the co-immunoprecipitation of overexpressed PS-1(M146V) and MAO-A proteins from N2a lysates. The PS-1(ΔEx9) and PS-1(D257A) variants, known to have low substrate-binding capacity, co-immunoprecipitated weakly with MAO-A. These combined data support a physical interaction between PS-1 and MAO-A that could influence MAO-A activity and contribute to the monoaminergic disruptions common to disorders as seemingly diverse as depression and AD.

Alzheimer disease-related presenilin-1 variants exert distinct effects on monoamine oxidase-A activity in vitro.

Monoamine oxidase-A (MAO-A) has been associated with both depression and Alzheimer disease (AD). Recently, carriers of AD-related presenilin-1 (PS-1) alleles have been found to be at higher risk for developing clinical depression. We chose to examine whether PS-1 could influence MAO-A function in vitro. Overexpression of selected AD-related PS-1 variants (wildtype, Y115H, ΔEx9 and M146V) in mouse hippocampal HT-22 cells affects MAO-A catalytic activity in a variant-specific manner. The ability of the PS-1 substrate-competitor DAPT to induce MAO-A activity in cells expressing either PS-1 wildtype or PS-1(M146V) suggests the potential for a direct influence of PS-1 on MAO-A function. In support of this, we were able to co-immunoprecipitate MAO-A with FLAG-tagged PS-1 wildtype and M146V proteins. This potential for a direct protein-protein interaction between PS-1 and MAO-A is not specific for HT-22 cells as we were also able to co-immunoprecipitate MAO-A with FLAG-PS-1 variants in N2a mouse neuroblastoma cells and in HEK293 human embryonic kidney cells. Finally, we demonstrate that the two PS-1 variants reported to be associated with an increased incidence of clinical depression [e.g., A431E and L235V] both induce MAO-A activity in HT-22 cells. A direct influence of PS-1 variants on MAO-A function could provide an explanation for the changes in monoaminergic tone observed in several neurodegenerative processes including AD. The ability to induce MAO-A catalytic activity with a PS-1/γ-secretase inhibitor should also be considered when designing secretase inhibitor-based therapeutics.

Serine 209 resides within a putative p38(MAPK) consensus motif and regulates monoamine oxidase-A activity.

The p38 mitogen-activated protein kinase (MAPK) cascade as well as the enzyme monoamine oxidase-A (MAO-A) have both been associated with oxidative stress. We observed that the specific inhibition of the p38(MAPK) protein [using either a chemical inhibitor or a dominant-negative p38(MAPK) clone] selectively induces MAO-A activity and MAO-A-sensitive toxicity in several neuronal cell lines, including primary cortical neurons. Over-expression of a constitutively active p38(MAPK) results in the phosphorylation of the MAO-A protein and inhibition of MAO-A activity. The MAO-A(Ser209Glu) phosphomimic - bearing a targeted substitution within a putative p38(MAPK) consensus motif - is neither active nor neurotoxic. In contrast, the MAO-A(Ser209Ala) variant (mimics dephosphorylation) does not associate with p38(MAPK), and is both very active and very toxic. Substitution of the homologous serine in the MAO-B isoform, i.e. Ser200, with either Glu or Ala does not affect the catalytic activity of the corresponding over-expressed proteins. These combined in vitro data strongly suggest a direct p38(MAPK)-dependent inhibition of MAO-A function. Based on published observations, this endogenous means of selectively regulating MAO-A function could provide for an adaptive response to oxidative stress associated with disorders as diverse as depression, reperfusion/ischemia, and the early stages of Alzheimer's disease.