Mònica Bosch-Morató

The blood-brain barrier: Structure, function and therapeutic approaches to cross it

Abstract The blood-brain barrier (BBB) is constituted by a specialized vascular endothelium that interacts directly with astrocytes, neurons and pericytes. It protects the brain from the molecules of the systemic circulation but it has to be overcome for the proper treatment of brain cancer, psychiatric disorders or neurodegenerative diseases, which are dramatically increasing as the population ages. In the present work we have revised the current knowledge on the cellular structure of the BBB and the different procedures utilized currently and those proposed to cross it. Chemical modifications of the drugs, such as increasing their lipophilicity, turn them more prone to be internalized in the brain. Other mechanisms are the use of molecular tools to bind the drugs such as small immunoglobulins, liposomes or nanoparticles that will act as Trojan Horses favoring the drug delivery in brain. This fusion of the classical pharmacology with nanotechnology has opened a wide field to many different approaches with promising results to hypothesize that BBB will not be a major problem for the new generation of neuroactive drugs. The present review provides an overview of all state-of-the-art of the BBB structure and function, as well as of the classic strategies and these appeared in recent years to deliver drugs into the brain for the treatment of Central Nervous System (CNS) diseases.

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.

The structure and function of actin cytoskeleton in mature glutamatergic dendritic spines

Dendritic spines are actin-rich protrusions from the dendritic shaft, considered to be the locus where most synapses occur, as they receive the vast majority of excitatory connections in the central nervous system (CNS). Interestingly, hippocampal spines are plastic structures that contain a dense array of molecules involved in postsynaptic signaling and synaptic plasticity. Since changes in spine shape and size are correlated with the strength of excitatory synapses, spine morphology directly reflects spine function. Therefore several neuropathologies are associated with defects in proteins located at the spines. The present work is focused on the spine actin cytoskeleton attending to its structure and function mainly in glutamatergic neurons. It addresses the study of the structural plasticity of dendritic spines associated with long-term potentiation (LTP) and the mechanisms that underlie learning and memory formation. We have integrated the current knowledge on synaptic proteins to relate this plethora of molecules with actin and actin-binding proteins. We further included recent findings that outline key uncharacterized proteins that would be useful to unveil the real ultrastructure and function of dendritic spines. Furthermore, this review is directed to understand how such spine diversity and interplay contributes to the regulation of spine morphogenesis and dynamics. It highlights their physiological relevance in the brain function, as well as it provides insights for pathological processes affecting dramatically dendritic spines, such as Alzheimers disease.

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.

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.

Methylglyoxal reduces mitochondrial potential and activates Bax and caspase-3 in neurons: Implications for Alzheimer's disease.

Alzheimers disease (AD) is characterized by the oxidative stress generated from amyloid β-peptide (Aβ) aggregates. It produces protein nitrotyrosination, after the reaction with nitric oxide to form peroxynitrite, being triosephosphate isomerase (TPI) one of the most affected proteins. TPI is a glycolytic enzyme that catalyzes the interconversion between glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP). Methylglyoxal (MG) is a by-product of TPI activity whose production is triggered when TPI is nitrotyrosinated. MG is harmful to cells because it glycates proteins. Here we found protein glycation when human neuroblastoma cells were treated with Aβ. Moreover glycation was also observed when neuroblastoma cells overexpressed mutated TPI where Tyr165 or Tyr209, the two tyrosines close to the catalytic center, were changed by Phe in order to mimic the effect of nitrotyrosination. The pathological relevance of these findings was studied by challenging cells with Aβ oligomers and MG. A significant decrease in mitochondrial transmembrane potential, one of the first apoptotic events, was obtained. Therefore, increasing concentrations of MG were assayed searching for MG effect in neuronal apoptosis. We found a decrease of the protective Bcl2 and an increase of the proapoptotic caspase-3 and Bax levels. Our results suggest that MG is triggering apoptosis in neurons and it would play a key role in AD neurodegeneration.

Posttranslational Nitro-Glycative Modifications of Albumin in Alzheimer's Disease: Implications in Cytotoxicity and Amyloid-β Peptide Aggregation

Glycation and nitrotyrosination are pathological posttranslational modifications that make proteins prone to losing their physiological properties. Since both modifications are increased in Alzheimers disease (AD) due to amyloid-β peptide (Aβ) accumulation, we have studied their effect on albumin, the most abundant protein in cerebrospinal fluid and blood. Brain and plasmatic levels of glycated and nitrated albumin were significantly higher in AD patients than in controls. In vitro turbidometry and electron microscopy analyses demonstrated that glycation and nitrotyrosination promote changes in albumin structure and biochemical properties. Glycated albumin was more resistant to proteolysis and less uptake by hepatoma cells occurred. Glycated albumin also reduced the osmolarity expected for a solution containing native albumin. Both glycation and nitrotyrosination turned albumin cytotoxic in a cell type-dependent manner for cerebral and vascular cells. Finally, of particular relevance to AD, these modified albumins were significantly less effective in avoiding Aβ aggregation than native albumin. In summary, nitrotyrosination and especially glycation alter albumin structural and biochemical properties, and these modifications might contribute for the progression of AD.

Methylglyoxal Produced by Amyloid-β Peptide-Induced Nitrotyrosination of Triosephosphate Isomerase Triggers Neuronal Death in Alzheimer's Disease

Amyloid-β peptide (Aβ) aggregates induce nitro-oxidative stress, contributing to the characteristic neurodegeneration found in Alzheimers disease (AD). One of the most strongly nitrotyrosinated proteins in AD is the triosephosphate isomerase (TPI) enzyme which regulates glycolytic flow, and its efficiency decreased when it is nitrotyrosinated. The main aims of this study were to analyze the impact of TPI nitrotyrosination on cell viability and to identify the mechanism behind this effect. In human neuroblastoma cells (SH-SY5Y), we evaluated the effects of Aβ42 oligomers on TPI nitrotyrosination. We found an increased production of methylglyoxal (MG), a toxic byproduct of the inefficient nitro-TPI function. The proapoptotic effects of Aβ42 oligomers, such as decreasing the protective Bcl2 and increasing the proapoptotic caspase-3 and Bax, were prevented with a MG chelator. Moreover, we used a double mutant TPI (Y165F and Y209F) to mimic nitrosative modifications due to Aβ action. Neuroblastoma cells transfected with the double mutant TPI consistently triggered MG production and a decrease in cell viability due to apoptotic mechanisms. Our data show for the first time that MG is playing a key role in the neuronal death induced by Aβ oligomers. This occurs because of TPI nitrotyrosination, which affects both tyrosines associated with the catalytic center.

Rare variants in calcium homeostasis modulator 1 (CALHM1) found in early onset Alzheimer's disease patients alter calcium homeostasis.

Calcium signaling in the brain is fundamental to the learning and memory process and there is evidence to suggest that its dysfunction is involved in the pathological pathways underlying Alzheimer’s disease (AD). Recently, the calcium hypothesis of AD has received support with the identification of the non-selective Ca2+-permeable channel CALHM1. A genetic polymorphism (p. P86L) in CALHM1 reduces plasma membrane Ca2+ permeability and is associated with an earlier age-at-onset of AD. To investigate the role of CALHM1 variants in early-onset AD (EOAD), we sequenced all CALHM1 coding regions in three independent series comprising 284 EOAD patients and 326 controls. Two missense mutations in patients (p.G330D and p.R154H) and one (p.A213T) in a control individual were identified. Calcium imaging analyses revealed that while the mutation found in a control (p.A213T) behaved as wild-type CALHM1 (CALHM1-WT), a complete abolishment of the Ca2+ influx was associated with the mutations found in EOAD patients (p.G330D and p.R154H). Notably, the previously reported p. P86L mutation was associated with an intermediate Ca2+ influx between the CALHM1-WT and the p.G330D and p.R154H mutations. Since neither expression of wild-type nor mutant CALHM1 affected amyloid ß-peptide (Aß) production or Aß-mediated cellular toxicity, we conclude that rare genetic variants in CALHM1 lead to Ca2+ dysregulation and may contribute to the risk of EOAD through a mechanism independent from the classical Aß cascade.

Amyloid-β Peptide Nitrotyrosination Stabilizes Oligomers and Enhances NMDAR-Mediated Toxicity.

Alzheimers disease (AD) is a neurodegenerative disorder characterized by the pathological aggregation of the amyloid-β peptide (Aβ). Monomeric soluble Aβ can switch from helicoidal to β-sheet conformation, promoting its assembly into oligomers and subsequently to amyloid fibrils. Oligomers are highly toxic to neurons and have been reported to induce synaptic transmission impairments. The progression from oligomers to fibrils forming senile plaques is currently considered a protective mechanism to avoid the presence of the highly toxic oligomers. Protein nitration is a frequent post-translational modification under AD nitrative stress conditions. Aβ can be nitrated at tyrosine 10 (Y10) by peroxynitrite. Based on our analysis of ThT binding, Western blot and electron and atomic force microscopy, we report that Aβ nitration stabilizes soluble, highly toxic oligomers and impairs the formation of fibrils. We propose a mechanism by which fibril elongation is interrupted upon Y10 nitration: Nitration disrupts fibril-forming folds by preventing H14-mediated bridging, as shown with an Aβ analog containing a single residue (H to E) replacement that mimics the behavior of nitrated Aβ related to fibril formation and neuronal toxicity. The pathophysiological role of our findings in AD was highlighted by the study of these nitrated oligomers on mouse hippocampal neurons, where an increased NMDAR-dependent toxicity of nitrated Aβ oligomers was observed. Our results show that Aβ nitrotyrosination is a post-translational modification that increases Aβ synaptotoxicity. SIGNIFICANCE STATEMENT We report that nitration (i.e., the irreversible addition of a nitro group) of the Alzheimer-related peptide amyloid-β (Aβ) favors the stabilization of highly toxic oligomers and inhibits the formation of Aβ fibrils. The nitrated Aβ oligomers are more toxic to neurons due to increased cytosolic calcium levels throughout their action on NMDA receptors. Sustained elevated calcium levels trigger excitotoxicity, a characteristic event in Alzheimers disease.