The role of heparan sulfates in protein aggregation and their potential impact on neurodegeneration.

Neurodegenerative disorders, such as Alzheimer's, Parkinson's, and prion diseases, are directly linked to the formation and accumulation of protein aggregates in the brain. These aggregates, principally made of proteins or peptides that clamp together after acquisition of ß-folded structures, also contain heparan sulfates. Several lines of evidence suggest that heparan sulfates centrally participate in the protein aggregation process. In vitro, they trigger misfolding, oligomerization, and fibrillation of amyloidogenic proteins, such as Aß, tau, α-synuclein, prion protein, etc. They participate in the stabilization of protein aggregates, protect them from proteolysis, and act as cell-surface receptors for the cellular uptake of proteopathic seeds during their spreading. This review focuses attention on the importance of heparan sulfates in protein aggregation in brain disorders including Alzheimer's, Parkinson's, and prion diseases. The presence of these sulfated polysaccharides in protein inclusions in vivo and their capacity to trigger protein aggregation in vitro strongly suggest that they might play critical roles in the neurodegenerative process. Further advances in glyco-neurobiology will improve our understanding of the molecular and cellular mechanisms leading to protein aggregation and neurodegeneration.

Heparan sulfate 3-O-sulfotransferase 2 (HS3ST2) displays an unexpected subcellular localization in the plasma membrane.

BACKGROUND: Heparan sulfate (HS) 3-O-sulfation can be catalysed by seven 3-O-sulfotransferases (HS3STs) in humans, still it is the rarest modification in HS and its biological function is yet misunderstood. HS3ST2 and HS3ST3B exhibit the same activity in vitro. They are however differently expressed in macrophages depending on cell environment, which suggests that they may be involved in distinct cellular processes. Here, we hypothesized that both isozymes might also display distinct subcellular localizations. METHODS: The subcellular distribution of HS3ST2 and HS3ST3B was analysed by using overexpression systems in HeLa cells. The localization of endogenous HS3ST2 was confirmed by immunostaining in primary macrophages. RESULTS: We found that HS3ST3B was only localized in the Golgi apparatus and no difference between full-length enzyme and truncated construct depleted of its catalytic domain was observed. In contrast, HS3ST2 was clearly visualized at the plasma membrane. Its truncated form remained in the Golgi apparatus, meaning that the catalytic domain might support correct addressing of HS3ST2 to cell surface. Moreover, we found a partial co-localization of HS3ST2 with syndecan-2 in HeLa cells and primary macrophages. Silencing the expression of this proteoglycan altered the localization of HS3ST2, which suggests that syndecan-2 is required to address the isozyme outside of the Golgi apparatus. CONCLUSIONS: We demonstrated that HS3ST3B is a Golgi-resident isozyme, while HS3ST2 is addressed to the plasma membrane with syndecan-2. GENERAL SIGNIFICANCE: The membrane localization of HS3ST2 suggests that this enzyme may participate in discrete processes that occur at the cell surface.

Reestablishment of the endothelial lining by endothelial cell therapy stabilizes experimental abdominal aortic aneurysms.

BACKGROUND: Loss of the endothelium and its replacement by a thick thrombus are structural features of human abdominal aortic aneurysms (AAAs). In AAAs, the relationship between aortic diameter expansion, the presence of thrombus, and the lack of endothelial cells (ECs) remains unexplored. We hypothesized that reendothelialization by cell therapy would modulate aortic wall destruction and ultimately stabilize AAAs. We evaluated the impact of local seeding of rat aortic ECs or peripheral blood-derived outgrowth ECs on AAA evolution. METHODS AND RESULTS: Rat aortic ECs (n=30) or serum-free medium (controls; n=29) were seeded endovascularly immediately (day 0) or 14 days after surgery in the rat xenograft model. Rat aortic EC seeding prevented AAA formation and stabilized formed AAAs at 28 days (diameter increase at day 0+28, 51±6% versus 83±6%; day 14+28, -1±4% versus 22±6% in rat aortic ECs and controls, respectively; P<0.01). This stabilizing effect was associated with the reestablishment of the endothelial lining, the suspension of proteolysis, and the reconstitution of new aortic wall rich in smooth muscle cells and extracellular matrix. Transplanted rat aortic ECs did not participate directly in aortic wall repair but exerted their healing properties through paracrine mechanisms involving the upregulation of endothelium-derived stabilizing factors and the recruitment of resident vascular cells. In rats, the transplantation of outgrowth ECs (n=7) significantly reduced by 30% the progression of AAAs and restored the abluminal endothelium at 28 days compared with controls (n=9). CONCLUSION: Our study demonstrates the potential of restoring the endothelial lining to control AAA dynamics and designates ECs as an efficient therapy to stop AAA expansion.

New ascending aortic aneurysm model in rats reproduces main structural features of degenerative ascending thoracic aortic aneurysms in human beings.

OBJECTIVES: The singularity of the ascending aorta regarding mechanisms driving aneurysm formation requires the development of specific animal models. We investigated if adventitial elastase application results in ascending aorta aneurysms in rats. METHODS: Adult Lewis rats (n = 26) were anesthetized, their ascending aortas measured by transthoracic ultrasound, and exposed via median sternotomy. Elastase or saline was applied on the ascending aortic adventitia. Ascending aorta diameters were monitored by ultrasound at 10 and 30 days, when the animals were killed. Wall area was measured on orcein stained sections. Matrix metalloproteinase-2 and matrix metalloproteinase-9 levels were quantified on gelatin zymography. RESULTS: Following elastase application, ascending aortic diameter increased at 10 and 30 days follow-up by 38% and 44%, respectively (P = .004). Despite thinning of the media secondary to vascular dilation, standardized medial area was not different between elastase-treated aortas and controls. Standardized total wall area had a significant increase in treated aortas compared with controls. Active matrix metalloproteinase-2 was significantly increased at 30 days in treated aortas, whereas active matrix metalloproteinase-9 was no different from controls. CONCLUSIONS: Elastase application on rat ascending aortic adventitia produced aneurysms, creating a reproducible model. Aortic wall remodeling evolved toward an increase in total wall area, reproducing the main structural features of this disease in human beings.

Cellular response to cetuximab in PTEN-silenced head and neck squamous cell carcinoma cell line.

The implication of loss of PTEN expression in resistance to targeted therapy has already been described in many tumor types. The absence of response to anti-EGFR agents in PTEN-deficient tumors relies on persistent activation of signaling pathways downstream of pEGFR. To investigate the role of PTEN loss of expression in head and neck squamous cell carcinoma (HNSCC) response to cetuximab, we used siRNA in Cal 27 cells and then evaluated key signaling protein activation (pAKT and pERK 1/2) as well as cell viability and proliferation. PTEN silencing in Cal 27 cells led to a constitutive activation of signaling pathways evidenced by a strong increase in pAKT and pERK 1/2 expression. Moreover, PTEN-silenced cells did not show any significant changes either in cell viability or proliferation, only slight modifications on cell cycle. Additionally and unpredictably, our results indicated that PTEN silencing, led to a drastic reduction in pEGFR expression whereas total EGFR level did not significantly vary. Strikingly, despite this overactivation of signaling pathways ruling cell survival and proliferation in siPTEN cells, cetuximab fully exerted pAKT and pERK 1/2 inhibition of expression, similarly to its effect in untransfected Cal 27 cells. In conclusion, our study established that in Cal 27 cells, cetuximab keeps full ability to inhibit EGFR-dependent mechanisms, as shown by a decreased pAKT and pERK 1/2 level of expression, despite a strong PTEN silencing-induced overactivation. In Cal 27 cells, loss of PTEN expression does not lead to a loss of cetuximab efficacy in inhibiting EGFR-downstream signaling pathways, contrarily to data shown in previous works conducted in other tumor types.

Pharmacological and physicochemical factors in the pressor effects of conjugated haemoglobin-based oxygen carriers in vivo.

BACKGROUND: The hypertension induced by haemoglobin-based oxygen carriers could be a result of different pharmacological and physicochemical factors. OBJECTIVE: To investigate whether production of superoxide anion (O2*-) and release of endothelin could be the factors responsible. METHODS: We studied the variation in mean arterial pressure (MAP) in guinea pigs by carrying out a 50% isovolaemic exchange transfusion with conjugated oxyhaemoglobin (non-oxidized form) or conjugated methaemoglobin (fully oxidized form) in the presence or absence of BQ-788 (5 nmol/l), an endothelin receptor type B (ETR-B) antagonist. At key timepoints of variation in MAP, the plasma concentrations of O2*- were measured. The presence of conjugated oxyhaemoglobin and increases in ETR-B concentrations inside the vascular wall were investigated in different vessels, using western blotting. RESULTS: We found that the administration of conjugated oxyhaemoglobin induced a significant increase in MAP, whereas conjugated methaemoglobin had no significant haemodynamic effect. Pretreatment with BQ-788 attenuated the increase in MAP induced by conjugated oxyhaemoglobin. This haemoglobin induced the production of high concentrations of O2*- that declined towards control values after 120 min and decreased in the presence of BQ-788. Western blot analysis showed that the presence of conjugated oxyhaemoglobin inside the vascular wall was time-dependent and correlated with increased ETR-B. CONCLUSION: These results show that the release of O2*- during auto-oxidation of conjugated oxyhaemoglobin is associated with the observed increase in MAP, which may be a result of the vasoconstriction caused by an increase in activation of ETR-B. This activation may be caused by the massive release of endothelin induced by the production of O2*-.

Eradication of p53-mutated head and neck squamous cell carcinoma xenografts using nonviral p53 gene therapy and photochemical internalization.

Photochemical internalization (PCI) technology has been used for PEI-mediated p53 gene transfer in mice bearing head and neck squamous cell carcinoma (HNSCC) xenografts. Using luciferase as a reporter gene, PCI led to a 20-fold increase in transgene expression 48 h after transfection and sustained transgene expression for 7 days. Therefore, iterative p53 gene transfer was performed by means of a weekly single injection of PEIGlu4/p53 complexes alone or with PCI for 5 (group A) or 7 (group B) weeks. The efficiency of p53 gene therapy was evaluated by following tumor growth and expression of P53-related downstream proteins (P21, MDM2, Bcl2, Bax). Apoptosis induction was evidenced through caspase-3 activation and PARP cleavage. Using PCI, tumor growth inhibition was observed in all transfected animals. Further, successful tumor cure was achieved in 17% (group A) and 83% (group B) of animals. PCI-mediated p53 gene transfer led to higher P53 protein expression that was correlated with induction of Bax and P21 proapoptotic proteins, repression of Bcl2 as well as activation of caspase-3, and cleavage of PARP. The present study demonstrates that PCI enhances the in vivo efficiency of PEI-mediated p53 gene transfer and can be proposed for p53 gene therapy in HNSCC.

Docosahexaenoic acid prevents neuronal apoptosis induced by soluble amyloid-beta oligomers.

A growing body of evidence supports the notion that soluble oligomers of amyloid-beta (Abeta) peptide interact with the neuronal plasma membrane, leading to cell injury and inducing death-signalling pathways that could account for the increased neurodegeneration occurring in Alzheimer's disease (AD). Docosahexaenoic acid (DHA, C22:6, n-3) is an essential polyunsaturated fatty acid in the CNS and has been shown in several epidemiological and in vivo studies to have protective effects against AD and cognitive alterations. However, the molecular mechanisms involved remain unknown. We hypothesized that DHA enrichment of plasma membranes could protect neurones from apoptosis induced by soluble Abeta oligomers. DHA pre-treatment was observed to significantly increase neuronal survival upon Abeta treatment by preventing cytoskeleton perturbations, caspase activation and apoptosis, as well as by promoting extracellular signal-related kinase (ERK)-related survival pathways. These data suggest that DHA enrichment probably induces changes in neuronal membrane properties with functional outcomes, thereby increasing protection from soluble Abeta oligomers. Such neuroprotective effects could be of major interest in the prevention of AD and other neurodegenerative diseases.

Microtubule-associated protein MAP1A, MAP1B, and MAP2 proteolysis during soluble amyloid beta-peptide-induced neuronal apoptosis. Synergistic involvement of calpain and caspase-3.

A growing body of evidence supports the notion that soluble oligomeric forms of the amyloid beta-peptide (Abeta) may be the proximate effectors of neuronal injuries and death in the early stages of Alzheimer disease. However, the molecular mechanisms associated with neuronal apoptosis induced by soluble Abeta remain to be elucidated. We recently demonstrated the involvement of an early reactive oxygen species-dependent perturbation of the microtubule network (Sponne, I., Fifre, A., Drouet, B., Klein, C., Koziel, V., Pincon-Raymond, M., Olivier, J.-L., Chambaz, J., and Pillot, T. (2003) J. Biol. Chem. 278, 3437-3445). Because microtubule-associated proteins (MAPs) are responsible for the polymerization, stabilization, and dynamics of the microtubule network, we investigated whether MAPs might represent the intracellular targets that would enable us to explain the microtubule perturbation involved in soluble Abeta-mediated neuronal apoptosis. The data presented here show that soluble Abeta oligomers induce a time-dependent degradation of MAP1A, MAP1B, and MAP2 involving a perturbation of Ca2+ homeostasis with subsequent calpain activation that, on its own, is sufficient to induce the proteolysis of isoforms MAP2a, MAP2b, and MAP2c. In contrast, MAP1A and MAP1B sequential proteolysis results from the Abeta-mediated activation of caspase-3 and calpain. The prevention of MAP1A, MAP1B, and MAP2 proteolysis by antioxidants highlights the early reactive oxygen species generation in the perturbation of the microtubule network induced by soluble Abeta. These data clearly demonstrate the impact of cytoskeletal perturbations on soluble Abeta-mediated cell death and support the notion of microtubule-stabilizing agents as effective Alzheimer disease drugs.

Cytosolic phospholipase A2 mediates neuronal apoptosis induced by soluble oligomers of the amyloid-beta peptide.

Recent data have revealed that soluble oligomeric forms of amyloid peptide (Abeta) may be the proximate effectors of the neuronal injury and death occurring in Alzheimer's disease (AD). However, the molecular mechanisms associated with the neuronal cell death induced by the nonfibrillar Abeta remain to be elucidated. In this study, we investigated the role of the cytosolic Ca2+-dependent phospholipase A2 (cPLA2), and its associated metabolic pathway, i.e., the arachidonic acid (AA) cascade, in the apoptotic cell death induced by soluble oligomers of Abeta. The treatment of rat cortical neurons with low concentrations of soluble Abeta(1-40) or Abeta(1-42) peptide resulted in an early calcium-dependent release of AA associated with a transient relocalization of cPLA2. Both cPLA2 antisense oligonucleotides and a selective inhibitor of cPLA2 activity abolished the release of AA from neurons and also protected cells against apoptosis induced by Abeta. Furthermore, inhibitors of the PKC, p38, and MEK/ERK pathways that are involved in cPLA2 phosphorylation and activation reduced Abeta-induced cell death. Finally, we demonstrate that inhibitors of cyclooxygenase-2 reduced the Abeta-induced cell death by 55%. Our studies suggest a novel neuronal response of soluble oligomers of Abeta, which occurs through a cPLA2 signaling cascade and an AA-dependent death pathway. This may prove to be crucial in AD processes and could provide important targets for drug development.

Oligodendrocytes are susceptible to apoptotic cell death induced by prion protein-derived peptides.

Neurodegenerative prion diseases, characterized by a progressive dementia, are associated with the accumulation of abnormal forms of the prion (PrPc) protein, potentially due to an aberrant regulation of PrPc biogenesis and/or topology. One of these forms, termed ctmPrP, displays a transmembrane conformation and might trigger neuronal cell death in Gerstmann-Straüssler-Scheinker (GSS) syndrome and other prion-associated diseases in humans. Although the primary target cells involved in the progression of prion diseases remain unidentified, it was recently suggested that modifications of the oligodendroglial cells occur early in prion diseases. In the present study, we demonstrate that a putative transmembrane domain of the human PrPc, i.e., amino acids 118-135, induces oligodendrocyte (OLG) death in vitro in a time- and dose-dependent manner. The process leading to OLG death and induced by the PrP[118-135] peptide was characterized by DNA fragmentation, cytoskeletal disruption, and caspase activation. Protection against the PrP[118-135] peptide-induced OLG apoptosis by several antioxidant molecules, such as probucol, propylgallate, and promethazine, suggests that oxidative injuries contribute to the PrP[118-135] cytotoxicity to OLGs. These results suggest a potential pathophysiological role of the ctmPrP- and/or PrP fragment-mediated OLG cytotoxicity in spongiform encephalopathies.

Membrane cholesterol interferes with neuronal apoptosis induced by soluble oligomers but not fibrils of amyloid-beta peptide.

Neuronal cell death in Alzheimer's disease (AD) is partly induced by the interaction of the amyloid-beta peptide (Abeta) with the plasma membrane of target cells. Accordingly, recent studies have suggested that cholesterol, an important component of membranes that controls their physical properties and functions, plays a critical role in neurodegenerative diseases. We report here that the enrichment of the neuronal plasma membrane with cholesterol protects cortical neurons from apoptosis induced by soluble oligomers of the Abeta(1-40) peptide. Conversely, cholesterol depletion using cyclodextrin renders cells more vulnerable to the cytotoxic effects of the Abeta-soluble oligomers. Increasing the cholesterol content of small unilamellar liposomes also decreases Abeta-dependent liposome fusion. We clearly demonstrate that cholesterol protection is specific to the soluble conformation of Abeta, because we observed no protective effects on cortical neurons treated by amyloid fibrils of the Abeta(1-40) peptide. This may provide a new opportunity for the development of an effective AD therapy as well as elucidate the impact of the cholesterol level during AD development.

Humanin rescues cortical neurons from prion-peptide-induced apoptosis.

We recently demonstrated that a soluble oligomeric prion peptide, the putative 118-135 transmembrane domain of prion protein (PrP), exhibited membrane fusogenic properties and induced apoptotic cell death both in vitro and in vivo. A recently discovered rescue factor humanin (HN) was shown to protect neuronal cells from various insults involved in human neurodegenerative diseases. We thus addressed the question of whether HN might modulate the apoptosis induced by the soluble PrP(118-135) fragment. We found that the incubation of rat cortical neurons with 10 microM HN prevented soluble PrP(118-135) fragment-induced cell death concomitantly with inhibition of apoptotic events. An HN variant, termed HNG, exhibited a 500-fold increase in the protective activity in cortical neurons, whereas the HNA variant displayed no protective effect. The effects of HN and HNG peptides did not require a preincubation with the PrP(118-135) fragment, strongly suggesting that these peptides rescue cells independently of a direct interaction with the prion peptide. By contrast, and in agreement with a previous study, HN had no effect on the fibrillar PrP(106-126) peptide-induced cell death. This protective effect for neurons from PrP(118-135)-induced cell death strongly suggests that PrP(118-135) and PrP(106-126) peptides may trigger different pathways leading to neuronal apoptosis.

Apoptotic neuronal cell death induced by the non-fibrillar amyloid-beta peptide proceeds through an early reactive oxygen species-dependent cytoskeleton perturbation.

In the present study, we have determined the nature and the kinetics of the cellular events triggered by the exposure of cells to non-fibrillar amyloid-beta peptide (A beta). When cortical neurons were treated with low concentrations of soluble A beta (1-40), an early reactive oxygen species (ROS)-dependent cytoskeleton disruption precedes caspase activation. Indeed, caspase activation and neuronal cell death were prevented by the microtubule-stabilizing drug taxol. A perturbation of the microtubule network was noticeable after being exposed to A beta for 1 h, as revealed by electron microscopy and immunocytochemistry. Microtubule disruption and neuronal cell death induced by A beta were inhibited in the presence of antioxidant molecules, such as probucol. These data highlight the critical role of ROS production in A beta-mediated cytoskeleton disruption and neuronal cell death. Finally, using FRAP (fluorescence recovery after photo bleaching) analysis, we observed a time-dependent biphasic modification of plasma membrane fluidity, as early as microtubule disorganization. Interestingly, molecules that inhibited neurotubule perturbation and cell death did not affect the membrane destabilizing properties of A beta, suggesting that the lipid phase of the plasma membrane might represent the earliest target for A beta. Altogether our results convey the idea that upon interaction with the plasma membrane, the non-fibrillar A beta induces a rapid ROS-dependent disorganization of the cytoskeleton, which results in apoptosis.