María Inés Barría

Activation of Wnt signaling rescues neurodegeneration and behavioral impairments induced by β -amyloid fibrils

Alzheimers disease (AD) is a progressive neurodegenerative disorder, which is probably caused by the cytotoxic effect of the amyloid β-peptide (Aβ). We report here molecular changes induced by Aβ, both in neuronal cells in culture and in rats injected in the dorsal hippocampus with preformed Aβ fibrils, as an in vivo model of the disease. Results indicate that in both systems, Aβ neurotoxicity resulted in the destabilization of endogenous levels of β-catenin, a key transducer of the Wnt signaling pathway. Lithium chloride, which mimics Wnt signaling by inhibiting glycogen synthase kinase-3β promoted the survival of post-mitotic neurons against Aβ neurotoxicity and recovered cytosolic β-catenin to control levels. Moreover, the neurotoxic effect of Aβ fibrils was also modulated with protein kinase C agonists/inhibitors and reversed with conditioned medium containing the Wnt-3a ligand. We also examined the spatial memory performance of rats injected with preformed Aβ fibrils in the Morris water maze paradigm, and found that chronic lithium treatment protected neurodegeneration by rescuing β-catenin levels and improved the deficit in spatial learning induced by Aβ. Our results are consistent with the idea that Aβ-dependent neurotoxicity induces a loss of function of Wnt signaling components and indicate that lithium or compounds that mimic this signaling cascade may be putative candidates for therapeutic intervention in Alzheimers patients.

β-sheet breaker peptide prevents Aβ-induced spatial memory impairments with partial reduction of amyloid deposits

Current evidence supports the notion that β-amyloid deposits or Aβ intermediates may be responsible for the pathogenesis in Alzheimers disease (AD) patients. In the present work, we have assessed the neuroprotective effect of the chronic intraperitoneal administration of a five-amino-acid β-sheet breaker peptide (iAβ5p) on the rat behavioral deficit induced by the intrahippocampal Aβ-fibrils injection. At 1 month after the injection, animals showed a partial reduction of the amyloid deposits formed and a decreased astrocytic response around the injection site. More importantly, we report that following the iAβ5p treatment, hippocampal-dependent spatial learning paradigms, including the standard Morris water maze and a working memory analysis, showed a significant prevention from impairments induced by Aβ deposits in the dorsal hippocampus. Thus, it is possible that a noninvasive treatment such as the one presented here with β-sheet breaker peptides may be used as a potential therapy for AD patients.

Copper reduction by copper binding proteins and its relation to neurodegenerative diseases

Increasing evidence supports an important role for metals in neurobiology. In fact, copper binding proteins that form bioinorganic complexes are able to display oxidant or anti-oxidant properties, which would impact on neuronal function or in the triggering of neurodegenerative process. Two proteins related to neurodegenerative diseases have been described as copper binding proteins: the amyloid precursor protein (APP), a protein related to Alzheimers disease, and the Prion protein (PrP), related to Creutzfeldt-Jakob disease. We used different synthetic peptides from APP and PrP sequences in order to evaluate the ability to reduce copper. We observed that APP135−156, amyloid-β-peptide (Aβ1−40), and PrP59−91 all have copper reducing ability, with the APP135−156 peptide being more potent than the other fragments. Moreover, we identify His, Cys and Trp residues as key amino acids involved in the copper reduction of Aβ, APP and PrP, respectively. We postulated, that in a cellular context, the interaction of these proteins with copper could be necessary to reduce copper on plasma membrane, possibly presenting Cu(I) to the copper transporter, driving the delivery of this metal to antioxidant enzymes. Moreover, protein-metal complexes could be the catalytic centers for the formation of reactive oxygen species involved in the oxidative damage present both in Alzheimers and Prion disease.

Wnt signaling involvement in β-amyloid-dependent neurodegeneration

Abstract Alzheimer’s disease (AD) is a progressive dementia paralleled by selective neuronal death, which is probably caused by the cytotoxic effects of the amyloid-β peptide (Aβ). We have observed that Aβ-dependent neurotoxicity induces a loss of function of Wnt signaling components and that activation of this signaling cascade prevent such cytotoxic effects. Therefore we propose that compounds which mimic this signaling cascade may be candidates for therapeutic intervention in Alzheimer’s patients.

Localized Mucosal Response to Intranasal Live Attenuated Influenza Vaccine in Adults

BACKGROUND Influenza virus infection is a major public health burden worldwide. Available vaccines include the inactivated intramuscular trivalent vaccine and, more recently, an intranasal live attenuated influenza vaccine (LAIV). The measure of successful vaccination with the inactivated vaccine is a systemic rise in immunoglobulin G (IgG) level, but for the LAIV no such correlate has been established. METHODS Seventy-nine subjects were given the LAIV FluMist. Blood was collected prior to vaccination and 3 days and 30 days after vaccination. Nasal wash was collected 3 days and 30 days after vaccination. Responses were measured systemically and in mucosal secretions for cytokines, cell activation profiles, and antibody responses. RESULTS Only 9% of subjects who received LAIV seroconverted, while 33% of patients developed at least a 2-fold increase in influenza virus-specific immunoglobulin A (IgA) antibodies in nasal wash. LAIV induced a localized inflammation, as suggested by increased expression of interferon-response genes in mucosal RNA and increased granulocyte colony-stimulating factor (G-CSF) and IP-10 in nasal wash. Interestingly, patients who seroconverted had significantly lower serum levels of G-CSF before vaccination. CONCLUSIONS Protection by LAIV is likely provided through mucosal IgA and not by increases in systemic IgG. LAIV induces local inflammation. Seroconversion is achieved in a small fraction of subjects with a lower serum G-CSF level.

ApoER2 expression increases Aβ production while decreasing Amyloid Precursor Protein (APP) endocytosis: Possible role in the partitioning of APP into lipid rafts and in the regulation of γ-secretase activity

BackgroundThe generation of the amyloid-β peptide (Aβ) through the proteolytic processing of the amyloid precursor protein (APP) is a central event in the pathogenesis of Alzheimers disease (AD). Recent studies highlight APP endocytosis and localization to lipid rafts as important events favoring amyloidogenic processing. However, the precise mechanisms underlying these events are poorly understood. ApoER2 is a member of the low density lipoprotein receptor (LDL-R) family exhibiting slow endocytosis rate and a significant association with lipid rafts. Despite the important neurophysiological roles described for ApoER2, little is known regarding how ApoER2 regulates APP trafficking and processing.ResultsHere, we demonstrate that ApoER2 physically interacts and co-localizes with APP. Remarkably, we found that ApoER2 increases cell surface APP levels and APP association with lipid rafts. The increase of cell surface APP requires the presence of ApoER2 cytoplasmic domain and is a result of decreased APP internalization rate. Unexpectedly, ApoER2 expression correlated with a significant increase in Aβ production and reduced levels of APP-CTFs. The increased Aβ production was dependent on the integrity of the NPxY endocytosis motif of ApoER2. We also found that expression of ApoER2 increased APP association with lipid rafts and increased γ-secretase activity, both of which might contribute to increased Aβ production.ConclusionThese findings show that ApoER2 negatively affects APP internalization. However, ApoER2 expression stimulates Aβ production by shifting the proportion of APP from the non-rafts to the raft membrane domains, thereby promoting β-secretase and γ-secretase mediated amyloidogenic processing and also by incrementing the activity of γ-secretase.

Analysis of natural variants of the hepatitis C virus internal ribosome entry site reveals that primary sequence plays a key role in cap-independent translation

The HCV internal ribosome entry site (IRES) spans a region of ∼340 nt that encompasses most of the 5′ untranslated region (5′UTR) of the viral mRNA and the first 24–40 nt of the core-coding region. To investigate the implication of altering the primary sequence of the 5′UTR on IRES activity, naturally occurring variants of the 5′UTR were isolated from clinical samples and analyzed. The impact of the identified mutations on translation was evaluated in the context of RLuc/FLuc bicistronic RNAs. Results show that depending on their location within the RNA structure, these naturally occurring mutations cause a range of effects on IRES activity. However, mutations within subdomain IIId hinder HCV IRES-mediated translation. In an attempt to explain these data, the dynamic behavior of the subdomain IIId was analyzed by means of molecular dynamics (MD) simulations. Despite the loss of function, MD simulations predicted that mutant G266A/G268U possesses a structure similar to the wt-RNA. This prediction was validated by analyzing the secondary structure of the isolated IIId RNAs by circular dichroism spectroscopy in the presence or absence of Mg2+ ions. These data strongly suggest that the primary sequence of subdomain IIId plays a key role in HCV IRES-mediated translation.

The N-terminal copper-binding domain of the amyloid precursor protein protects against Cu2+ neurotoxicity in vivo

The amyloid precursor protein (APP) contains a Cu binding domain (CuBD) localized between amino acids 135 and 156 (APP135‐156), which can reduce Cu2+ to Cu1+ in vitro. The physiological function of this APP domain has not yet being established; nevertheless several studies support the notion that the CuBD of APP is involved in Cu homeostasis. We used APP synthetic peptides to evaluate their protective properties against Cu2+ neurotoxicity in a bilateral intra‐hippocampal injection model. We found that human APP135‐156 protects against Cu2+‐induced neurotoxic effects, such as, impairment of spatial memory, neuronal cell loss, and astrogliosis. APP135‐156 lacking two histidine residues showed protection against Cu2+; however, APP135‐156 mutated in cysteine 144, a key residue in the reduction of Cu2+ to Cu1+, did not protect against Cu2+ neurotoxicity. In accordance with recent reports, the CuBD of the Caenorhabditis elegans, APL‐1, protected against Cu2+ neurotoxicity in vivo. We also found that Cu2+ neurotoxicity is associated with an increase in nitrotyrosine immunofluorescence as well as with a decrease in Cu2+ uptake. The CuBD of APP therefore may play a role in the detoxification of brain Cu.

A human prion protein peptide (PrP59-91) protects against copper neurotoxicity

Human cellular prion protein (PrPC) is involved in several neurodegenerative disorders; however, its normal function is unknown. We report here that a synthetic peptide corresponding to the four-octarepeat sequence of the PrPC (PrP59–91) protects hippocampal neurons against copper neurotoxic effects in vivo. Using a rat bilateral intrahippocampal injection model, we found that PrP59–91 protects against copper-induced neurotoxicity, including a recovery in spatial learning performance and a reduced neuronal cell loss and astrogliosis. Previous studies from our laboratory indicated that a tryptophan (Trp) residue plays a key role in the reduction of copper(II) to copper(I); therefore several PrP59–91 fragments lacking histidine (His) and Trp residues were tested for their capacity to protect from copper toxicity. A PrP59–91 peptide lacking His residue shows as much neuroprotection as the native peptide; however, PrP59–91 without Trp residues only partially protected against copper toxicity. The neuroprotective effect not only occurs with PrP59–91, in fact a full neuroprotection was also observed using just one octamer of the N-terminal region of prion protein. We conclude that the N-terminal tandem octarepeat of the human PrPC protects neurons against copper toxicity by a differential contribution of the binding (His) and reducing (Trp) copper activities of PrP59–91. Our results are consistent with the idea that PrPC function is related to copper homeostasis.

Hepatitis C virus quasispecies in plasma and peripheral blood mononuclear cells of treatment naïve chronically infected patients.

Summary.  Peripheral blood mononuclear cells (PBMCs) from 45 treatment naïve, HIV‐negative, chronically hepatitis C virus (HCV)‐infected patients were analyzed for the presence of HCV RNA. Viral RNA was detected in 73% of the studied patients. Single‐strand conformation polymorphism assays and sequence analysis of the HCV 5′untranslated regions amplified from RNA recovered from both Plasma and PBMCs suggested virus compartmentalization in 57.6% of patients studied. In summary, our study presents evidence that HCV RNA can be found in PBMCs of treatment naïve chronically infected patients that are not immunocompromised or co‐infected with the human immunodeficiency virus.