Mitofusin 2 knock-down enhances endoplasmic reticulum-mitochondria contact and decreases amyloid \u3b2-peptide production.

Mitochondria are physically and biochemically in contact with other organelles including the endoplasmic reticulum (ER). Such contacts are formed between mitochondria-associated ER membranes (MAM), specialized subregions of ER, and the outer mitochondrial membrane (OMM). We have previously shown increased expression of MAM-associated proteins and enhanced ER to mitochondria Ca2+ transfer in Alzheimer\u2019s disease (AD) and amyloid \u3b2- peptide (A\u3b2) related neuronal models. Here, we report that siRNA knockdown of mitofusin-2 (Mfn2), a protein that is involved in the tethering of ER and mitochondria, leads to increased contact between the two organelles. Cells depleted in Mfn2 showed increased ER to mitochondria Ca2+ transfer and longer stretches of ER forming contacts with OMM. Interestingly, increased contact resulted in decreased concentrations of intra- and extracellular A\u3b240 and A\u3b242. Analysis of \u3b3-secretase protein expression, maturation and activity revealed that the low A\u3b2 concentrations were a result of impaired \u3b3-secretase complex function. APP, BACE1 and neprilysin expression as well as neprilysin activity were not affected by Mfn2 siRNA treatment. In summary, our data show that modulation of ER-mitochondria contact affects \u3b3-secretase activity and A\u3b2 generation. Increased ER-mitochondria contact results in lower \u3b3-secretase activity suggesting a new mechanism by which A\u3b2 generation can be controlled

Monoamine oxidase B is elevated in Alzheimer disease neurons, is associated with γ-secretase and regulates neuronal amyloid β-peptide levels

Background Increased levels of the pathogenic amyloid β-peptide (Aβ), released from its precursor by the transmembrane protease γ-secretase, are found in Alzheimer disease (AD) brains. Interestingly, monoamine oxidase B (MAO-B) activity is also increased in AD brain, but its role in AD pathogenesis is not known. Recent neuroimaging studies have shown that the increased MAO-B expression in AD brain starts several years before the onset of the disease. Here, we show a potential connection between MAO-B, γ-secretase and Aβ in neurons. Methods MAO-B immunohistochemistry was performed on postmortem human brain. Affinity purification of γ-secretase followed by mass spectrometry was used for unbiased identification of γ-secretase-associated proteins. The association of MAO-B with γ-secretase was studied by coimmunoprecipitation from brain homogenate, and by in-situ proximity ligation assay (PLA) in neurons as well as mouse and human brain sections. The effect of MAO-B on Aβ production and Notch processing in cell cultures was analyzed by siRNA silencing or overexpression experiments followed by ELISA, western blot or FRET analysis. Methodology for measuring relative intraneuronal MAO-B and Aβ42 levels in single cells was developed by combining immunocytochemistry and confocal microscopy with quantitative image analysis. Results Immunohistochemistry revealed MAO-B staining in neurons in the frontal cortex, hippocampus CA1 and entorhinal cortex in postmortem human brain. Interestingly, the neuronal staining intensity was higher in AD brain than in control brain in these regions. Mass spectrometric data from affinity purified γ-secretase suggested that MAO-B is a γ-secretase-associated protein, which was confirmed by immunoprecipitation and PLA, and a neuronal location of the interaction was shown. Strikingly, intraneuronal Aβ42 levels correlated with MAO-B levels, and siRNA silencing of MAO-B resulted in significantly reduced levels of intraneuronal Aβ42. Furthermore, overexpression of MAO-B enhanced Aβ production. Conclusions This study shows that MAO-B levels are increased not only in astrocytes but also in pyramidal neurons in AD brain. The study also suggests that MAO-B regulates Aβ production in neurons via γ-secretase and thereby provides a key to understanding the relationship between MAO-B and AD pathogenesis. Potentially, the γ-secretase/MAO-B association may be a target for reducing Aβ levels using protein–protein interaction breakers

Additional file 1: Supplementary figures. of Monoamine oxidase B is elevated in Alzheimer disease neurons, is associated with γ-secretase and regulates neuronal amyloid β-peptide levels

Figure S1. MAO-B antibody validation, precipitation and expression experiments. Figure S2. Western blot (WB) of postmortem AD and control human brain homogenate with MAO-B antibody. Figure S3. Validation of the Aβ42-specific antibody G2-11 by immunocytochemistry. Figure S4. MAO-B and Ab42 quantification in single cells from MAO-B silenced cortex neurons. Figure S5. Treatment of primary cortical neurons with fluorescently labeled siRNA. (PDF 1472 kb