AAV-mediated neuronal expression of an scFv antibody selective for Aß oligomers protects synapses and rescues memory in Alzheimer models.

The accumulation of soluble oligomers of the amyloid-ß peptide (AßOs) in the brain has been implicated in synapse failure and memory impairment in Alzheimer's disease. Here, we initially show that treatment with NUsc1, a single-chain variable-fragment antibody (scFv) that selectively targets a subpopulation of AßOs and shows minimal reactivity to Aß monomers and fibrils, prevents the inhibition of long-term potentiation in hippocampal slices and memory impairment induced by AßOs in mice. As a therapeutic approach for intracerebral antibody delivery, we developed an adeno-associated virus vector to drive neuronal expression of NUsc1 (AAV-NUsc1) within the brain. Transduction by AAV-NUsc1 induced NUsc1 expression and secretion in adult human brain slices and inhibited AßO binding to neurons and AßO-induced loss of dendritic spines in primary rat hippocampal cultures. Treatment of mice with AAV-NUsc1 prevented memory impairment induced by AßOs and, remarkably, reversed memory deficits in aged APPswe/PS1ΔE9 Alzheimer's disease model mice. These results support the feasibility of immunotherapy using viral vector-mediated gene delivery of NUsc1 or other AßO-specific single-chain antibodies as a potential therapeutic approach in Alzheimer's disease.

Neural Infection by Oropouche Virus in Adult Human Brain Slices Induces an Inflammatory and Toxic Response.

Oropouche virus (OROV) is an emerging arbovirus in South and Central Americas with high spreading potential. OROV infection has been associated with neurological complications and OROV genomic RNA has been detected in cerebrospinal fluid from patients, suggesting its neuroinvasive potential. Motivated by these findings, neurotropism and neuropathogenesis of OROV have been investigated in vivo in murine models, which do not fully recapitulate the complexity of the human brain. Here we have used slice cultures from adult human brains to investigate whether OROV is capable of infecting mature human neural cells in a context of preserved neural connections and brain cytoarchitecture. Our results demonstrate that human neural cells can be infected ex vivo by OROV and support the production of infectious viral particles. Moreover, OROV infection led to the release of the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α) and diminished cell viability 48 h post-infection, indicating that OROV triggers an inflammatory response and tissue damage. Although OROV-positive neurons were observed, microglia were the most abundant central nervous system (CNS) cell type infected by OROV, suggesting that they play an important role in the response to CNS infection by OROV in the adult human brain. Importantly, we found no OROV-infected astrocytes. To the best of our knowledge, this is the first direct demonstration of OROV infection in human brain cells. Combined with previous data from murine models and case reports of OROV genome detection in cerebrospinal fluid from patients, our data shed light on OROV neuropathogenesis and help raising awareness about acute and possibly chronic consequences of OROV infection in the human brain.

Antibody Fragments as Tools for Elucidating Structure-Toxicity Relationships and for Diagnostic/Therapeutic Targeting of Neurotoxic Amyloid Oligomers.

The accumulation of amyloid protein aggregates in tissues is the basis for the onset of diseases known as amyloidoses. Intriguingly, many amyloidoses impact the central nervous system (CNS) and usually are devastating diseases. It is increasingly apparent that neurotoxic soluble oligomers formed by amyloidogenic proteins are the primary molecular drivers of these diseases, making them lucrative diagnostic and therapeutic targets. One promising diagnostic/therapeutic strategy has been the development of antibody fragments against amyloid oligomers. Antibody fragments, such as fragment antigen-binding (Fab), scFv (single chain variable fragments), and VHH (heavy chain variable domain or single-domain antibodies) are an alternative to full-length IgGs as diagnostics and therapeutics for a variety of diseases, mainly because of their increased tissue penetration (lower MW compared to IgG), decreased inflammatory potential (lack of Fc domain), and facile production (low structural complexity). Furthermore, through the use of in vitro-based ligand selection, it has been possible to identify antibody fragments presenting marked conformational selectivity. In this review, we summarize significant reports on antibody fragments selective for oligomers associated with prevalent CNS amyloidoses. We discuss promising results obtained using antibody fragments as both diagnostic and therapeutic agents against these diseases. In addition, the use of antibody fragments, particularly scFv and VHH, in the isolation of unique oligomeric assemblies is discussed as a strategy to unravel conformational moieties responsible for neurotoxicity. We envision that advances in this field may lead to the development of novel oligomer-selective antibody fragments with superior selectivity and, hopefully, good clinical outcomes.

Simvastatin Prevents Long-Term Cognitive Deficits in Sepsis Survivor Rats by Reducing Neuroinflammation and Neurodegeneration.

Sepsis-associated encephalopathy causes brain dysfunction that can result in cognitive impairments in sepsis survivor patients. In previous work, we showed that simvastatin attenuated oxidative stress in brain structures related to memory in septic rats. However, there is still a need to evaluate the long-term impact of simvastatin administration on brain neurodegenerative processes and cognitive damage in sepsis survivors. Here, we investigated the possible neuroprotective role of simvastatin in neuroinflammation, and neurodegeneration conditions of brain structures related to memory in rats at 10 days after sepsis survival. Male Wistar rats (250-300 g) were submitted to cecal ligation and puncture (CLP, n = 42) or remained as non-manipulated (naïve, n = 30). Both groups were treated (before and after the surgery) by gavage with simvastatin (20 mg/kg) or an equivalent volume of saline and observed for 10 days. Simvastatin-treated rats that survived to sepsis showed a reduction in the levels of nitrate, IL1-ß, and IL-6 and an increase in Bcl-2 protein expression in the prefrontal cortex and hippocampus, and synaptophysin only in the hippocampus. Immunofluorescence revealed a reduction of glial activation, neurodegeneration, apoptosis, and amyloid aggregates confirmed by quantification of GFAP, Iba-1, phospho Ser396-tau, total tau, cleaved caspase-3, and thioflavin-S in the prefrontal cortex and hippocampus. In addition, treated animals presented better performance in tasks involving habituation memory, discriminative, and aversive memory. These results suggest that statins exert a neuroprotective role by upregulation of the Bcl-2 and gliosis reduction, which may prevent the cognitive deficit observed in sepsis survivor animals.

Short-Term Free-Floating Slice Cultures from the Adult Human Brain.

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of slice cultures in neuroscience, studies using adult nervous tissue to prepare such cultures are still scarce, particularly those from human subjects. The use of adult human tissue to prepare slice cultures is particularly attractive to enhance the understanding of human neuropathologies, as they hold unique properties typical of the mature human brain lacking in slices produced from rodent (usually neonatal) nervous tissue. This protocol describes how to use brain tissue collected from living human donors submitted to resective brain surgery to prepare short-term, free-floating slice cultures. Procedures to maintain and perform biochemical and cell biology assays using these cultures are also presented. Representative results demonstrate that the typical human cortical lamination is preserved in slices after 4 days in vitro (DIV4), with expected presence of the main neural cell types. Moreover, slices at DIV4 undergo robust cell death when challenged with a toxic stimulus (H2O2), indicating the potential of this model to serve as a platform in cell death assays. This method, a simpler and cost-effective alternative to the widely used protocol using membrane inserts, is mainly recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases. Finally, although the protocol is devoted to using cortical tissue collected from patients submitted to surgical treatment of pharmacoresistant temporal lobe epilepsy, it is argued that tissue collected from other brain regions/conditions should also be considered as sources to produce similar free-floating slice cultures.

A phylogenetically conserved hnRNP type A/B protein from squid brain.

Eukaryotic mRNA precursors are co-transcriptionally assembled into ribonucleoprotein complexes. Heterogeneous nuclear ribonucleoprotein (hnRNP) complexes are involved in mRNA translocation, stability, subcellular localization and regulation of mRNA translation. About 20 major classes of hnRNPs have been identified in mammals. In a previous work, we characterized a novel, strongly-basic, RNA-binding protein (p65) in presynaptic terminals of squid neurons presenting homology with human hnRNPA/B type proteins, likely involved in local mRNA processing. We have identified and sequenced two hnRNPA/B-like proteins associated with tissue purified squid p65: Protein 1 (36.3 kDa, IP 7.1) and Protein 2 (37.6 kDa, IP 8.9). In the present work we generated an in silico, tridimensional, structural model of squid hnRNPA/B-like Protein 2, which showed highly conserved secondary and tertiary structure of RNA recognition motifs with human hnRNPA1 protein, as well as illustrated the potential for squid Protein 2 stable homodimerization. This was supported by biophysical measurements of bacterially expressed, recombinant protein. In addition, we induced expression of squid hnRNPA/B-like Protein 2 in human neuroblastoma cells (SH-SY5Y) and observed an exclusively nuclear localization, which depended on an intact C-terminal amino acid sequence and which relocated to cytoplasm particles containing PABP when the cells were challenged with sorbitol, suggesting an involvement with stress granule function.

Amyloid ß-peptide oligomers stimulate RyR-mediated Ca2+ release inducing mitochondrial fragmentation in hippocampal neurons and prevent RyR-mediated dendritic spine remodeling produced by BDNF.

Soluble amyloid ß-peptide oligomers (AßOs), increasingly recognized as causative agents of Alzheimer's disease (AD), disrupt neuronal Ca(2+) homeostasis and synaptic function. Here, we report that AßOs at sublethal concentrations generate prolonged Ca(2+) signals in primary hippocampal neurons; incubation in Ca(2+)-free solutions, inhibition of ryanodine receptors (RyRs) or N-methyl-d-aspartate receptors (NMDARs), or preincubation with N-acetyl-l-cysteine abolished these signals. AßOs decreased (6 h) RyR2 and RyR3 mRNA and RyR2 protein, and promoted mitochondrial fragmentation after 24 h. NMDAR inhibition abolished the RyR2 decrease, whereas RyR inhibition prevented significantly the RyR2 protein decrease and mitochondrial fragmentation induced by AßOs. Incubation with AßOs (6 h) eliminated the RyR2 increase induced by brain-derived nerve factor (BDNF) and the dendritic spine remodeling induced within minutes by BDNF or the RyR agonist caffeine. Addition of BDNF to neurons incubated with AßOs for 24 h, which had RyR2 similar to and slightly higher RyR3 protein content than those of controls, induced dendritic spine growth but at slower rates than in controls. These combined effects of sublethal AßOs concentrations (which include redox-sensitive stimulation of RyR-mediated Ca(2+) release, decreased RyR2 protein expression, mitochondrial fragmentation, and prevention of RyR-mediated spine remodeling) may contribute to impairing the synaptic plasticity in AD.

Expression profile of rat hippocampal neurons treated with the neuroprotective compound 2,4-dinitrophenol: up-regulation of cAMP signaling genes.

2,4-Dinitrophenol (DNP) is classically known as a mitochondrial uncoupler and, at high concentrations, is toxic to a variety of cells. However, it has recently been shown that, at subtoxic concentrations, DNP protects neurons against a variety of insults and promotes neuronal differentiation and neuritogenesis. The molecular and cellular mechanisms underlying the beneficial neuroactive properties of DNP are still largely unknown. We have now used DNA microarray analysis to investigate changes in gene expression in rat hippocampal neurons in culture treated with low micromolar concentrations of DNP. Under conditions that did not affect neuronal viability, high-energy phosphate levels or mitochondrial oxygen consumption, DNP induced up-regulation of 275 genes and down-regulation of 231 genes. Significantly, several up-regulated genes were linked to intracellular cAMP signaling, known to be involved in neurite outgrowth, synaptic plasticity, and neuronal survival. Differential expression of specific genes was validated by quantitative RT-PCR using independent samples. Results shed light on molecular mechanisms underlying neuroprotection by DNP and point to possible targets for development of novel therapeutics for neurodegenerative disorders.

Soluble oligomers from a non-disease related protein mimic Abeta-induced tau hyperphosphorylation and neurodegeneration.

Protein aggregation and amyloid accumulation in different tissues are associated with cellular dysfunction and toxicity in important human pathologies, including Alzheimer's disease and various forms of systemic amyloidosis. Soluble oligomers formed at the early stages of protein aggregation have been increasingly recognized as the main toxic species in amyloid diseases. To gain insight into the mechanisms of toxicity instigated by soluble protein oligomers, we have investigated the aggregation of hen egg white lysozyme (HEWL), a normally harmless protein. HEWL initially aggregates into beta-sheet rich, roughly spherical oligomers which appear to convert with time into protofibrils and mature amyloid fibrils. HEWL oligomers are potently neurotoxic to rat cortical neurons in culture, while mature amyloid fibrils are little or non-toxic. Interestingly, when added to cortical neuronal cultures HEWL oligomers induce tau hyperphosphorylation at epitopes that are characteristically phosphorylated in neurons exposed to soluble oligomers of the amyloid-beta peptide. Furthermore, injection of HEWL oligomers in the cerebral cortices of adult rats induces extensive neurodegeneration in different brain areas. These results show that soluble oligomers from a non-disease related protein can mimic specific neuronal pathologies thought to be induced by soluble amyloid-beta peptide oligomers in Alzheimer's disease and support the notion that amyloid oligomers from different proteins may share common structural determinants that would explain their generic cytotoxicities.

Heparin-binding sites in granulocyte-macrophage colony-stimulating factor. Localization and regulation by histidine ionization.

The biological activity of granulocyte-macrophage colony-stimulating factor (GM-CSF) is modulated by the sulfated glycosaminoglycans (GAGs) heparan sulfate and heparin. However, the molecular mechanisms involved in such interactions are still not completely understood. We have proposed previously that helix C, one of the four alpha-helices of human GM-CSF (hGM-CSF), contains a GAG-binding site in which positively charged residues are spatially positioned for interaction with the sulfate moieties of the GAGs (Wettreich, A., Sebollela, A., Carvalho, M. A., Azevedo, S. P., Borojevic, R., Ferreira, S. T., and Coelho-Sampaio, T. (1999) J. Biol. Chem. 274, 31468-31475). Protonation of two histidine residues (His83 and His87) in helix C of hGM-CSF appears to act as a pH-dependent molecular switch to control the interaction with GAGs. Based on these findings, we have now generated a triple mutant form of murine GM-CSF (mGM-CSF) in which three noncharged residues in helix C of the murine factor (Tyr83, Gln85, and Tyr87) were replaced by the corresponding basic residues present in hGM-CSF (His83, Lys85, and His87). Binding assays on heparin-Sepharose showed that, at acidic pH, the triple mutant mGM-CSF binds to immobilized heparin with significantly higher affinity than wild type (WT) mGM-CSF and that neither protein binds to the column at neutral pH. The fact that even WT mGM-CSF binds to heparin at acidic pH indicates the existence of a distinct, lower affinity heparin-binding site in the protein. Chemical modification of the single histidine residue (His15) located in helix A of WT mGM-CSF with diethyl pyrocarbonate totally abolished binding to immobilized heparin. Moreover, replacement of His15 for an alanine residue significantly reduced the affinity of mGM-CSF for heparin at pH 5.0 and completely blocked heparin binding to a synthetic peptide corresponding to helix A of GM-CSF. These results indicate a major role of histidine residues in the regulation of the binding of GM-CSF to GAGs, supporting the notion that an acidic microenvironment is required for GM-CSF-dependent regulation of target cells. In addition, our results provide insight into the molecular basis of the strict species specificity of the biological activity of GM-CSF.

Amyloidogenicity and cytotoxicity of recombinant mature human islet amyloid polypeptide (rhIAPP).

Pancreatic amyloid plaques formed by the pancreatic islet amyloid polypeptide (IAPP) are present in more than 95% of type II diabetes mellitus patients, and their abundance correlates with the severity of the disease. IAPP is currently considered the most amyloidogenic peptide known, but the molecular bases of its aggregation are still incompletely understood. Detailed characterization of the mechanisms of amyloid formation requires large quantities of pure material. Thus, availability of recombinant IAPP in sufficient amounts for such studies constitutes an important step toward elucidation of the mechanisms of amyloidogenicity. Here, we report, for the first time, the successful expression, purification and characterization of the amyloidogenicity and cytotoxicity of recombinant human mature IAPP. This approach is likely to be useful for the production of other amyloidogenic peptides or proteins that are difficult to obtain by chemical synthesis.

Inhibition of yeast glutathione reductase by trehalose: possible implications in yeast survival and recovery from stress.

Accumulation of trehalose has been implicated in the tolerance of yeast cells to several forms of stress, including heat-shock and high ethanol levels. However, yeast lacking trehalase, the enzyme that degrades trehalose, exhibit poor survival after exposure to stress conditions. This suggests that optimal cell viability also depends on the capacity to rapidly degrade the high levels of trehalose that build up under stress. Here, we initially examined the effects of trehalose on the activity of an important antioxidant enzyme, glutathione reductase (GR), from Saccharomyces cerevisiae. At 25 degrees C, GR was inhibited by trehalose in a dose-dependent manner, with 70% inhibition at 1.5M trehalose. The inhibition was practically abolished at 40 degrees C, a temperature that induces a physiological response of trehalose accumulation in yeast. The inhibition of GR by trehalose was additive to the inhibition caused by ethanol, indicating that enzyme function is drastically affected upon ethanol-induced stress. Moreover, two other yeast enzymes, cytosolic pyrophosphatase and glucose 6-phosphate dehydrogenase, showed temperature dependences on inhibition by trehalose that were similar to the temperature dependence of GR inhibition. These results are discussed in terms of the apparent paradox represented by the induction of enzymes involved in both synthesis and degradation of trehalose under stress, and suggest that the persistence of high levels of trehalose after recovery from stress could lead to the inactivation of important yeast enzymes.