SSAO/VAP-1 in Cerebrovascular Disorders: A Potential Therapeutic Target for Stroke and Alzheimer's Disease.

The semicarbazide-sensitive amine oxidase (SSAO), also known as vascular adhesion protein-1 (VAP-1) or primary amine oxidase (PrAO), is a deaminating enzyme highly expressed in vessels that generates harmful products as a result of its enzymatic activity. As a multifunctional enzyme, it is also involved in inflammation through its ability to bind and promote the transmigration of circulating leukocytes into inflamed tissues. Inflammation is present in different systemic and cerebral diseases, including stroke and Alzheimer's disease (AD). These pathologies show important affectations on cerebral vessels, together with increased SSAO levels. This review summarizes the main roles of SSAO/VAP-1 in human physiology and pathophysiology and discusses the mechanisms by which it can affect the onset and progression of both stroke and AD. As there is an evident interrelationship between stroke and AD, basically through the vascular system dysfunction, the possibility that SSAO/VAP-1 could be involved in the transition between these two pathologies is suggested. Hence, its inhibition is proposed to be an interesting therapeutical approach to the brain damage induced in these both cerebral pathologies.

Synthesis, biological evaluation, and molecular modeling of nitrile-containing compounds: Exploring multiple activities as anti-Alzheimer agents.

Based on the monoamine oxidase (MAO) inhibition properties of aminoheterocycles with a carbonitrile group we have carried out a systematic exploration to discover new classes of carbonitriles endowed with dual MAO and AChE inhibitory activities, and Aß anti-aggregating properties. Eighty-three nitrile-containing compounds, 13 of which are new, were synthesized and evaluated. in vitro screening revealed that 31, a new compound, presented the best lead for trifunctional inhibition against MAO A (0.34 µM), MAO B (0.26 µM), and AChE (52 µM), while 32 exhibited a lead for selective MAO A (0.12 µM) inhibition coupled to AChE (48 µM) inhibition. Computational analysis revealed that the malononitrile group can find an advantageous position with the aromatic cleft and FAD of MAO A or MAO B. However, the total binding energy can be handicapped by an internal penalty caused by twisting of the ligand molecule and subsequent disruption of the conjugation (32 in MAO B compared to the conjugated 31). Conjugation is also important for AChE as well as the hydrophilic character of malononitrile that allows this group to be in close contact with the aqueous environment as seen for 83. Although the effect of 31 and 32 against Aß1-42 , was very weak, the effect of 63 and 65, and of the new compound 75, indicated that these compounds were able to disaggregate Aß1-42 fibrils. The most effective was 63, a (phenylhydrazinylidene)propanedinitrile derivative that also inhibited MAO A (1.65 µM), making it a potential lead for Alzheimer's disease application.

Simvastatin blocks soluble SSAO/VAP-1 release in experimental models of cerebral ischemia: Possible benefits for stroke-induced inflammation control.

Beyond cholesterol reduction, statins mediate their beneficial effects on stroke patients through pleiotropic actions. They have shown anti-inflammatory properties by a number of different mechanisms, including the inhibition of NF-κB transcriptional activity and the consequent increase and release of adhesion molecules. We have studied simvastatin's effects on the vascular enzyme semicarbazide-sensitive amine oxidase/vascular adhesion protein 1 (SSAO/VAP-1), which is involved in stroke-mediated brain injury. SSAO/VAP-1 has leukocyte-binding capacity and mediates the expression of other adhesion proteins through signaling molecules generated by its catalytic activity. Our results indicate that soluble SSAO/VAP-1 is released into the bloodstream after an ischemic stimulus, in parallel with an increase in E-selectin and VCAM-1 and correlating with infarct volume. Simvastatin blocks soluble SSAO/VAP-1 release and prevents E-selectin and VCAM-1 overexpression as well. Simvastatin also effectively blocks SSAO/VAP-1-mediated leukocyte adhesion, although it is not an enzymatic inhibitor of SSAO in vitro. In addition, simvastatin-induced changes in adhesion molecules are greater in human brain endothelial cell cultures expressing SSAO/VAP-1, compared to those not expressing it, indicating some synergic effect with SSAO/VAP-1. We think that part of the beneficial effect of simvastatin in stroke is mediated by the attenuation of the SSAO/VAP-1-dependent inflammatory response.

The proof-of-concept of ASS234: Peripherally administered ASS234 enters the central nervous system and reduces pathology in a male mouse model of Alzheimer disease.

BACKGROUND: The heterogeneity of Alzheimer disease requires the development of multitarget drugs for treating the symptoms of the disease and its progression. Both cholinergic and monoamine oxidase dysfunctions are involved in the pathological process. Thus, we hypothesized that the development of therapies focused on these targets might be effective. We have developed and assessed a new product, coded ASS234, a multipotent acetyl and butyrylcholinesterase/monoamine oxidase A-B inhibitor with a potent inhibitory effect on amyloid-ß aggregation as well as antioxidant and antiapoptotic properties. But there is a need to reliably correlate in vitro and in vivo drug release data. METHODS: We examined the effect of ASS234 on cognition in healthy adult C57BL/6J mice in a model of scopolamine-induced cognitive impairment that often accompanies normal and pathological aging. Also, in a characterized transgenic APPswe/PS1ΔE9 mouse model of Alzheimer disease, we examined the effects of short-term ASS234 treatment on plaque deposition and gliosis using immunohistochemistry. Toxicology of ASS234 was assessed using a quantitative high-throughput in vitro cytotoxicity screening assay following the MTT assay method in HepG2 liver cells. RESULTS: In vivo, ASS234 significantly decreased scopolamine-induced learning deficits in C57BL/6J mice. Also, reduction of amyloid plaque burden and gliosis in the cortex and hippocampus was assessed. In vitro, ASS234 exhibited lesser toxicity than donepezil and tacrine. LIMITATIONS: The study was conducted in male mice only. Although the Alzheimer disease model does not recapitulate all features of the human disease, it exhibits progressive monoaminergic neurodegeneration. CONCLUSION: ASS234 is a promising alternative drug of choice to treat the cognitive decline and neurodegeneration underlying Alzheimer disease.

Design, synthesis and biological evaluation of N-methyl-N-[(1,2,3-triazol-4-yl)alkyl]propargylamines as novel monoamine oxidase B inhibitors.

Different azides and alkynes have been coupled via Cu-catalyzed 1,3-dipolar Huisgen cycloaddition to afford a novel family of N1- and C5-substituted 1,2,3-triazole derivatives that feature the propargylamine group typical of irreversible MAO-B inhibitors at the C4-side chain of the triazole ring. All the synthesized compounds were evaluated against human MAO-A and MAO-B. Structure-activity relationships and molecular modeling were utilized to gain insight into the structural and chemical features that enhance the binding affinity and selectivity between the two enzyme isoforms. Several lead compounds, in terms of potency (submicromolar to low micromolar range), MAO-B selective recognition, and brain permeability, were identified. One of these leads (MAO-B IC50 of 3.54µM, selectivity MAO-A/MAO-B index of 27.7) was further subjected to reversibility and time-dependence inhibition studies, which disclosed a slow and irreversible inhibition of human MAO-B. Overall, the results support the suitability of the 4-triazolylalkyl propargylamine scaffold for exploring the design of multipotent anti-Alzheimer compounds endowed with irreversible MAO-B inhibitory activity.

Kinetic and structural analysis of the irreversible inhibition of human monoamine oxidases by ASS234, a multi-target compound designed for use in Alzheimer's disease.

Monoamine oxidases (MAO) and cholinesterases are validated targets in the design of drugs for the treatment of Alzheimer's disease. The multi-target compound N-((5-(3-(1-benzylpiperidin-4-yl)propoxy)-1-methyl-1H-indol-2-yl)methyl)-N-methylprop-2-yn-1-amine (ASS234), bearing the MAO-inhibiting propargyl group attached to a donepezil moiety that inhibits cholinesterases, retained activity against human acetyl- and butyryl-cholinesterases. The inhibition of MAO A and MAO B by ASS234 was characterized and compared to other known MAO inhibitors. ASS234 was almost as effective as clorgyline (kinact/KI=3×10(6) min(-1)M(-1)) and was shown by structural studies to form the same N5 covalent adduct with the FAD cofactor.

Involvement of SSAO/VAP-1 in oxygen-glucose deprivation-mediated damage using the endothelial hSSAO/VAP-1-expressing cells as experimental model of cerebral ischemia.

BACKGROUND: In the acute phase of ischemic stroke, endothelial cells are activated and induce the expression of adhesion molecules. Vascular adhesion protein 1 (VAP-1) is a proinflammatory protein that mediates leukocyte recruitment through its semicarbazide-sensitive amine oxidase (SSAO) activity (EC 1.4.3.21). Plasmatic SSAO activity predicts the appearance of parenchymal hemorrhages after tissue plasminogen activator treatment in ischemic stroke patients, and it is increased as well in hemorrhagic stroke patients. The aim of this study has been to elucidate the role of SSAO/VAP-1 present in endothelial cells during ischemic stroke conditions. METHODS: Based on the use of endothelial cells expressing, or not expressing, the human SSAO/VAP-1 protein, we have set up an easy ischemic model using oxygen-glucose deprivation (OGD) as an experimental approach to the stroke process. Different OGD and reoxygenation conditions have been analyzed. Western blotting has been used to analyze the activated apoptotic pathways. Several metalloproteinase inhibitors were also used to determine their role in the SSAO/VAP-1 release from the membrane of endothelial cells to the culture media, as a soluble form. Adhesion assays were also performed in order to assess the SSAO/VAP-1-dependent leukocyte adhesion to the endothelia under different OGD and reoxygenation conditions. RESULTS: Our results show that SSAO/VAP-1 expression increases the susceptibility of endothelial cells to OGD, and that its enzymatic activity, through specific substrate oxidation, increases vascular cell damage under these experimental conditions. Caspase-3 and caspase-8 are activated during the death process. In addition, OGD constitutes a stimulus for soluble SSAO/VAP-1 release, partly mediated by metalloproteinase-2-dependent shedding. Short-time OGD induces SSAO/VAP-1-dependent leukocyte binding on endothelial cells, which is partly dependent on its enzymatic activity. CONCLUSIONS: Our results show that SSAO/VAP-1 could participate in some of the processes occurring during stroke. Its expression in endothelial cells increases the OGD-associated cell damage. SSAO/VAP-1 mediates also part of the tissue damage during the reoxygenation process by oxidizing its known enzymatic substrate, methylamine. Also, OGD constitutes a stimulus for its soluble-form release, found elevated in many pathological conditions including stroke. OGD induces SSAO-dependent leukocyte-binding activity, which may have consequences in disease progression, since leukocyte infiltration has shown a determinant role in cerebral ischemia. For all the stroke-related processes in which SSAO/VAP-1 participates, it would be an interesting therapeutic target. Therefore, this model will be a very useful tool for the screening of new molecules as therapeutic agents for cerebral ischemia.

Induction of atypical EAE mediated by transgenic production of IL-6 in astrocytes in the absence of systemic IL-6.

Interleukin (IL)-6 is crucial for the induction of many murine models of autoimmunity including experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. While IL-6-deficient mice (IL-6 KO) are resistant to EAE, we showed previously that in transgenic mice with astrocyte-targeted production of IL-6-restricted to the cerebellum (GFAP-IL6), EAE induced with MOG(35-55) was redirected away from the spinal cord to the cerebellum. To further establish the importance of IL-6 produced in the central nervous system, we have generated mice producing IL-6 essentially only in the brain by crossing the GFAP-IL6 mice with IL-6 KO mice. Interestingly, GFAP-IL6-IL-6 KO mice showed a milder but almost identical phenotype as the GFAP-IL6 mice, which correlated with a lower load of inflammatory cells and decreased microglial reactivity. These results indicate that not only is cerebellar IL-6 production and eventual leakage into the peripheral compartment the dominating factor controlling this type of EAE but that it can also facilitate induction of autoimmunity in the absence of normal systemic IL-6 production.

Propargylamine-derived multitarget-directed ligands: fighting Alzheimer's disease with monoamine oxidase inhibitors.

Alzheimer's disease (AD) is a complex neurodegenerative disorder with a multifaceted pathogenesis. There are at present three Food and Drug Administration-approved drugs based on the "one drug, one target" paradigm (donepezil, galantamine and rivastigmine) that improve symptoms by inhibiting acetylcholinesterase. However, apart from the beneficial palliative properties, cholinergic drugs have shown little efficacy to prevent the progression of the disease evidencing the unsuitability of this strategy for the complex nature of AD. By contrast, the multifactorial nature of this neurodegenerative disorder supports the most current innovative therapeutic approach based on the "one drug, multiple targets" paradigm, which suggests the use of compounds with multiple activities at different target sites. Accordingly, the also called multitarget-directed ligand (MTDL) approach has been the subject of increasing attention by many research groups, which have developed a variety of hybrid compounds acting on very diverse targets. The therapeutic potential of monoamine oxidase inhibitors (MAOI) in AD has been suggested due to their demonstrated neuroprotective properties besides their enhancing effect on monoaminergic transmission. Especially, those containing a propargylamine moiety are of particular interest due to their reported beneficial actions. Therefore, targeting MAO enzymes should be considered in therapeutic interventions. This review makes a special emphasis on MTDLs that commonly target MAO enzymes. There is at present an urgent need for real disease-modifying therapies for AD and the MTDL approach makes a breakthrough for the development of new drugs capable of addressing the biological complexity of this disorder.

A comparison between radiometric and fluorimetric methods for measuring SSAO activity.

Semicarbazide-sensitive amine oxidase (SSAO) metabolizes the oxidative deamination of primary aromatic and aliphatic amines. The final cytotoxic products of its catalysis contribute to diseases involving vascular degeneration. The increasing interest in measuring SSAO activity has led to the development of several different methods. Herein, we compare SSAO activity results obtained with radiometric and fluorimetric methods in 49 plasma samples. Although not interchangeable, a significant correlation was obtained between methods. Considering these limitations, the fluorimetric method might replace the radioisotopic one.

A therapeutic approach to cerebrovascular diseases based on indole substituted hydrazides and hydrazines able to interact with human vascular adhesion protein-1, monoamine oxidases (A and B), AChE and BuChE.

Herein, we report the biological evaluation of a series of indole substituted hydrazides and hydrazines throughout the assessment of their multipotent inhibitory potency towards monoamine oxidase (MAO) A and B, semicarbazide-sensitive amine oxidase/vascular adhesion protein-1 (SSAO/VAP-1), and the cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Hydrazine JL72 (3-(3-hydrazinylpropyl)-1H-indole) showed a potent, reversible and non-time-dependent inhibition of MAO-A, which suggests its capacity in restoring serotoninergic neurotransmission being devoid of the side effects observed for classic MAO-A inhibitors. In addition, JL72 behaved as a moderate BuChE inhibitor. Finally, both hydrazines and hydrazides derivatives showed high affinity towards SSAO/VAP-1. Among them, JL72 behaved as a noncompetitive and the most potent inhibitor (IC50 = 0.19 ± 0.04 µM), possessing also a significant anti-inflammatory activity. The combined inhibition of SSAO/VAP-1, MAO (A and B), AChE and BuChE appear as an important therapeutic target to be considered in the treatment of cerebrovascular and neurological disorders such as Alzheimer's disease.

Immunohistochemical study of semicarbazide-sensitive amine oxidase/vascular adhesion protein-1 in the hippocampal vasculature: pathological synergy of Alzheimer's disease and diabetes mellitus.

Semicarbazide-sensitive amine oxidase/vascular adhesion protein-1 (SSAO/VAP-1) is involved in vascular endothelial damage as well as in the vascular degeneration underlying diabetes mellitus and Alzheimer's disease (AD). Recent evidence suggests that classic pathological features of AD are more pronounced in diabetic mellitus patients. To investigate the expression and distribution of SSAO/VAP-1 in the two pathologies, we have performed an immunohistochemical study in human hippocampal vessels of AD, AD with diabetic mellitus (ADD), diabetic mellitus (DM), and nondemented (ND) patients. The present results demonstrate major vessel accumulation of both SSAO/VAP-1 and amyloid-ß immunolabeling intensity in ADD compared with AD patients. Interestingly, nearly damaged vessels with high levels of SSAO/VAP-1 also showed increased oxidative damage markers (AGE, RAGE, and SOD-1) and glial activation (GFAP and HLA). Overall, this work suggests that high vascular SSAO/VAP-1 levels in human hippocampus may contribute to vascular degeneration, which can explain the severe progression in patients with both pathologies.

VAP-1/SSAO plasma activity and brain expression in human hemorrhagic stroke.

BACKGROUND: Vascular adhesion protein-1 (VAP-1) is a cell surface and circulating enzyme that belongs to the semicarbazide-sensitive amine oxidase (SSAO) family, which oxidatively deaminates primary amines and is implicated in leukocyte extravasation. Our aim was to investigate the alteration of soluble VAP-1/SSAO activity in plasma samples after acute intracerebral hemorrhage (ICH) and its presence in human ICH brain tissue. METHODS: VAP-1/SSAO activity was determined in plasma of 66 ICH patients and 58 healthy controls. In addition, we assessed the expression of VAP-1/SSAO in postmortem brain tissue from hemorrhagic stroke patients by Western blot and immunohistochemistry. RESULTS: We observed significantly higher levels of plasma VAP-1/SSAO activity in patients with ICH compared to matched elderly controls (p = 0.001). Plasma VAP-1/SSAO activity <2.7 pmol/min·mg and baseline ICH volume <17 ml were independent predictors of neurological improvement after 48 h (OR 6.8, 95% CI 1.14-41.67, p = 0.035, and OR 10.64, 95% CI 1.1-100, p = 0.041, respectively), after adjustment for baseline stroke severity. We also found that membrane-bound VAP-1/SSAO levels were lower in the perihematoma region than in the corresponding contralateral brain areas of patients deceased due to ICH (p = 0.024). CONCLUSIONS: Our data demonstrate that plasma VAP-1/SSAO activity is increased in ICH and predicts neurological outcome, suggesting a possible contribution of the soluble protein in secondary brain damage. Furthermore, anti-VAP-1/SSAO strategies might be a promising approach to prevent neurological worsening following ICH.

Vascular cell lines expressing SSAO/VAP-1: a new experimental tool to study its involvement in vascular diseases.

BACKGROUND INFORMATION: PrAO (primary amine oxidase), also known as SSAO (semicarbazide-sensitive amine oxidase)/VAP-1 (vascular adhesion protein-1), is an enzyme (EC 1.4.3.21) that is highly expressed in blood vessels and participates in many cell processes, including glucose handling or inflammatory leucocyte recruitment. High activity levels of this enzyme are associated with diabetes, atherosclerosis, AD (Alzheimer's disease) or stroke, among others, thus meaning that studies concerning SSAO as a therapeutic target are becoming more frequent. However, the study of this enzyme is difficult, owing to its loss of expression in cell cultures. RESULTS: We have developed an endothelial cell line that stably expresses the human SSAO/VAP-1 to be used as endothelial cell model for the study of this enzyme. The transfected protein is mainly expressed as a dimer in the membrane of these cells, and we demonstrate its specific localization in the lipid rafts of endothelial cells. The protein shows levels of enzymatic activity and kinetic parameters comparable with those observed in vivo by the same cell type. The transfected SSAO/VAP-1 is also able to mediate the adhesion of leucocytes to the endothelium, a known function of this protein under inflammatory conditions. This distinctive function is not exerted by the SSAO/VAP-1 transfected protein in a smooth muscle cell line that expresses 3-fold higher protein levels. These differences have been widely reported to exist in vivo. Furthermore, using this endothelial cell model, we describe for the first time the involvement of the leucocyte-adhesion activity of SSAO/VAP-1 in the Aß (amyloid ß-peptide)-mediated pro-inflammatory effect. CONCLUSIONS: The characterization of this new cell line shows the correct behaviour of the transfected protein and endorses the use of these cellular models for the in-depth study of the currently poorly understood functions of SSAO/VAP-1 and its involvement in the above-mentioned pathologies. This cellular model will be also useful for the evaluation of potential compounds that could modulate its activity for therapeutic purposes.

Plasma VAP-1/SSAO activity predicts intracranial hemorrhages and adverse neurological outcome after tissue plasminogen activator treatment in stroke.

BACKGROUND AND PURPOSE: Vascular adhesion protein-1 (VAP-1) is a cell surface and circulating enzyme involved in recruitment of lymphocytes and neutrophils through its semicarbazide-sensitive amine oxidase (SSAO) activity. We aimed to study plasma VAP-1/SSAO activity in relation to the risk for intracranial bleeding complications in patients with stroke treated with tissue plasminogen activator (tPA), the greatest safety concern with this treatment. METHODS: In 141 patients with ischemic stroke, we measured VAP-1/SSAO activity in plasma taken before tPA administration. Hemorrhagic events were classified according to brain CT criteria and functional outcomes evaluated using the National Institutes of Health Stroke Scale. We also assessed the potential therapeutic effect of blocking VAP-1/SSAO activity in a rat embolic stroke model treated with tPA. RESULTS: We saw significantly higher levels of plasma VAP-1/SSAO activity in patients who subsequently experienced hemorrhagic transformation. Elevated plasma VAP-1/SSAO activity also predicted worse neurological outcome in these patients. In the rat model, we confirmed that use of the inhibitor semicarbazide prevented adverse effects caused by delayed tPA administration, leading to a smaller infarct volume. CONCLUSIONS: Our data demonstrate that baseline VAP-1/SSAO activity predicts parenchymal hemorrhage after tPA, suggesting the safety of thrombolytic agents could be improved by considering VAP-1/SSAO activity. Furthermore, anti-VAP-1/SSAO drugs given with tPA may prevent neurological worsening in patients with ischemic stroke.

Immunohistochemical analysis of human brain suggests pathological synergism of Alzheimer's disease and diabetes mellitus.

It has been extensively reported that diabetes mellitus (DM) patients have a higher risk of developing Alzheimer's disease (AD), but a mechanistic connection between both pathologies has not been provided so far. Carbohydrate-derived advanced glycation endproducts (AGEs) have been implicated in the chronic complications of DM and have been reported to play an important role in the pathogenesis of AD. The earliest histopathological manifestation of AD is the apparition of extracellular aggregates of the amyloid beta peptide (Abeta). To investigate possible correlations between AGEs and Abeta aggregates with both pathologies, we have performed an immuhistochemical study in human post-mortem samples of AD, AD with diabetes (ADD), diabetic and nondemented controls. ADD brains showed increased number of Abeta dense plaques and receptor for AGEs (RAGE)-positive and Tau-positive cells, higher AGEs levels and major microglial activation, compared to AD brain. Our results indicate that ADD patients present a significant increase of cell damage through a RAGE-dependent mechanism, suggesting that AGEs may promote the generation of an oxidative stress vicious cycle, which can explain the severe progression of patients with both pathologies.

PF9601N [N-(2-propynyl)-2-(5-benzyloxy-indolyl) methylamine] confers MAO-B independent neuroprotection in ER stress-induced cell death.

Endoplasmic reticulum (ER) stress has recently been proposed as one of the factors contributing to apoptotic cell death in Parkinson's disease (PD). Although MAO-B inhibitors have been suggested to exert neuroprotective effects in several experimental models of PD, their effectiveness against ER stress has not been fully determined. Therefore, we have studied the potential usefulness of PF9601N, a non-amphetamine-like MAO-B inhibitor, in preventing cell death in a cell culture model of ER stress. Exposure of human dopaminergic cell line SH-SY5Y to the ER stressor brefeldin A led to Golgi disassembly, activation of the unfolded protein response (UPR), and subsequent expression of the proapoptotic mediator GADD153/CHOP. In this context, PF9601N pretreatment prevented brefeldin A-induced UPR responses, thus blocking the expression of GADD153/CHOP and resulting apoptotic features. In summary, our data suggests that PF9601N is able to block the responses elicited by ER stress, thus preventing apoptotic cell death in brefeldin A-treated cells.

Sensing of nutrients by CPT1C controls SAC1 activity to regulate AMPA receptor trafficking.

Carnitine palmitoyltransferase 1C (CPT1C) is a sensor of malonyl-CoA and is located in the ER of neurons. AMPA receptors (AMPARs) mediate fast excitatory neurotransmission in the brain and play a key role in synaptic plasticity. In the present study, we demonstrate across different metabolic stress conditions that modulate malonyl-CoA levels in cortical neurons that CPT1C regulates the trafficking of the major AMPAR subunit, GluA1, through the phosphatidyl-inositol-4-phosphate (PI(4)P) phosphatase SAC1. In normal conditions, CPT1C down-regulates SAC1 catalytic activity, allowing efficient GluA1 trafficking to the plasma membrane. However, under low malonyl-CoA levels, such as during glucose depletion, CPT1C-dependent inhibition of SAC1 is released, facilitating SAC1's translocation to ER-TGN contact sites to decrease TGN PI(4)P pools and trigger GluA1 retention at the TGN. Results reveal that GluA1 trafficking is regulated by CPT1C sensing of malonyl-CoA and provide the first report of a SAC1 inhibitor. Moreover, they shed light on how nutrients can affect synaptic function and cognition.

Proteasomal-Mediated Degradation of AKAP150 Accompanies AMPAR Endocytosis during cLTD.

The number and function of synaptic AMPA receptors (AMPARs) tightly regulates excitatory synaptic transmission. Current evidence suggests that AMPARs are inserted into the postsynaptic membrane during long-term potentiation (LTP) and are removed from the membrane during long-term depression (LTD). Dephosphorylation of GluA1 at Ser-845 and enhanced endocytosis are critical events in the modulation of LTD. Moreover, changes in scaffold proteins from the postsynaptic density (PSD) could be also related to AMPAR regulation in LTD. In the present study we analyzed the effect of chemical LTD (cLTD) on A-kinase anchoring protein (AKAP)150 and AMPARs levels in mouse-cultured neurons. We show that cLTD induces AKAP150 protein degradation via proteasome, coinciding with GluA1 dephosphorylation at Ser-845 and endocytosis of GluA1-containing AMPARs. Pharmacological inhibition of proteasome activity, but not phosphatase calcineurin (CaN), reverted cLTD-induced AKAP150 protein degradation. Importantly, AKAP150 silencing induced dephosphorylation of GluA1 Ser-845 and GluA1-AMPARs endocytosis while AKAP150 overexpression blocked cLTD-mediated GluA1-AMPARs endocytosis. Our results provide direct evidence that cLTD-induced AKAP150 degradation by the proteasome contributes to synaptic AMPARs endocytosis.

p53 phosphorylation is involved in vascular cell death induced by the catalytic activity of membrane-bound SSAO/VAP-1.

Semicarbazide sensitive amine oxidase (SSAO) is a multifunctional enzyme present mainly in adipocytes, endothelial and smooth muscle cells. It metabolizes primary aliphatic and aromatic amines generating products able to contribute to cellular oxidative stress. SSAO is expressed in a membrane-bound form and is also present as a soluble enzyme in plasma. Both isoforms are increased in several pathologies, and the catalytic products generated by the soluble enzymatic activity can induce cytotoxicity of vascular cells in culture. We have analyzed whether the transmembrane form of the enzyme is able to produce a cytotoxic effect through methylamine oxidation. Since cells in culture lose the expression of this enzyme, we used an SSAO stably transfected smooth muscle cell line. Herein we report that cell treatment with the substrate methylamine induced a dose and time dependent cytotoxic effect. The tumor suppressor protein p53 played an important role in the molecular pathway involved in this cell death. Moreover, we also observed the induction of PUMA-alpha expression with mitochondrial Bcl-2 family proteins being affected, and final effector caspases being activated.