Amyloid-beta peptide and tau protein crosstalk in Alzheimer's disease.

Alzheimer's disease is a neurodegenerative disease that accounts for most of the 50-million dementia cases worldwide in 2018. A large amount of evidence supports the amyloid cascade hypothesis, which states that amyloid-beta accumulation triggers tau hyperphosphorylation and aggregation in form of neurofibrillary tangles, and these aggregates lead to inflammation, synaptic impairment, neuronal loss, and thus to cognitive decline and behavioral abnormalities. The poor correlation found between cognitive decline and amyloid plaques, have led the scientific community to question whether amyloid-beta accumulation is actually triggering neurodegeneration in Alzheimer's disease. The occurrence of tau neurofibrillary tangles better correlates to neuronal loss and clinical symptoms and, although amyloid-beta may initiate the cascade of events, tau impairment is likely the effector molecule of neurodegeneration. Recently, it has been shown that amyloid-beta and tau cooperatively work to impair transcription of genes involved in synaptic function and, more importantly, that downregulation of tau partially reverses transcriptional perturbations. Despite mounting evidence points to an interplay between amyloid-beta and tau, some factors could independently affect both pathologies. Thus, the dual pathway hypothesis, which states that there are common upstream triggers causing both amyloid-beta and tau abnormalities has been proposed. Among others, the immune system seems to be strongly involved in amyloid-beta and tau pathologies. Other factors, as the apolipoprotein E ε4 isoform has been suggested to act as a link between amyloid-beta and tau hyperphosphorylation. Interestingly, amyloid-beta-immunotherapy reduces not only amyloid-beta but also tau levels in animal models and in clinical trials. Likewise, it has been shown that tau-immunotherapy also reduces amyloid-beta levels. Thus, even though amyloid-beta immunotherapy is more advanced than tau-immunotherapy, combined amyloid-beta and tau-directed therapies at early stages of the disease have recently been proposed as a strategy to stop the progression of Alzheimer's disease.

Production of Therapeutic Single-Chain Variable Fragments (ScFv) in Pichia pastoris.

The interest in the use of monoclonal antibodies as therapeutic molecules has raised in the recent years. Due to their high affinity and specificity towards other biological molecules, antibodies are being widely used to treat a broad range of human diseases such as cancer, rheumatism, and cardiovascular diseases. Currently, the production of IgG-like antibodies is mainly obtained from stable or transient mammalian expression systems that allow proper folding and posttranslational modifications. Despite the technological advances of the last decade, the use of these systems still has a rather high production cost and long processing times. For these reasons, researchers are increasingly interested in alternative antibody production methods as well as alternative antibody formats. Bacterial systems, such as Escherichia coli, are extensively being used for recombinant protein production because their easy manipulation and cheap costs. However, the presence of lipopolysaccharides (LPS) traces in the already fractionated recombinant protein makes these systems not good candidates for the preparation of therapeutic molecules. Yeast systems, such as Pichia pastoris, present the convenient easy manipulation of microbial systems but show some key advantages of eukaryotic expression systems, like improved folding machinery and absence of LPS. They are especially suitable for the production of antibody fragments, which do not need human-like glycosylation, avoiding the high costs of mammalian systems. Here, the protocol for the expression and purification of a single-chain antibody fragment (scFv) in P. pastoris is provided, in deep detail for lab manipulation and briefly for a 5L-bioreactor production.

Cognitive Impairment in the 3xTg-AD Mouse Model of Alzheimer's Disease is Affected by Aß-ImmunoTherapy and Cognitive Stimulation.

Clinical symptoms of Alzheimer's Disease (AD) include behavioral alterations and cognitive impairment. These functional phenotypes early occur in triple-transgenic (3xTg-AD) mice. Specifically, behavioral alterations are first detected when mice are at around 2.5 months old and cognitive impairment in between 3- and 5-month-old mice. In this work, the effect of chronic Aß-immunotherapy on behavioral and cognitive abilities was tested by monthly administering the antibody fragment scFv-h3D6 to 3xTg-AD female mice from 5 to 9 months of age. An untreated group was used as a reference, as well as to attain some information on the effect of training during the longitudinal study. Behavioral and psychological symptoms of dementia (BPSD)-like symptoms were already evident in 5-month-old mice, in the form of neophobia and anxious-like behavior. The exploratory activity decreased over the longitudinal study, not only for 3xTgAD mice but also for the corresponding non-transgenic mice (NTg). Learning abilities of 3xTg-AD mice were not seriously compromised but an impairment in long-term spatial memory was evident at 5 months of age. Interestingly, scFv-h3D6-treatment affected the cognitive impairment displayed by 5-month-old 3xTg-AD mice. It is worth noting that training also reduced cognitive impairment of 3xTg-AD mice over the longitudinal study, suggesting that to properly quantify the isolated therapeutic potential of any drug on cognition using this model it is convenient to perform a prompt, age-matched study rather than a longitudinal study. In addition, a combination of both training and Aß-immunotherapy could constitute a possible approach to treat Alzheimer's disease.

Both Amyloid-ß Peptide and Tau Protein Are Affected by an Anti-Amyloid-ß Antibody Fragment in Elderly 3xTg-AD Mice.

Alzheimer's disease (AD) is the most common dementia worldwide. According to the amyloid hypothesis, the early accumulation of the Aß-peptide triggers tau phosphorylation, synaptic dysfunction, and eventually neuronal death leading to cognitive impairment, as well as behavioral and psychological symptoms of dementia. ScFv-h3D6 is a single-chain variable fragment that has already shown its ability to diminish the amyloid burden in 5-month-old 3xTg-AD mice. However, tau pathology is not evident at this early stage of the disease in this mouse model. In this study, the effects of scFv-h3D6 on Aß and tau pathologies have been assessed in 22-month-old 3xTg-AD mice. Briefly, 3xTg-AD female mice were treated for 2 weeks with scFv-h3D6 and compared with 3xTg-AD and non-transgenic (NTg) mice treated with PBS. The treatment with scFv-h3D6 was unequivocally effective in reducing the area of Aß staining. Furthermore, a tendency for a reduction in tau levels was also observed after treatment that points to the interplay between Aß and tau pathologies. The pro-inflammatory state observed in the 3xTg-AD mice did not progress after scFv-h3D6 treatment. In addition, the treatment did not alter the levels of apolipoprotein E or apolipoprotein J. Thus, a 2-week treatment with scFv-h3D6 was able to reduce AD-like pathology in elderly 3xTg-AD female mice.

Low-density lipoprotein aggregation is inhibited by apolipoprotein J-derived mimetic peptide D-[113-122]apoJ.

Mimetic peptides are promising therapeutic agents for atherosclerosis prevention. A 10-residue class G* peptide from apolipoprotein J (apoJ), namely, D-[113-122]apoJ, possesses anti-inflammatory and anti-atherogenic properties. This prompted us to determine its effect on the aggregation process of low-density lipoprotein (LDL) particles, an early event in the development of atherosclerosis. LDL particles with and without [113-122]apoJ peptide were incubated at 37 °C with sphingomyelinase (SMase) or were left to aggregate spontaneously at room temperature. The aggregation process was analyzed by size-exclusion chromatography (SEC), native gradient gel electrophoresis (GGE), absorbance at 405 nm, dynamic light scattering (DLS), and transmission electronic microscopy (TEM). In addition, circular dichroism was used to determine changes in the secondary structure of apoB, and SDS-PAGE was performed to assess apoB degradation. At an equimolar ratio of [113-122]apoJ peptide to apoB-100, [113-122]apoJ inhibited both SMase-induced or spontaneous LDL aggregation. All methods showed that [113-122]apoJ retarded the progression of SMase-induced LDL aggregation at long incubation times. No effect of [113-122]apoJ on apoB secondary structure was observed. Binding experiments showed that [113-122]apoJ presents low affinity for native LDL but binds readily to LDL during the first stages of aggregation. Laurdan fluorescence experiments showed that mild aggregation of LDL resulted in looser lipid packaging, which was partially prevented by D-[113-122]apoJ. These results demonstrate that [113-122]apoJ peptide prevents SMase-induced LDL aggregation at an equimolar ratio and opens the possibility for the use of this peptide as a therapeutic tool.

Treatment with scFv-h3D6 Prevented Neuronal Loss and Improved Spatial Memory in Young 3xTg-AD Mice by Reducing the Intracellular Amyloid-ß Burden.

The intracellular deposition of amyloid-ß (Aß) peptides has been described in the brains of both Alzheimer's disease (AD) patients and animal models. A correlation between the intracellular amyloid burden and neurodegeneration has recently been reported in a triple-transgenic AD (3xTg-AD) murine model. In the present study, we assessed the effect of scFv-h3D6, an anti-Aß single-chain variable fragment (scFv) derived from the antibody bapineuzumab, on amyloid pathology in 5-month-old 3xTg-AD female mice, focusing on intracellular Aß clearance, neuronal survival, and functional abilities. We also examined neuroinflammation and the histology of peripheral organ samples to detect any adverse effects. A single intraperitoneal injection of scFv-h3D6 dramatically reduced intracellular Aß burden in the deep layers of the cerebral cortex, pyramidal cells layer of the hippocampus, and basolateral amygdalar nucleus. The treatment prevented neuronal loss in the hippocampus and amygdala, while neither astrogliosis nor microgliosis was induced. Instead, an increase in the size of the white pulp after the treatment indicated that the spleen could be involved in the clearance mechanism. Although the treatment did not ameliorate behavioral and psychological symptoms of dementia-like symptoms, the results of cognitive testing pointed to a noticeable improvement in spatial memory. These findings indicated that the mechanism underlying the therapeutic effect of scFv-h3D6 was the clearance of intracellular Aß, with subsequent prevention of neuronal loss and amelioration of cognitive disabilities. The treatment was safe in terms of neuroinflammation and kidney and liver function, whereas some effects on the spleen were observed.

Pharmacokinetic parameters and mechanism of action of an efficient anti-Aß single chain antibody fragment.

The success of the targeting of amyloid-ß (Aß) oligomers through immunotherapy in Alzheimer's disease (AD) mouse models has not been translated into the clinics. The use of single-chain variable fragments (scFvs) has been proposed to prevent the potential severe effects of full-length mAbs by precluding crystallizable fraction-mediated microglia activation. The efficacy of scFv-h3D6, a bapineuzumab-derived anti-Aß scFv, has been extensively proven. In this work, we compared scFv-h3D6-EL, an elongated variant of the scFv-h3D6, with its original version to assess whether its characteristic higher thermodynamic stability improved its pharmacokinetic parameters. Although scFv-h3D6-EL had a longer half-life than its original version, its absorption from the peritoneal cavity into the systemic compartment was lower than that of the original version. Moreover, we attempted to determine the mechanism underlying the protective effect of scFv-h3D6. We found that scFv-h3D6 showed compartmental distribution and more interestingly crossed the blood-brain barrier. In the brain, scFv-h3D6 was engulfed by glial cells or internalized by Aß peptide-containing neurons in the early phase post-injection, and was colocalized with the Aß peptide almost exclusively in glial cells in the late phase post-injection. Aß peptide levels in the brain decreased simultaneously with an increase in scFv-h3D6 levels. This observation in addition to the increased tumor necrosis factor-α levels in the late phase post-injection suggested that the engulfment of Aß peptide/scFv-h3D6 complex extruded from large neurons by phagocytic cells was the mechanism underlying Aß peptide withdrawal. The mechanism of action of scFv-h3D6 demonstrates the effectivity of Aß-immunotherapy and lays the background for other studies focused on the finding of a treatment for AD.

Molecular basis for the protective effects of low-density lipoprotein receptor-related protein 1 (LRP1)-derived peptides against LDL aggregation.

Aggregated LDL is the first ligand reported to interact with the cluster II CR9 domain of low-density lipoprotein receptor-related protein 1 (LRP1). In particular, the C-terminal half of domain CR9, comprising the region Gly1127-Cys1140 exclusively recognizes aggregated LDL and it is crucial for aggregated LDL binding. Our aim was to study the effect of the sequence Gly1127-Cys1140 (named peptide LP3 and its retro-enantio version, named peptide DP3) on the structural characteristics of sphingomyelinase- (SMase) and phospholipase 2 (PLA2)-modified LDL particles. Turbidimetry, gel filtration chromatography (GFC) and transmission electronic microscopy (TEM) analysis showed that LP3 and DP3 peptides strongly inhibited SMase- and PLA2-induced LDL aggregation. Nondenaturing polyacrylamide gradient gel electrophoresis (GGE), agarose gel electrophoresis and high-performance thin-layer chromatography (HPTLC) indicated that LP3 and DP3 prevented SMase-induced alterations in LDL particle size, electric charge and phospholipid content, respectively, but not those induced by PLA2. Western blot analysis showed that LP3 and DP3 counteracted changes in ApoB-100 conformation induced by the two enzymes. LDL proteomics (LDL trypsin digestion followed by mass spectroscopy) and computational modeling methods evidenced that peptides preserve ApoB-100 conformation due to their electrostatic interactions with a basic region of ApoB-100. These results demonstrate that LRP1-derived peptides are protective against LDL aggregation, even in conditions of extreme lipolysis, through their capacity to bind to ApoB-100 regions critical for ApoB-100 conformational preservation. These results suggests that these LRP1(CR9) derived peptides could be promising tools to prevent LDL aggregation induced by the main proteolytic enzymes acting in the arterial intima.

The Role of Apolipoprotein E Isoforms in Alzheimer's Disease.

Alzheimer's disease (AD), the most common type of dementia worldwide, is characterized by high levels of amyloid-ß (Aß) peptide and hyperphosphorylated tau protein. Genetically, the ɛ4 allele of apolipoprotein E (ApoE) has been established as the major risk factor for developing late-onset AD (LOAD), the most common form of the disease. Although the role ApoE plays in AD is still not completely understood, a differential role of its isoforms has long been known. The current review compiles the involvement of ApoE isoforms in amyloid-ß protein precursor transcription, Aß aggregation and clearance, synaptic plasticity, neuroinflammation, lipid metabolism, mitochondrial function, and tau hyperphosphorylation. Due to the complexity of LOAD, an accurate description of the interdependence among all the related molecular mechanisms involved in the disease is needed for developing successful therapies.

Differential effects of apoE and apoJ mimetic peptides on the action of an anti-Aß scFv in 3xTg-AD mice.

Anti-Aß immunotherapy has emerged as a promising approach to treat Alzheimer's disease (AD). The single-chain variable fragment scFv-h3D6 is an anti-Aß antibody fragment that lacks the Fc region, which is associated with the induction of microglial reactivity by the full-length monoclonal antibody bapineuzumab. ScFv-h3D6 was previously shown to restore the levels of apolipoprotein E (apoE) and apolipoprotein J (apoJ) in a triple-transgenic-AD (3xTg-AD) mouse model. Since apoE and apoJ play an important role in the development of AD, we aimed to study the in vivo effect of the combined therapy of scFv-h3D6 with apoE and apoJ mimetic peptides (MPs). Four-and-a-half-month-old 3xTg-AD mice were treated for six weeks with scFv-h3D6, apoE-MP, apoJ-MP, or a combination of scFv-h3D6 with each of the MPs, or a vehicle, and then the results were compared to non-transgenic mice. Magnetic Resonance Imaging showed a general tendency of the different treatments to protect against the reduction in brain volume. Aß burden decreased after treatment with scFv-h3D6, apoE-MP, or apoJ-MP, but the effect was not as evident with the combined therapies. In terms of glial reactivity, apoE-MP showed a potent anti-inflammatory effect that was eased by the presence of scFv-h3D6, whereas the combination of apoJ-MP and scFv-h3D6 was not detrimental. ScFv-h3D6 alone did not induce microglial reactivity, as full-length antibodies do; rather, it reduced it. Endogenous apoE and apoJ levels were decreased by scFv-h3D6, but the MPs lead to a simultaneous increase of both apolipoproteins. While apoE-MP and apoJ-MP demonstrated different effects in the combined therapies with scFv-h3D6, they did not improve the overall protective effect of scFv-h3D6 in reducing the Aß burden, apolipoproteins levels or microglial reactivity.

Aß-oligomer uptake and the resulting inflammatory response in adult human astrocytes are precluded by an anti-Aß single chain variable fragment in combination with an apoE mimetic peptide.

An imbalance between production and clearance of soluble amyloid-ß (Aß) initiates the pathological process in sporadic Alzheimer's disease (AD). Aß-specific antibodies seemed promising as therapeutic option in AD mouse models. In patients, however, vascular side-effects and Aß-antibody complex-induced microglial and/or perivascular macrophage inflammatory responses were encountered. To prevent inflammatory reactions, we designed a single chain variable fragment (scFv-h3D6), based on monoclonal antibody bapineuzumab (mAb-h3D6), but lacking the Fc region. ScFv-h3D6 reduced Aß-oligomer burden and prevented AD-associated behavioral and cellular changes in 3xTg-AD mice. As scFv-h3D6 lacks the Fc-tail, it cannot enhance Fc-receptor mediated Aß clearance by microglia and probably exerts its beneficial effects in 3xTg-AD mice through other mechanisms. ScFv-h3D6 restored the increased apoE and apoJ levels in 3xTg-AD brains back to normal. ApoE and apoJ influence cholesterol transport, Aß aggregation and clearance, and their genetic variants are risk factors for sporadic AD. Astrocytes are constitutive scavengers of soluble Aß from the CNS. We previously found apoE and apoJ to inhibit Aß uptake by adult human astrocytes, in vitro, and thus to potentially protect astrocytes from Aß cytotoxicity. In the present study, scFv-h3D6 and mAb-h3D6 inhibited Aß-oligomer uptake by adult human astrocytes. ApoE- and apoJ- mimetic peptides (MP) affected Aß uptake as well as Aß-induced cytokine release similar to intact apoE and apoJ, without interfering with the strong inhibitory effects of scFv-h3D6 on Aß-oligomer uptake. These results suggest that combining Aß-specific scFv and apoE-MP, that inhibits Aß oligomer-induced cytokine release by astrocytes, could offer advantages over currently used therapeutics.

Long-term efficacy and safety after corneal collagen crosslinking in pediatric patients: Three-year follow-up.

PURPOSE: To evaluate the effectiveness of standard corneal collagen crosslinking for children with progressive keratoconus. METHODS: Prospective study including 26 eyes of 26 patients younger than 18 years old with progressive keratoconus at Oftalmosalud Instituto de Ojos, Lima, Peru. Standard epi-off corneal crosslinking was performed in all eyes between January 2012 and January 2013. Pre- and postoperative evaluation (at 3 years) included uncorrected and best-corrected visual acuity and Scheimpflug analysis. Crosslinking failure was defined as an increase in maximum keratometry (Kmax) of more than 1 diopter after 1 year or more. RESULTS: Mean uncorrected visual acuity improvement was 0.24 LogMAR (p = 0.07) and mean best-corrected visual acuity improvement was 0.18 LogMAR (p = 0.01). None of the eyes lost more than one line in the best-corrected visual acuity. Four eyes (15.38%) lost two lines in the uncorrected visual acuity at 3 years postoperative. Mean steeper keratometry improvement was 1.14 diopters (p = 0.60). Progression rate was 23.07%. CONCLUSION: Standard epi-off corneal collagen crosslinking is safe and effective to halt the progression of the keratoconus with significant improvement in the best-corrected visual acuity at 3-year follow-up.

Effects of an Aß-antibody fragment on Aß aggregation and astrocytic uptake are modulated by apolipoprotein E and J mimetic peptides.

Aß-Immunotherapy has long been studied in the treatment of Alzheimer's disease (AD), but not how other molecules involved in the disease can affect antibody performance. We previously designed an antibody fragment, scFv-h3D6, and showed that it precludes Aß-induced cytotoxicity by withdrawing Aß oligomers from the amyloid pathway towards a non-toxic, worm-like pathway. ScFv-h3D6 was effective at the behavioral, cellular, and molecular levels in the 3xTg-AD mouse model. Because scFv-h3D6 treatment restored apolipoprotein E (apoE) and J (apoJ) concentrations to non-pathological values, and Aß internalization by glial cells was found to be decreased in the presence of these apolipoproteins, we now aimed to test the influence of scFv-h3D6 on Aß aggregation and cellular uptake by primary human astrocytes in the presence of therapeutic apoE and apoJ mimetic peptides (MPs). Firstly, we demonstrated by CD and FTIR that the molecules used in this work were well folded. Next, interactions between apoE or apoJ-MP, scFv-h3D6 and Aß were studied by CD. The conformational change induced by the interaction of Aß with apoE-MP was much bigger than the induced with apoJ-MP, in line with the observed formation of protective worm-like fibrils by the scFv-h3D6/Aß complex in the presence of apoJ-MP, but not of apoE-MP. ScFv-h3D6, apoJ-MP, and apoE-MP to a different extent reduced Aß uptake by astrocytes, and apoE-MP partially interfered with the dramatic reduction by scFv-h3D6 while apoJ-MP had no effect on scFv-h3D6 action. As sustained Aß uptake by astrocytes may impair their normal functions, and ultimately neuronal viability, this work shows another beneficence of scFv-h3D6 treatment, which is not further improved by the use of apoE or apoJ mimetic peptides.

Production of an anti-Aß antibody fragment in Pichia pastoris and in vitro and in vivo validation of its therapeutic effect.

ScFv-h3D6 has been shown as an efficient therapy in the 3xTg-AD mouse model of Alzheimer's Disease. Because one of the major bottlenecks for the therapeutic uses of proteins produced in Escherichia coli is their potential contamination with endotoxins, LPS were extensively removed by a rather low-efficient, expensive, and time-consuming purification step. In addition, disulfide scrambling is favored in the reducing bacterial cytoplasm albeit the use of reductase deficient strains. To overcome these hurdles, as well as to improve the yield, the yeast Pichia pastoris, an endotoxin-free host system for recombinant protein production, has been used to produce scFv-h3D6, both in flask and in a fed-batch bioreactor. Comparison of the thermal stability of the obtained protein with that from E. coli showed no differences. Opposite to the case of the protein obtained from E. coli, no disulfide scrambled conformations or LPS traces were detected in that produced in P. pastoris. Cytotoxicity assays in SH-SY5Y neuroblastoma cell-cultures demonstrated that proteins from both expression systems were similarly efficient in precluding Aß-induced toxicity. Finally, the 3xTg-AD mouse model was used to test the therapeutic effect of both proteins. Quantification of Aß levels from cortex and hippocampus protein extracts by ELISA, and Aß-immunohistochemistry, showed that both proteins reduced Aß burden. This work demonstrates that scFv-h3D6 obtained from P. pastoris shows the same benefits as those already known for that obtained from E. coli, with multiple advantages in terms of recombinant production and safety.

An Intracellular Amyloid-ß/AßPP Epitope Correlates with Neurodegeneration in those Neuronal Populations Early Involved in Alzheimer's Disease.

The main histopathological hallmarks of Alzheimer's disease (AD) are the extracellular deposition of neuritic amyloid plaques, composed of amyloid-ß (Aß) peptide, and the intracellular accumulation of neurofibrillary tangles, composed of hyperphosphorylated tau. Both traits are emulated in the 3xTg-AD mouse model. Because the relevance of this model in the bibliography and the main role of Aß in neuronal impairment, here we have detailed the brain Aß/AßPP distribution to subsequently quantify cellular density and intracellular burden for specific neuronal populations in the early stages of the disease. 6E10 immunoreactivity was evident in the deep layers of the cerebral cortex, in the pyramidal cell layer of the hippocampus, in the basolateral amygdala nucleus, and in the deep cerebellar nuclei macroneurons; whereas the specific neuronal populations with decreased cellular density were the large pyramidal neurons from the layers V-VI in the cerebral cortex, the pyramidal neurons from the CA2-3 region in the hippocampus, and the large neurons from the basolateral nucleus in the amygdala, apart from the already reported deep cerebellar nuclei. Interestingly, we found a strong correlation between intracellular Aß/AßPP burden and cellular density in all these populations. In addition, behavioral testing showed the functional consequences of such a neuronal depletion. Concretely, anxious-like behavior is manifested in the corner and open-field tests, as well as cognitive functions shown to be impaired in the novel object recognition test and Morris water maze paradigm. To our knowledge, this is the first deep characterization of the specific neuronal populations affected in the 3xTg-AD mouse model.

Towards the improvement in stability of an anti-Aß single-chain variable fragment, scFv-h3D6, as a way to enhance its therapeutic potential.

ScFv-h3D6 is a single-chain variable fragment derived from the monoclonal antibody bapineuzumab that prevents Aß-induced cytotoxicity by capturing Aß oligomers. The benefits of scFv-h3D6 treatment in Alzheimer's disease are known at the behavioural, cellular and molecular levels in the 3xTg-AD mouse model. Antibody-based therapeutics are only stable in a limited temperature range, so their benefit in vivo depends on their capability for maintaining the proper fold. Here, we have stabilized the scFv-h3D6 folding by introducing the mutation VH-K64R and combining it with the previously described elongation of the VL domain (C3). The stabilities of the different scFv-h3D6 constructs were calculated from urea and thermal denaturation followed by Trp-fluorescence, CD and DSC and resulted in the order C3 > K64R/C3 > VH-K64R ≥ scFv-h3D6; showing that the combination of both mutations was not additive, instead they partially cancelled each other. The three mutants assayed showed a decreased aggregation tendency but maintained their capability to aggregate in the form of worm-like fibrils, basis of the protective effect of scFv-h3D6. Cytotoxicity assays showed that all the mutants recovered cell viability of Aß-treated neuroblastoma cell cultures in a dose-dependent manner and with efficiencies that correlated with stability, therefore improving the therapeutic ability of this antibody.

Understanding the contribution of disulfide bridges to the folding and misfolding of an anti-Aß scFv.

ScFv-h3D6 is a single chain variable fragment that precludes Aß peptide-induced cytotoxicity by withdrawing Aß oligomers from the amyloid pathway to the worm-like pathway. Production of scFv molecules is not a straightforward procedure because of the occurrence of disulfide scrambled conformations generated in the refolding process. Here, we separately removed the disulfide bond of each domain and solved the scrambling problem; and then, we intended to compensate the loss of thermodynamic stability by adding three C-terminal elongation mutations, previously described to stabilize the native fold of scFv-h3D6. Such stabilization occurred through stabilization of the intermediate state in the folding pathway and destabilization of a different, ß-rich, intermediate state driving to worm-like fibrils. Elimination of the disulfide bridge of the less stable domain, VL , deeply compromised the yield and increased the aggregation tendency, but elimination of the disulfide bridge of the more stable domain, VH , solved the scrambling problem and doubled the production yield. Notably, it also changed the aggregation pathway from the protective worm-like morphology to an amyloid one. This was so because a partially unfolded intermediate driving to amyloid aggregation was present, instead of the ß-rich intermediate driving to worm-like fibrils. When combining with the elongation mutants, stabilization of the partially unfolded intermediate driving to amyloid fibrils was the only effect observed. Therefore, the same mutations drove to completely different scenarios depending on the presence of disulfide bridges and this illustrates the relevance of such linkages in the stability of different intermediate states for folding and misfolding.

Mouse Models of Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disorder that nowadays affects more than 40 million people worldwide and it is predicted to exponentially increase in the coming decades. Because no curative treatment exists, research on the pathophysiology of the disease, as well as the testing of new drugs, are mandatory. For these purposes, animal models constitute a valuable, although perfectible tool. This review takes a tour through several aspects of mouse models of AD, such as the generation of transgenic models, the relevance of the promoter driving the expression of the transgenes, and the concrete transgenes used to simulate AD pathophysiology. Then, transgenic mouse lines harboring mutated human genes at several loci such as APP, PSEN1, APOEɛ4, and ob (leptin) are reviewed. Therefore, not only the accumulation of the Aß peptide is emulated but also cholesterol and insulin metabolism. Further novel information about the disease will allow for the development of more accurate animal models, which in turn will undoubtedly be helpful for bringing preclinical research closer to clinical trials in humans.

Reversible Nuclear-Lipid-Droplet Morphology Induced by Oleic Acid: A Link to Cellular-Lipid Metabolism.

Neutral lipids-involved in many cellular processes-are stored as lipid droplets (LD), those mainly cytosolic (cLD) along with a small nuclear population (nLD). nLD could be involved in nuclear-lipid homeostasis serving as an endonuclear buffering system that would provide or incorporate lipids and proteins involved in signalling pathways as transcription factors and as enzymes of lipid metabolism and nuclear processes. Our aim was to determine if nLD constituted a dynamic domain. Oleic-acid (OA) added to rat hepatocytes or HepG2 cells in culture produced cellular-phenotypic LD modifications: increases in TAG, CE, C, and PL content and in cLD and nLD numbers and sizes. LD increments were reversed on exclusion of OA and were prevented by inhibition of acyl-CoA synthetase (with Triacsin C) and thus lipid biosynthesis. Under all conditions, nLD corresponded to a small population (2-10%) of total cellular LD. The anabolism triggered by OA, involving morphologic and size changes within the cLD and nLD populations, was reversed by a net balance of catabolism, upon eliminating OA. These catabolic processes included lipolysis and the mobilization of hydrolyzed FA from the LD to cytosolic-oxidation sites. These results would imply that nLD are actively involved in nuclear processes that include lipids. In conclusion, nLD are a dynamic nuclear domain since they are modified by OA through a reversible mechanism in combination with cLD; this process involves acyl-CoA-synthetase activity; ongoing TAG, CE, and PL biosynthesis. Thus, liver nLD and cLD are both dynamic cellular organelles.

Inhibition of Mevalonate Pathway and Synthesis of the Storage Lipids in Human Liver-Derived and Non-liver Cell Lines by Lippia alba Essential Oils.

The essential oils (EOs) of Lippia alba, an herb extensively used as a folk medicine in Latin America, are today promoted as an effective means of eliminating problems caused by hyperlipemia. We hypothesized that L.alba EOs inhibited cholesterol and triacylglycerols synthesis and decreased the intracellular depots of those lipids (lipid droplets), mechanisms involving the induction of a hypolipidemic response. Our aim was, therefore, to evaluate the hypolipogenic capability of the EOs of four L. alba chemotypes on liver-derived (HepG2) and non-liver (A549) human cell lines and to identify the potential biochemical targets of those chemotypes, particularly within the mevalonate pathway (MP). [14C]Acetate was used as radioactive precursor for assays. Lipid analyses were performed by thin-layer and capillary gas chromatography, lipid droplets analyzed by fluorescence microscopy, and HMGCR levels determined by Western blot. In both cell lines, all four chemotypes exerted hypocholesterogenic effects within a concentration range of 3.2-32 µg/mL. Nonsaponifiable lipids manifested a decrease in incorporation of [14C]acetate into squalene, lanosterol, lathosterol, and cholesterol, but not into ubiquinone, thus suggesting an inhibition of enzymes in the MP downstream from farnesyl pyrophosphate. The tagetenone chemotype, the most efficacious hypocholesterogenic L. alba EO, lowered HMGCR protein levels; inhibited triacylglycerols, cholesteryl esters, and phospholipids synthesis; and diminished lipid droplets in size and volume. These results revealed that L. alba EOs inhibited different lipogenic pathways and such lipid-lowering effects could prove essential to prevent cardiovascular diseases.