Genetic deletion of astrocytic calcineurin B1 prevents cognitive impairment and neuropathology development in acute and chronic mouse models of Alzheimer's disease.

Alzheimer's disease (AD) represents an urgent yet unmet challenge for modern society, calling for exploration of innovative targets and therapeutic approaches. Astrocytes, main homeostatic cells in the CNS, represent promising cell-target. Our aim was to investigate if deletion of the regulatory CaNB1 subunit of calcineurin in astrocytes could mitigate AD-related memory deficits, neuropathology, and neuroinflammation. We have generated two, acute and chronic, AD mouse models with astrocytic CaNB1 ablation (ACN-KO). In the former, we evaluated the ability of ß-amyloid oligomers (AßOs) to impair memory and activate glial cells once injected in the cerebral ventricle of conditional ACN-KO mice. Next, we generated a tamoxifen-inducible astrocyte-specific CaNB1 knock-out in 3xTg-AD mice (indACNKO-AD). CaNB1 was deleted, by tamoxifen injection, in 11.7-month-old 3xTg-AD mice for 4.4 months. Spatial memory was evaluated using the Barnes maze; ß-amyloid plaques burden, neurofibrillary tangle deposition, reactive gliosis, and neuroinflammation were also assessed. The acute model showed that ICV injected AßOs in 2-month-old wild type mice impaired recognition memory and fostered a pro-inflammatory microglia phenotype, whereas in ACN-KO mice, AßOs were inactive. In indACNKO-AD mice, 4.4 months after CaNB1 depletion, we found preservation of spatial memory and cognitive flexibility, abolishment of amyloidosis, and reduction of neurofibrillary tangles, gliosis, and neuroinflammation. Our results suggest that ACN is crucial for the development of cognitive impairment, AD neuropathology, and neuroinflammation. Astrocyte-specific CaNB1 deletion is beneficial for both the abolishment of AßO-mediated detrimental effects and treatment of ongoing AD-related pathology, hence representing an intriguing target for AD therapy.

Aß1-6A2V(D) peptide, effective on Aß aggregation, inhibits tau misfolding and protects the brain after traumatic brain injury.

Alzheimer's disease (AD), the leading cause of dementia in older adults, is a double proteinopathy characterized by amyloid-ß (Aß) and tau pathology. Despite enormous efforts that have been spent in the last decades to find effective therapies, late pharmacological interventions along the course of the disease, inaccurate clinical methodologies in the enrollment of patients, and inadequate biomarkers for evaluating drug efficacy have not allowed the development of an effective therapeutic strategy. The approaches followed so far for developing drugs or antibodies focused solely on targeting Aß or tau protein. This paper explores the potential therapeutic capacity of an all-D-isomer synthetic peptide limited to the first six amino acids of the N-terminal sequence of the A2V-mutated Aß, Aß1-6A2V(D), that was developed following the observation of a clinical case that provided the background for its development. We first performed an in-depth biochemical characterization documenting the capacity of Aß1-6A2V(D) to interfere with the aggregation and stability of tau protein. To tackle Aß1-6A2V(D) in vivo effects against a neurological decline in genetically predisposed or acquired high AD risk mice, we tested its effects in triple transgenic animals harboring human PS1(M146 V), APP(SW), and MAPT(P301L) transgenes and aged wild-type mice exposed to experimental traumatic brain injury (TBI), a recognized risk factor for AD. We found that Aß1-6A2V(D) treatment in TBI mice improved neurological outcomes and reduced blood markers of axonal damage. Exploiting the C. elegans model as a biosensor of amyloidogenic proteins' toxicity, we observed a rescue of locomotor defects in nematodes exposed to the brain homogenates from TBI mice treated with Aß1-6A2V(D) compared to TBI controls. By this integrated approach, we demonstrate that Aß1-6A2V(D) not only impedes tau aggregation but also favors its degradation by tissue proteases, confirming that this peptide interferes with both Aß and tau aggregation propensity and proteotoxicity.

Evolution toward beta common chain receptor usage links the matrix proteins of HIV-1 and its ancestors to human erythropoietin.

The HIV-1 matrix protein p17 (p17) is a pleiotropic molecule impacting on different cell types. Its interaction with many cellular proteins underlines the importance of the viral protein as a major determinant of human specific adaptation. We previously showed the proangiogenic capability of p17. Here, by integrating functional analysis and receptor binding, we identify a functional epitope that displays molecular mimicry with human erythropoietin (EPO) and promotes angiogenesis through common beta chain receptor (ßCR) activation. The functional EPO-like epitope was found to be present in the matrix protein of HIV-1 ancestors SIV originated in chimpanzees (SIVcpz) and gorillas (SIVgor) but not in that of HIV-2 and its ancestor SIVsmm from sooty mangabeys. According to biological data, evolution of the EPO-like epitope showed a clear differentiation between HIV-1/SIVcpz-gor and HIV-2/SIVsmm branches, thus highlighting this epitope on p17 as a divergent signature discriminating HIV-1 and HIV-2 ancestors. P17 is known to enhance HIV-1 replication. Similarly to other ßCR ligands, p17 is capable of attracting and activating HIV-1 target cells and promoting a proinflammatory microenvironment. Thus, it is tempting to speculate that acquisition of an epitope on the matrix proteins of HIV-1 ancestors capable of triggering ßCR may have represented a critical step to enhance viral aggressiveness and early human-to-human SIVcpz/gor dissemination. The hypothesis that the p17/ßCR interaction and ßCR abnormal stimulation may also play a role in sustaining chronic activation and inflammation, thus marking the difference between HIV-1 and HIV-2 in term of pathogenicity, needs further investigation.

A novel bio-inspired strategy to prevent amyloidogenesis and synaptic damage in Alzheimer's disease.

Alzheimer's disease (AD) is an irreversible neurodegenerative disorder that affects millions of people worldwide. AD pathogenesis is intricate. It primarily involves two main molecular players-amyloid-ß (Aß) and tau-which actually have an intrinsic trend to generate molecular assemblies that are toxic to neurons. Incomplete knowledge of the molecular mechanisms inducing the onset and sustaining the progression of the disease, as well as the lack of valid models to fully recapitulate the pathogenesis of human disease, have until now hampered the development of a successful therapy for AD. The overall experience with clinical trials with a number of potential drugs-including the recent outcomes of studies with monoclonal antibodies against Aß-seems to indicate that Aß-targeting is not effective if it is not accompanied by an efficient challenge of Aß neurotoxic properties. We took advantage from the discovery of a naturally-occurring variant of Aß (AßA2V) that has anti-amyloidogenic properties, and designed a novel bio-inspired strategy for AD based on the intranasal delivery of a six-mer peptide (Aß1-6A2V) retaining the anti-amyloidogenic abilities of the full-length AßA2V variant. This approach turned out to be effective in preventing the aggregation of wild type Aß and averting the synaptic damage associated with amyloidogenesis in a mouse model of AD. The results of our preclinical studies inspired by a protective model already existing in nature, that is the human heterozygous AßA2V carriers which seem to be protected from AD, open the way to an unprecedented and promising approach for the prevention of the disease in humans.

Chronic cholesterol administration to the brain supports complete and long-lasting cognitive and motor amelioration in Huntington's disease.

Evidence that Huntington's disease (HD) is characterized by impaired cholesterol biosynthesis in the brain has led to strategies to increase its level in the brain of the rapidly progressing R6/2 mouse model, with a positive therapeutic outcome. Here we tested the long-term efficacy of chronic administration of cholesterol to the brain of the slowly progressing zQ175DN knock-in HD mice in preventing ("early treatment") or reversing ("late treatment") HD symptoms. To do this we used the most advanced formulation of cholesterol loaded brain-permeable nanoparticles (NPs), termed hybrid-g7-NPs-chol, which were injected intraperitoneally. We show that one cycle of treatment with hybrid-g7-NPs-chol, administered in the presymptomatic ("early treatment") or symptomatic ("late treatment") stages is sufficient to normalize cognitive defects up to 5 months, as well as to improve other behavioral and neuropathological parameters. A multiple cycle treatment combining both early and late treatments ("2 cycle treatment") lasting 6 months generates therapeutic effects for more than 11 months, without severe adverse reactions. Sustained cholesterol delivery to the brain of zQ175DN mice also reduces mutant Huntingtin aggregates in both the striatum and cortex and completely normalizes synaptic communication in the striatal medium spiny neurons compared to saline-treated HD mice. Furthermore, through a meta-analysis of published and current data, we demonstrated the power of hybrid-g7-NPs-chol and other strategies able to increase brain cholesterol biosynthesis, to reverse cognitive decline and counteract the formation of mutant Huntingtin aggregates. These results demonstrate that cholesterol delivery via brain-permeable NPs is a therapeutic option to sustainably reverse HD-related behavioral decline and neuropathological signs over time, highlighting the therapeutic potential of cholesterol-based strategies in HD patients. DATA AVAILABILITY: This study does not include data deposited in public repositories. Data are available on request to the corresponding authors.

Selenoprotein N is an endoplasmic reticulum calcium sensor that links luminal calcium levels to a redox activity.

The endoplasmic reticulum (ER) is the reservoir for calcium in cells. Luminal calcium levels are determined by calcium-sensing proteins that trigger calcium dynamics in response to calcium fluctuations. Here we report that Selenoprotein N (SEPN1) is a type II transmembrane protein that senses ER calcium fluctuations by binding this ion through a luminal EF-hand domain. In vitro and in vivo experiments show that via this domain, SEPN1 responds to diminished luminal calcium levels, dynamically changing its oligomeric state and enhancing its redox-dependent interaction with cellular partners, including the ER calcium pump sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). Importantly, single amino acid substitutions in the EF-hand domain of SEPN1 identified as clinical variations are shown to impair its calcium-binding and calcium-dependent structural changes, suggesting a key role of the EF-hand domain in SEPN1 function. In conclusion, SEPN1 is a ER calcium sensor that responds to luminal calcium depletion, changing its oligomeric state and acting as a reductase to refill ER calcium stores.

A Comprehensive Technology Platform for the Rapid Discovery of Peptide Inhibitors against SARS-CoV-2 Pseudovirus Infection.

We developed and validated a technology platform for designing and testing peptides inhibiting the infectivity of SARS-CoV-2 spike protein-based pseudoviruses. This platform integrates target evaluation, in silico inhibitor design, peptide synthesis, and efficacy screening. We generated a cyclic peptide library derived from the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and the angiotensin-converting enzyme 2 (ACE2) receptor. The cell-free validation process by ELISA competition assays and Surface Plasmon Resonance (SPR) studies revealed that the cyclic peptide c9_05, but not its linear form, binds well to ACE2. Moreover, it effectively inhibited the transduction in HEK293, stably expressing the human ACE2 receptor of pseudovirus particles displaying the SARS-CoV-2 spike in the Wuhan or UK variants. However, the inhibitory efficacy of c9_05 was negligible against the Omicron variant, and it failed to impede the entry of pseudoviruses carrying the B.1.351 (South African) spike. These variants contain three or more mutations known to increase affinity to ACE2. This suggests further refinement is needed for potential SARS-CoV-2 inhibition. Our study hints at a promising approach to develop inhibitors targeting viral infectivity receptors, including SARS-CoV-2's. This platform also promises swift identification and evaluation of inhibitors for other emergent viruses.

Nonphosphorylated tau slows down Aß1-42 aggregation, binds to Aß1-42 oligomers, and reduces Aß1-42 toxicity.

The formation of neurofibrillary tangles and amyloid plaques accompanies the progression of Alzheimer's disease. Tangles are made of fibrillar aggregates formed by the microtubule-associated protein tau, whereas plaques comprise fibrillar forms of amyloid-beta (Aß). Both form toxic oligomers during aggregation and are thought to interact synergistically to each promote the accumulation of the other. Recent in vitro studies have suggested that the monomeric nonphosphorylated full-length tau protein hinders the aggregation of Aß1-40 peptide, but whether the same is true for the more aggregation-prone Aß1-42 was not determined. We used in vitro and in vivo techniques to explore this question. We have monitored the aggregation kinetics of Aß1-42 by thioflavine T fluorescence in the presence or the absence of different concentrations of nonphosphorylated tau. We observed that elongation of Aß1-42 fibrils was inhibited by tau in a dose-dependent manner. Interestingly, the fibrils were structurally different in the presence of tau but did not incorporate tau. Surface plasmon resonance indicated that tau monomers bound to Aß1-42 oligomers (but not monomers) and hindered their interaction with the anti-Aß antibody 4G8, suggesting that tau binds to the hydrophobic central core of Aß recognized by 4G8. Tau monomers also antagonized the toxic effects of Aß oligomers in Caenorhabditis elegans. This suggests that nonphosphorylated tau might have a neuroprotective effect by binding Aß1-42 oligomers formed during the aggregation and shielding their hydrophobic patches.

The mode of dexamethasone decoration influences avidin-nucleic-acid-nano-assembly organ biodistribution and in vivo drug persistence.

Avidin-Nucleic-Acid-NanoASsemblies (ANANAS) possess natural tropism for the liver and, when loaded with dexamethasone, reduce clinical progression in an autoimmune hepatitis murine model. Here, we investigated the linker chemistry (hydrazide-hydrazone, Hz-Hz, or carbamate hydrazide-hydrazone, Cb-Hz bond) and length (long, 5 kDa PEG, or short, 5-6 carbons) in biotin-dexamethasone conjugates used for nanoparticle decoration through in vitro and in vivo studies. All four newly synthesized conjugates released the drug at acidic pH only. In vitro, the Hz-Hz and the PEG derivatives were less stable than the Cb-Hz and the short chain ones, respectively. Once injected in healthy mice, dexamethasone location in the PEGylated ANANAS outer layer favors liver penetration and resident macrophages uptake, while drug Hz-Hz, but not Cb-Hz, short spacing prolongs drug availability. In conclusion, the tight modulation of ANANAS decoration can significantly influence the host interaction, paving the way for the development of steroid nanoformulations suitable for different pharmacokinetic profiles.

Cytoskeleton Elements Contribute to Prion Peptide-Induced Endothelial Barrier Breakdown in a Blood-Brain Barrier In Vitro System.

The mechanisms involved in the interaction of PrP 106-126, a peptide corresponding to the prion protein amyloidogenic region, with the blood-brain barrier (BBB) were studied. PrP 106-126 treatment that was previously shown to impair BBB function, reduced cAMP levels in cultured brain endothelial cells, increased nitric oxide (NO) levels, and changed the activation mode of the small GTPases Rac1 (inactivation) and RhoA (activation). The latter are well established regulators of endothelial barrier properties that act via cytoskeletal elements. Indeed, liquid chromatography-mass spectrometry (LC-MS)-based proteomic profiling study revealed extensive changes in expression of cytoskeleton-related proteins. These results shed light on the nature of the interaction between the prion peptide PrP 106-126 and the BBB and emphasize the importance of the cytoskeleton in endothelium response to prion- induced stress.

Micro- and Nanoplastics' Effects on Protein Folding and Amyloidosis.

A significant portion of the world's plastic is not properly disposed of and, through various processes, is degraded into microscopic particles termed micro- and nanoplastics. Marine and terrestrial faunae, including humans, inevitably get in contact and may inhale and ingest these microscopic plastics which can deposit throughout the body, potentially altering cellular and molecular functions in the nervous and other systems. For instance, at the cellular level, studies in animal models have shown that plastic particles can cross the blood-brain barrier and interact with neurons, and thus affect cognition. At the molecular level, plastics may specifically influence the folding of proteins, induce the formation of aberrant amyloid proteins, and therefore potentially trigger the development of systemic and local amyloidosis. In this review, we discuss the general issue of plastic micro- and nanoparticle generation, with a focus on their effects on protein folding, misfolding, and their possible clinical implications.

Sleep inhibition induced by amyloid-ß oligomers is mediated by the cellular prion protein.

Sleep is severely impaired in patients with Alzheimer's disease. Amyloid-ß deposition in the brain of Alzheimer's disease patients is a key event in its pathogenesis and is associated with disrupted sleep, even before the appearance of cognitive decline. Because soluble amyloid-ß oligomers are the key mediators of synaptic and cognitive dysfunction in Alzheimer's disease and impair long-term memory in rodents, the first aim of this study was to test the hypothesis that amyloid-ß oligomers would directly impair sleep in mice. The cellular prion protein is a cell surface glycoprotein of uncertain function. Because cellular prion protein binds oligomeric amyloid-ß with high affinity and mediates some of its neurotoxic effects, the second aim of the study was to test whether amyloid-ß oligomer-induced sleep alterations were mediated by cellular prion protein. To address these aims, wild-type and cellular prion protein-deficient mice were given acute intracerebroventricular injections (on different days, at lights on) of vehicle and synthetic amyloid-ß oligomers. Compared to vehicle, amyloid-ß oligomers significantly reduced the amount of time spent in non-rapid eye movement sleep by wild-type mice during both the light and dark phases of the light-dark cycle. The amount of time spent in rapid eye movement sleep was reduced during the dark phase. Sleep was also fragmented by amyloid-ß oligomers, as the number of transitions between states increased in post-injection hours 9-24. No such effects were observed in cellular prion protein-deficient mice. These results show that amyloid-ß oligomers do inhibit and fragment sleep, and that these effects are mediated by cellular prion protein.

Increased transcription of transglutaminase 1 mediates neuronal death in in vitro models of neuronal stress and Aß1-42-mediated toxicity.

Alzheimer's disease (AD) is the most common cause of dementia. At the pre-symptomatic phase of the disease, the processing of the amyloid precursor protein (APP) produces toxic peptides, called amyloid-ß 1-42 (Aß 1-42). The downstream effects of Aß 1-42 production are not completely uncovered. Here, we report the involvement of transglutaminase 1 (TG1) in in vitro AD models of neuronal toxicity. TG1 was increased at late stages of the disease in the hippocampus of a mouse model of AD and in primary cortical neurons undergoing stress. Silencing of TGM1 gene was sufficient to prevent Aß-mediated neuronal death. Conversely, its overexpression enhanced cell death. TGM1 upregulation was mediated at the transcriptional level by an activator protein 1 (AP1) binding site that when mutated halted TGM1 promoter activation. These results indicate that TG1 acts downstream of Aß-toxicity, and that its stress-dependent increase makes it suitable for pharmacological intervention.

Aß Beyond the AD Pathology: Exploring the Structural Response of Membranes Exposed to Nascent Aß Peptide.

The physiological and pathological roles of nascent amyloid beta (Aß) monomers are still debated in the literature. Their involvement in the pathological route of Alzheimer's Disease (AD) is currently considered to be the most relevant, triggered by their aggregation into structured oligomers, a toxic species. Recently, it has been suggested that nascent Aß, out of the amyloidogenic pathway, plays a physiological and protective role, especially in the brain. In this emerging perspective, the study presented in this paper investigated whether the organization of model membranes is affected by contact with Aß in the nascent state, as monomers. The outcome is that, notably, the rules of engagement and the resulting structural outcome are dictated by the composition and properties of the membrane, rather than by the Aß variant. Interestingly, Aß monomers are observed to favor the tightening of adjacent complex membranes, thereby affecting a basic structural event for cell-cell adhesion and cell motility.

Synthesis and Molecular Modelling Studies of New 1,3-Diaryl-5-Oxo-Proline Derivatives as Endothelin Receptor Ligands.

The synthesis of seventeen new 1,3-diaryl-5-oxo-proline derivatives as endothelin receptor (ETR) ligands is described. The structural configuration of the new molecules was determined by analyzing selected signals in proton NMR spectra. In vitro binding assays of the human ETA and ETB receptors allowed us to identify compound 31h as a selective ETAR ligand. The molecular docking of the selected compounds and the ETA antagonist atrasentan in the ETAR homology model provided insight into the structural elements required for the affinity and the selectivity of the ETAR subtype.

p17 from HIV induces brain endothelial cell angiogenesis through EGFR-1-mediated cell signalling activation.

HIV-associated neurocognitive disorder in HIV patients substantially reduces their quality of life. We previously showed that the HIV matrix protein, p17 could stimulate lymph-angiogenesis in vitro potentially contributing to lymphoma tumour growth and in addition is associated with vascular activation in neuro-degenerating brain tissue; here, therefore, we have investigated the detailed molecular mechanisms of this action. We performed in vitro cell culture, angiogenesis experiments, phospho-protein microarrays and Western blotting to identify cellular signalling induced by p17 within human brain endothelial cells (HbMEC), and inhibitor studies to block p17-induced vascular growth. We also characterised the effects of hippocampal CA1 injection of p17 on epidermal growth factor receptor-1 (EGFR1) expression linked to our murine model of dementia. p17 strongly induced angiogenesis of HbMEC (migration, tube formation and spheroid growth). p17 concomitantly increased phosphorylation of EGFR1 as well as down-stream intermediates ERK1/2, FAK, PLC-γ and PKC-ß whilst an inhibitor peptide of EGFR, blocked cell signalling and angiogenesis. Finally, Mice that showed reduced cognitive function and behavioural deficiencies after p17 injection, demonstrated that p17 localised in cortical microvessels and also neurones many of which stained positive for p-EGFR1 by histology/IHC. This work provides strong support that p17 may be involved in initiating and/or perpetuating vascular tissue pathophysiology associated with comorbidity in HIV patients.

Plasma and Brain Concentrations of Doxycycline after Single and Repeated Doses in Wild-Type and APP23 Mice.

Repurposing doxycycline for the treatment of amyloidosis has recently been put forward because of the antiaggregating and anti-inflammatory properties of the drug. Most of the investigations of the therapeutic potential of doxycycline for neurodegenerative amyloidosis, e.g., prion and Alzheimer disease (AD), have been carried out in mouse models, but surprisingly no data are available regarding the concentrations reached in the brain after systemic administration. We filled this gap by analyzing the pharmacokinetic profile of doxycycline in plasma and brain after single and repeated intraperitoneal injections of 10 and 100 mg/kg, in wild-type mice and the APP23 mouse model of AD. The main outcomes of our study are: 1) Peak plasma concentrations ranged from 2 to10 µg/ml, superimposable to those in humans; 2) brain-to-plasma ratio was ∼0.2, comparable to the cerebrospinal fluid/serum ratios in humans; 3) brain Cmax 4-6 hours after a single dose was ∼0.5 (10 mg/kg) and ∼5 µM (100 mg/kg). Notably, these concentrations are lower than those required for the drug's antiaggregating properties as observed in cell-free studies, suggesting that other features underlie the positive cognitive effects in AD mice; 4) elimination half-life was shorter than in humans (3-6 vs. 15-30 hours), therefore no significant accumulation was observed in mouse brain following repeated treatments; and 5) there were no differences between doxycycline concentrations in brain areas of age-matched wild-type and APP23 mice. These data are useful for planning preclinical studies with translational validity, and to identify more reliably the mechanism(s) of action underlying the central in vivo effects of doxycycline.

bioNMR-based identification of natural anti-Aß compounds in Peucedanum ostruthium.

The growing interest in medicinal plants for the identification of new bioactive compounds and the formulation of new nutraceuticals and drugs prompted us to develop a powerful experimental approach allowing the detailed metabolic profiling of complex plant extracts, the identification of ligands of macromolecular targets of biomedical relevance and a preliminary characterization of their biological activity. To this end, we selected Peucedanum ostruthium, a plant traditionally employed in Austria and Italy for its several potential therapeutic applications, as case study. We combined the use of NMR and UPLC-HR-MS for the identification of the metabolites present in its leaves and rhizome extracts. Due to the significant content of polyphenols, particularly chlorogenic acids, recently identified as anti-amyloidogenic compounds, polyphenols-enriched fractions were prepared and tested for their ability to prevent Aß1-42 peptide aggregation and neurotoxicity in a neuronal human cell line. STD-NMR experiments allowed the detailed identification of Aß oligomers' ligands responsible for the anti-amyloidogenic activity. These data provide experimental protocols and structural information suitable for the development of innovative molecular tools for prevention, therapy and diagnosis of Alzheimer's disease.

The Anti-Amyloidogenic Action of Doxycycline: A Molecular Dynamics Study on the Interaction with Aß42.

The pathological aggregation of amyloidogenic proteins is a hallmark of many neurological diseases, including Alzheimer's disease and prion diseases. We have shown both in vitro and in vivo that doxycycline can inhibit the aggregation of Aß42 amyloid fibrils and disassemble mature amyloid fibrils. However, the molecular mechanisms of the drug's anti-amyloidogenic property are not understood. In this study, a series of molecular dynamics simulations were performed to explain the molecular mechanism of the destabilization of Aß42 fibrils by doxycycline and to compare the action of doxycycline with those of iododoxorubicin (a toxic structural homolog of tetracyclines), curcumin (known to have anti-amyloidogenic activity) and gentamicin (an antibiotic with no experimental evidence of anti-amyloidogenic properties). We found that doxycycline tightly binds the exposed hydrophobic amino acids of the Aß42 amyloid fibrils, partly leading to destabilization of the fibrillar structure. Clarifying the molecular determinants of doxycycline binding to Aß42 may help devise further strategies for structure-based drug design for Alzheimer's disease.

V363I and V363A mutated tau affect aggregation and neuronal dysfunction differently in C. elegans.

Mutations in the microtubule-associated protein tau (MAPT) gene have been linked to a heterogeneous group of progressive neurodegenerative disorders commonly called tauopathies. From patients with frontotemporal lobar degeneration with distinct atypical clinical phenotypes, we recently identified two new mutations on the same codon, in position 363 of the MAPT gene, which resulted in the production of Val-to-Ala (tauV363A) or Val-to-Ile (tauV363I) mutated tau. These substitutions specifically affected microtubule polymerization and propensity of tau to aggregate in vitro suggesting that single amino acid modification may dictate the fate of the neuropathology. To clarify whether tauV363A and tauV363I affect protein misfolding differently in vivo driving certain phenotypes, we generated new transgenic C. elegans strains. Human 2N4R tau carrying the mutation was expressed in all the neurons of worms. The behavioral defects, misfolding and proteotoxicity caused by the tauV363A and tauV363I mutated proteins were compared to that induced by the expression of wild-type tau (tauwt). Pan-neuronal expression of human 2N4R tauWT in worms resulted in a neuromuscular defect with characteristics of a neurodegenerative phenotype. This defect was worsened by the expression of mutated proteins which drive distinct neuronal dysfunctions and synaptic impairments involving, in transgenic worms expressing tauV363A (V363A) also a pharyngeal defect never linked before to other mutations. The two mutations differently affected the tau phosphorylation and misfolding propensities: tauV363I was highly phosphorylated on epitopes corresponding to Thr231 and Ser202/Thr205, and accumulated as insoluble tau assemblies whereas tauV363A showed a greater propensity to form soluble oligomeric assemblies. These findings uphold the role of a single amino acid substitution in specifically affecting the ability of tau to form soluble and insoluble assemblies, opening up new perspectives in the pathogenic mechanism underlying tauopathies.