Francesc X. Guix

Amyloid-dependent triosephosphate isomerase nitrotyrosination induces glycation and tau fibrillation

Alzheimers disease neuropathology is characterized by neuronal death, amyloid beta-peptide deposits and neurofibrillary tangles composed of paired helical filaments of tau protein. Although crucial for our understanding of the pathogenesis of Alzheimers disease, the molecular mechanisms linking amyloid beta-peptide and paired helical filaments remain unknown. Here, we show that amyloid beta-peptide-induced nitro-oxidative damage promotes the nitrotyrosination of the glycolytic enzyme triosephosphate isomerase in human neuroblastoma cells. Consequently, nitro-triosephosphate isomerase was found to be present in brain slides from double transgenic mice overexpressing human amyloid precursor protein and presenilin 1, and in Alzheimers disease patients. Higher levels of nitro-triosephosphate isomerase (P < 0.05) were detected, by Western blot, in immunoprecipitates from hippocampus (9 individuals) and frontal cortex (13 individuals) of Alzheimers disease patients, compared with healthy subjects (4 and 9 individuals, respectively). Triosephosphate isomerase nitrotyrosination decreases the glycolytic flow. Moreover, during its isomerase activity, it triggers the production of the highly neurotoxic methylglyoxal (n = 4; P < 0.05). The bioinformatics simulation of the nitration of tyrosines 164 and 208, close to the catalytic centre, fits with a reduced isomerase activity. Human embryonic kidney (HEK) cells overexpressing double mutant triosephosphate isomerase (Tyr164 and 208 by Phe164 and 208) showed high methylglyoxal production. This finding correlates with the widespread glycation immunostaining in Alzheimers disease cortex and hippocampus from double transgenic mice overexpressing amyloid precursor protein and presenilin 1. Furthermore, nitro-triosephosphate isomerase formed large beta-sheet aggregates in vitro and in vivo, as demonstrated by turbidometric analysis and electron microscopy. Transmission electron microscopy (TEM) and atomic force microscopy studies have demonstrated that nitro-triosephosphate isomerase binds tau monomers and induces tau aggregation to form paired helical filaments, the characteristic intracellular hallmark of Alzheimers disease brains. Our results link oxidative stress, the main etiopathogenic mechanism in sporadic Alzheimers disease, via the production of peroxynitrite and nitrotyrosination of triosephosphate isomerase, to amyloid beta-peptide-induced toxicity and tau pathology.

Plasma membrane voltage-dependent anion channel mediates antiestrogen-activated maxi Cl- currents in C1300 neuroblastoma cells.

The cell membrane large conductance voltage-dependent chloride channel (Maxi Cl– channel) has been recorded in different cell types following excision of membrane patches or stimulation by antiestrogens under whole-cell recording conditions. However, both its molecular nature and relevance to cell physiology await elucidation. Its electrophysiological properties resemble those of the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane. This observation has led to the controversial hypothesis that VDAC could be the molecular correlate of the plasma membrane Maxi Cl– channel. We have investigated the cellular localization of VDAC and its relationship with the antiestrogen-activated Maxi Cl– current in C1300 neuroblastoma cells. The presence of a plasma membrane VDAC was demonstrated by immunoblotting of membrane fractions with monoclonal antibodies against the VDAC and by reverse transcription-PCR using primers that hybridize to a VDAC sequence coding for an N-terminal leader peptide required for its plasma membrane sorting. Besides, VDAC colocalized with markers of plasma membrane lipid rafts (cholera toxin β subunit) but not caveolin-1. Transfection of C1300 cells with an antisense oligonucleotide directed against the specific membrane leader sequence of VDAC markedly reduced both VDAC immunostaining and antiestrogen-activated Maxi Cl– currents, suggesting that VDAC forms the plasma membrane Maxi Cl– channel or a part thereof.

Oxidative stress triggers the amyloidogenic pathway in human vascular smooth muscle cells.

Cerebral amyloid angiopathy, associated to most cases of Alzheimers disease (AD), is characterized by the deposition of amyloid ss-peptide (Ass) in brain vessels, although the origin of the vascular amyloid deposits is still controversial: neuronal versus vascular. In the present work, we demonstrate that primary cultures of human cerebral vascular smooth muscle cells (HC-VSMCs) have all the secretases involved in amyloid ss-protein precursor (APP) cleavage and produce Ass(1-40) and Ass(1-42). Oxidative stress, a key factor in the etiology and pathophysiology of AD, up-regulates ss-site APP cleaving enzyme 1 (BACE1) expression, as well as Ass(1-40) and Ass(1-42) secretion in HC-VSMCs. This process is mediated by c-Jun N-terminal Kinase and p38 MAPK signaling and appears restricted to BACE1 regulation as no changes in the other secretases were observed. In conclusion, oxidative stress-mediated up-regulation of the amyloidogenic pathway in human cerebral vascular smooth muscle cells may contribute to the overall cerebrovascular amyloid angiopathy observed in AD patients.

Modification of γ-secretase by nitrosative stress links neuronal ageing to sporadic Alzheimer's disease

Inherited familial Alzheimers disease (AD) is characterized by small increases in the ratio of Aβ42 versus Aβ40 peptide which is thought to drive the amyloid plaque formation in the brain of these patients. Little is known however whether ageing, the major risk factor for sporadic AD, affects amyloid beta‐peptide (Aβ) generation as well. Here we demonstrate that the secretion of Aβ is enhanced in an in vitro model of neuronal ageing, correlating with an increase in γ‐secretase complex formation. Moreover we found that peroxynitrite (ONOO−), produced by the reaction of superoxide anion with nitric oxide, promoted the nitrotyrosination of presenilin 1 (PS1), the catalytic subunit of γ‐secretase. This was associated with an increased association of the two PS1 fragments, PS1‐CTF and PS1‐NTF, which constitute the active catalytic centre. Furthermore, we found that peroxynitrite shifted the production of Aβ towards Aβ42 and increased the Aβ42/Aβ40 ratio. Our work identifies nitrosative stress as a potential mechanistic link between ageing and AD.

Amyloid-β Peptide Fibrils Induce Nitro-Oxidative Stress in Neuronal Cells

Different mechanisms including oxidative stress are proposed for amyloid-β peptide (Aβ) neurotoxicity, and here we contribute to demonstrate that nitro-oxidative stress is playing a key role. Yeasts are a well-known model for H2O2 toxicity. Interestingly, yeast cell wall prevents interaction of Aβ fibrils with membrane receptors or calcium channels and we found a significant viability reduction in yeasts when challenged with Aβ fibrils. Furthermore, iron and copper chelators, as well as the antioxidants glutathione and trolox, were neuroprotective on neuroblastoma cells and mouse hippocampal neurons challenged with Aβ fibrils. Glutathione prevents the oxidation, glycation and nitrotyrosination of cell proteins induced by Aβ. Trolox protected neurons in cell viability studies, maintaining the vesicular transport integrity and preventing the trigger of apoptotic mechanisms. Interestingly, we have also found that brain derived neuronal factor (BDNF) and neurotrophin-3 (NT-3) were able to protect mouse hippocampal and cortical neurons against H2O2 and Aβ fibrils. Considering that superoxide anion, produced by Aβ cell damage, and nitric oxide, whose production is altered in AD, react to form the highly reactive peroxynitrite anion, we studied the role of trolox to ameliorate the peroxynitrite cell damage. Finally, one of the major proteins to be nitrotyrosinated in AD, the triose phosphate isomerase (TPI) was assayed searching for a denitrase activity that could reverse intracellular nitrotyrosination. We have found that human neuroblastoma SH-SY5Y cells express a constitutive denitrase activity that partially denitrated nitro-TPI. Altogether, our results support a key role of nitro-oxidative stress in the neuronal damage induced by Aβ fibrils.

Activation of PKR Causes Amyloid ß-Peptide Accumulation via De-Repression of BACE1 Expression

BACE1 is a key enzyme involved in the production of amyloid ß-peptide (Aß) in Alzheimers disease (AD) brains. Normally, its expression is constitutively inhibited due to the presence of the 5′untranslated region (5′UTR) in the BACE1 promoter. BACE1 expression is activated by phosphorylation of the eukaryotic initiation factor (eIF)2-alpha, which reverses the inhibitory effect exerted by BACE1 5′UTR. There are four kinases associated with different types of stress that could phosphorylate eIF2-alpha. Here we focus on the double-stranded (ds) RNA-activated protein kinase (PKR). PKR is activated during viral infection, including that of herpes simplex virus type 1 (HSV1), a virus suggested to be implicated in the development of AD, acting when present in brains of carriers of the type 4 allele of the apolipoprotein E gene. HSV1 is a dsDNA virus but it has genes on both strands of the genome, and from these genes complementary RNA molecules are transcribed. These could activate BACE1 expression by the PKR pathway. Here we demonstrate in HSV1-infected neuroblastoma cells, and in peripheral nervous tissue from HSV1-infected mice, that HSV1 activates PKR. Cloning BACE1 5′UTR upstream of a luciferase (luc) gene confirmed its inhibitory effect, which can be prevented by salubrinal, an inhibitor of the eIF2-alpha phosphatase PP1c. Treatment with the dsRNA analog poly (I∶C) mimicked the stimulatory effect exerted by salubrinal over BACE1 translation in the 5′UTR-luc construct and increased Aß production in HEK-APPsw cells. Summarizing, our data suggest that PKR activated in brain by HSV1 could play an important role in the development of AD.

Down-regulation of the ATP-binding Cassette Transporter 2 (Abca2) Reduces Amyloid-β Production by Altering Nicastrin Maturation and Intracellular Localization

Background: The intracellular sterol transporter Abca2 has been genetically linked to Alzheimer disease. Results: Abca2 depletion reduces γ-secretase cleavage of APP without affecting γ-cleavage of Notch and alters maturation and intracellular localization of Nicastrin, a member of the γ-secretase complex. Conclusion: Abca2 depletion affects γ-secretase cleavage in a substrate-distinctive manner. Significance: Abca2 is an important regulator of γ-secretase-mediated APP proteolysis and therefore Aβ generation. Clinical, pharmacological, biochemical, and genetic evidence support the notion that alteration of cholesterol homeostasis strongly predisposes to Alzheimer disease (AD). The ATP-binding cassette transporter-2 (Abca2), which plays a role in intracellular sterol trafficking, has been genetically linked to AD. It is unclear how these two processes are related. Here we demonstrate that down-regulation of Abca2 in mammalian cells leads to decreased amyloid-β (Aβ) generation. In vitro studies revealed altered γ-secretase complex formation in Abca2 knock-out cells due to the altered levels, post-translational modification, and subcellular localization of Nicastrin. Reduced Abca2 levels in mammalian cells in vitro, in Drosophila melanogaster and in mice resulted in altered γ-secretase processing of APP, and thus Aβ generation, without affecting Notch cleavage.

Nitro-Oxidative Stress after Neuronal Ischemia Induces Protein Nitrotyrosination and Cell Death

Ischemic stroke is an acute vascular event that obstructs blood supply to the brain, producing irreversible damage that affects neurons but also glial and brain vessel cells. Immediately after the stroke, the ischemic tissue produces nitric oxide (NO) to recover blood perfusion but also produces superoxide anion. These compounds interact, producing peroxynitrite, which irreversibly nitrates protein tyrosines. The present study measured NO production in a human neuroblastoma (SH-SY5Y), a murine glial (BV2), a human endothelial cell line (HUVEC), and in primary cultures of human cerebral myocytes (HC-VSMCs) after experimental ischemia in vitro. Neuronal, endothelial, and inducible NO synthase (NOS) expression was also studied up to 24 h after ischemia, showing a different time course depending on the NOS type and the cells studied. Finally, we carried out cell viability experiments on SH-SY5Y cells with H2O2, a prooxidant agent, and with a NO donor to mimic ischemic conditions. We found that both compounds were highly toxic when they interacted, producing peroxynitrite. We obtained similar results when all cells were challenged with peroxynitrite. Our data suggest that peroxynitrite induces cell death and is a very harmful agent in brain ischemia.

Alterations in phosphatidylethanolamine levels affect the generation of Aβ

Several studies suggest that the generation of Aβ is highly dependent on the levels of cholesterol within membranes’ detergent‐resistant microdomains (DRM). Indeed, the β‐amyloid precursor protein (APP) cleaving machinery, namely β‐ and γ‐secretases, has been shown to be present in DRM and its activity depends on membrane cholesterol levels. Counterintuitive to the localization of the cleavage machinery, the substrate, APP, localizes to membranes’ detergent‐soluble microdomains enriched in phospholipids (PL), indicating that Aβ generation is highly dependent on the capacity of enzyme and substrate to diffuse along the lateral plane of the membrane and therefore on the internal equilibrium of the different lipids of DRM and non‐DRM domains. Here, we studied to which extent changes in the content of a main non‐DRM lipid might affect the proteolytic processing of APP. As phosphatidylethanolamine (PE) accounts for the majority of PL, we focused on its impact on the regulation of APP proteolysis. In mammalian cells, siRNA‐mediated knock‐down of PE synthesis resulted in decreased Aβ owing to a dual effect: promoted α‐secretase cleavage and decreased γ‐secretase processing of APP. In vivo, in Drosophila melanogaster, genetic reduction in PL synthesis results in decreased γ‐secretase‐dependent cleavage of APP. These results suggest that modulation of the membrane‐soluble domains could be a valuable alternative to reduce excessive Aβ generation.

Tetraspanin 6: a pivotal protein of the multiple vesicular body determining exosome release and lysosomal degradation of amyloid precursor protein fragments

BackgroundThe mechanisms behind Aβ-peptide accumulation in non-familial Alzheimer’s disease (AD) remain elusive. Proteins of the tetraspanin family modulate Aβ production by interacting to γ-secretase.MethodsWe searched for tetraspanins with altered expression in AD brains. The function of the selected tetraspanin was studied in vitro and the physiological relevance of our findings was confirmed in vivo.ResultsTetraspanin-6 (TSPAN6) is increased in AD brains and overexpression in cells exerts paradoxical effects on Amyloid Precursor Protein (APP) metabolism, increasing APP-C-terminal fragments (APP-CTF) and Aβ levels at the same time. TSPAN6 affects autophagosome-lysosomal fusion slowing down the degradation of APP-CTF. TSPAN6 recruits also the cytosolic, exosome-forming adaptor syntenin which increases secretion of exosomes that contain APP-CTF.ConclusionsTSPAN6 is a key player in the bifurcation between lysosomal-dependent degradation and exosome mediated secretion of APP-CTF. This corroborates the central role of the autophagosomal/lysosomal pathway in APP metabolism and shows that TSPAN6 is a crucial player in APP-CTF turnover.