Different responses of mice and rats hippocampus CA1 pyramidal neurons to in vitro and in vivo-like inputs.

The fundamental role of any neuron within a network is to transform complex spatiotemporal synaptic input patterns into individual output spikes. These spikes, in turn, act as inputs for other neurons in the network. Neurons must execute this function across a diverse range of physiological conditions, often based on species-specific traits. Therefore, it is crucial to determine the extent to which findings can be extrapolated between species and, ultimately, to humans. In this study, we employed a multidisciplinary approach to pinpoint the factors accounting for the observed electrophysiological differences between mice and rats, the two species most used in experimental and computational research. After analyzing the morphological properties of their hippocampal CA1 pyramidal cells, we conducted a statistical comparison of rat and mouse electrophysiological features in response to somatic current injections. This analysis aimed to uncover the parameters underlying these distinctions. Using a well-established computational workflow, we created ten distinct single-cell computational models of mouse CA1 pyramidal neurons, ready to be used in a full-scale hippocampal circuit. By comparing their responses to a variety of somatic and synaptic inputs with those of rat models, we generated experimentally testable hypotheses regarding species-specific differences in ion channel distribution, kinetics, and the electrophysiological mechanisms underlying their distinct responses to synaptic inputs during the behaviorally relevant Gamma and Sharp-Wave rhythms.

αS1-Casein-Loaded Proteo-liposomes as Potential Inhibitors in Amyloid Fibrillogenesis: In Vivo Effects on a C. elegans Model of Alzheimer's Disease.

According to the amyloid hypothesis, in the early phases of Alzheimer's disease (AD), small soluble prefibrillar aggregates of the amyloid ß-peptide (Aß) interact with neuronal membranes, causing neural impairment. Such highly reactive and toxic species form spontaneously and transiently in the amyloid building pathway. A therapeutic strategy consists of the recruitment of these intermediates, thus preventing aberrant interaction with membrane components (lipids and receptors), which in turn may trigger a cascade of cellular disequilibria. Milk αs1-Casein is an intrinsically disordered protein that is able to inhibit Aß amyloid aggregation in vitro, by sequestering transient species. In order to test αs1-Casein as an inhibitor for the treatment of AD, it needs to be delivered in the place of action. Here, we demonstrate the use of large unilamellar vesicles (LUVs) as suitable nanocarriers for αs1-Casein. Proteo-LUVs were prepared and characterized by different biophysical techniques, such as multiangle light scattering, atomic force imaging, and small-angle X-ray scattering; αs1-Casein loading was quantified by a fluorescence assay. We demonstrated on a C. elegans AD model the effectiveness of the proposed delivery strategy in vivo. Proteo-LUVs allow efficient administration of the protein, exerting a positive functional readout at very low doses while avoiding the intrinsic toxicity of αs1-Casein. Proteo-LUVs of αs1-Casein represent an effective proof of concept for the exploitation of partially disordered proteins as a therapeutic strategy in mild AD conditions.

The Possible Role of the Type I Chaperonins in Human Insulin Self-Association.

Insulin is a hormone that attends to energy metabolism by regulating glucose levels in the bloodstream. It is synthesised within pancreas beta-cells where, before being released into the serum, it is stored in granules as hexamers coordinated by Zn2+ and further packaged in microcrystalline structures. The group I chaperonin cpn60, known for its assembly-assisting function, is present, together with its cochaperonin cpn10, at each step of the insulin secretory pathway. However, the exact function of the heat shock protein in insulin biosynthesis and processing is still far from being understood. Here we explore the possibility that the molecular machine cpn60/cpn10 could have a role in insulin hexameric assembly and its further crystallization. Moreover, we also evaluate their potential protective effect in pathological insulin aggregation. The experiments performed with the cpn60 bacterial homologue, GroEL, in complex with its cochaperonin GroES, by using spectroscopic methods, microscopy and hydrodynamic techniques, reveal that the chaperonins in vitro favour insulin hexameric organisation and inhibit its aberrant aggregation. These results provide new details in the field of insulin assembly and its related disorders.

Scaling Concepts in Serpin Polymer Physics.

α1-Antitrypsin is a protease inhibitor belonging to the serpin family. Serpin polymerisation is at the core of a class of genetic conformational diseases called serpinopathies. These polymers are known to be unbranched, flexible, and heterogeneous in size with a beads-on-a-string appearance viewed by negative stain electron microscopy. Here, we use atomic force microscopy and time-lapse dynamic light scattering to measure polymer size and shape for wild-type (M) and Glu342→Lys (Z) α1-antitrypsin, the most common variant that leads to severe pathological deficiency. Our data for small polymers deposited onto mica and in solution reveal a power law relation between the polymer size, namely the end-to-end distance or the hydrodynamic radius, and the polymer mass, proportional to the contour length. We use the scaling concepts of polymer physics to assess that α1-antitrypsin polymers are random linear chains with a low persistence length.

Nanoalgosomes: Introducing extracellular vesicles produced by microalgae.

Cellular, inter-organismal and cross kingdom communication via extracellular vesicles (EVs) is intensively studied in basic science with high expectation for a large variety of bio-technological applications. EVs intrinsically possess many attributes of a drug delivery vehicle. Beyond the implications for basic cell biology, academic and industrial interests in EVs have increased in the last few years. Microalgae constitute sustainable and renewable sources of bioactive compounds with a range of sectoral applications, including the formulation of health supplements, cosmetic products and food ingredients. Here we describe a newly discovered subtype of EVs derived from microalgae, which we named nanoalgosomes. We isolated these extracellular nano-objects from cultures of microalgal strains, including the marine photosynthetic chlorophyte Tetraselmis chuii, using differential ultracentrifugation or tangential flow fractionation and focusing on the nanosized small EVs (sEVs). We explore different biochemical and physical properties and we show that nanoalgosomes are efficiently taken up by mammalian cell lines, confirming the cross kingdom communication potential of EVs. This is the first detailed description of such membranous nanovesicles from microalgae. With respect to EVs isolated from other organisms, nanoalgosomes present several advantages in that microalgae are a renewable and sustainable natural source, which could easily be scalable in terms of nanoalgosome production.

Valorization of Apple Peels through the Study of the Effects on the Amyloid Aggregation Process of κ-Casein.

Waste valorization represents one of the main social challenges when promoting a circular economy and environmental sustainability. Here, we evaluated the effect of the polyphenols extracted from apple peels, normally disposed of as waste, on the amyloid aggregation process of κ-casein from bovine milk, a well-used amyloidogenic model system. The effect of the apple peel extract on protein aggregation was examined using a thioflavin T fluorescence assay, Congo red binding assay, circular dichroism, light scattering, and atomic force microscopy. We found that the phenolic extract from the peel of apples of the cultivar "Fuji", cultivated in Sicily (Caltavuturo, Italy), inhibited κ-casein fibril formation in a dose-dependent way. In particular, we found that the extract significantly reduced the protein aggregation rate and inhibited the secondary structure reorganization that accompanies κ-casein amyloid formation. Protein-aggregated species resulting from the incubation of κ-casein in the presence of polyphenols under amyloid aggregation conditions were reduced in number and different in morphology.

Isolation of extracellular vesicles from microalgae: towards the production of sustainable and natural nanocarriers of bioactive compounds.

Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeutics, precision medicine and other biotechnology sectors. Novel bio-based nanotechnologies have recently arisen, which are based on the exploitation of extracellular vesicles (EVs). In this context, it has become essential to identify suitable organisms or cellular types to act as reliable sources of EVs and to develop their pilot- to large-scale production. The discovery of new biosources and the optimisation of related bioprocesses for the isolation and functionalisation of nano-delivery vehicles are fundamental to further develop therapeutic and biotechnological applications. Microalgae constitute sustainable sources of bioactive compounds with a range of sectorial applications including for example the formulation of health supplements, cosmetic products or food ingredients. In this study, we demonstrate that microalgae are promising producers of EVs. By analysing the nanosized extracellular nano-objects produced by eighteen microalgal species, we identified seven promising EV-producing strains belonging to distinct lineages, suggesting that the production of EVs in microalgae is an evolutionary conserved trait. Here we report the selection process and focus on one of this seven species, the glaucophyte Cyanophora paradoxa, which returned a protein yield in the small EV fraction of 1 µg of EV proteins per mg of dry weight of microalgal biomass (corresponding to 109 particles per mg of dried biomass) and EVs with a diameter of 130 nm (mode), as determined by the micro bicinchoninic acid assay, nanoparticle tracking and dynamic light scattering analyses. Moreover, the extracellular nanostructures isolated from the conditioned media of microalgae species returned positive immunoblot signals for some commonly used EV-biomarkers such as Alix, Enolase, HSP70, and ß-actin. Overall, this work establishes a platform for the efficient production of EVs from a sustainable bioresource and highlights the potential of microalgal EVs as novel biogenic nanovehicles.

Small Angle X-ray Scattering Sensing Membrane Composition: The Role of Sphingolipids in Membrane-Amyloid ß-Peptide Interaction.

The early impairments appearing in Alzheimer's disease are related to neuronal membrane damage. Both aberrant Aß species and specific membrane components play a role in promoting aggregation, deposition, and signaling dysfunction. Ganglioside GM1, present with cholesterol and sphingomyelin in lipid rafts, preferentially interacts with the Aß peptide. GM1 at physiological conditions clusters in the membrane, the assembly also involves phospholipids, sphingomyelin, and cholesterol. The structure of large unilamellar vesicles (LUV), made of a basic POPC:POPS matrix in a proportion of 9:1, and containing different amounts of GM1 (1%, 3%, and 4% mol/mol) in the presence of 5% mol/mol sphingomyelin and 15% mol/mol cholesterol, was studied using small angle X-ray scattering (SAXS). The effect of the membrane composition on the LUVs-Aß-peptide interaction, both for Aß1-40 and Aß1-42 variants, was, thus, monitored. The presence of GM1 leads to a significant shift of the main peak, towards lower scattering angles, up to 6% of the initial value with SM and 8% without, accompanied by an opposite shift of the first minimum, up to 21% and 24% of the initial value, respectively. The analysis of the SAXS spectra, using a multi-Gaussian model for the electronic density profile, indicated differences in the bilayer of the various compositions. An increase in the membrane thickness, by 16% and 12% when 2% and 3% mol/mol GM1 was present, without and with SM, respectively, was obtained. Furthermore, in these cases, in the presence of Aß40, a very small decrease of the bilayer thickness, less than 4% and 1%, respectively, was derived, suggesting the inhibiting effect that the presence of sphingomyelin has on the GM1-Aß interaction.

Light Scattering as an Easy Tool to Measure Vesicles Weight Concentration.

Over the last few decades, liposomes have emerged as promising drug delivery systems and effective membrane models for studying biophysical and biological processes. For all applications, knowing their concentration after preparation is crucial. Thus, the development of methods for easily controlling vesicles concentration would be of great utility. A new assay is presented here, based on a suitable analysis of light scattering intensity from liposome dispersions. The method, tested for extrusion preparations, is precise, easy, fast, non-destructive and uses a tiny amount of sample. Furthermore, the scattering intensity can be measured indifferently at different angles, or even by using the elastic band obtained from a standard spectrofluorimeter. To validate the method, the measured concentrations of vesicles of different matrix compositions and sizes, measured by light scattering with different angles and instruments, were compared to the data obtained by the standard Stewart assay. Consistent results were obtained. The light scattering assay is based on the assessment of the mass fraction lost in the preparation, and can be applied for methods such as extrusion, homogenization, French press and other microfluidic procedures.

Inhibition of Aß1-42 Fibrillation by Chaperonins: Human Hsp60 Is a Stronger Inhibitor than Its Bacterial Homologue GroEL.

Alzheimer's disease is a chronic neurodegenerative disease characterized by the accumulation of pathological aggregates of amyloid beta peptide. Many efforts have been focused on understanding peptide aggregation pathways and on identification of molecules able to inhibit aggregation in order to find an effective therapy. As a result, interest in neuroprotective proteins, such as molecular chaperones, has increased as their normal function is to assist in protein folding or to facilitate the disaggregation and/or clearance of abnormal aggregate proteins. Using biophysical techniques, we evaluated the effects of two chaperones, human Hsp60 and bacterial GroEL, on the fibrillogenesis of Aß1-42. Both chaperonins interfere with Aß1-42 aggregation, but the effect of Hsp60 is more significant and correlates with its more pronounced flexibility and stronger interaction with ANS, an indicator of hydrophobic regions. Dose-dependent ThT fluorescence kinetics and SAXS experiments reveal that Hsp60 does not change the nature of the molecular processes stochastically leading to the formation of seeds, but strongly delays them by recognition of hydrophobic sites of some peptide species crucial for triggering amyloid formation. Hsp60 reduces the initial chaotic heterogeneity of Aß1-42 sample at high concentration regimes. The understanding of chaperone action in counteracting pathological aggregation could be a starting point for potential new therapeutic strategies against neurodegenerative diseases.

Amyloid ß-Peptide Interaction with Membranes: Can Chaperones Change the Fate?

The understanding of amyloid ß-peptide (Aß) interactions with cellular membranes is a crucial molecular challenge against Alzheimer's disease. Indeed, Aß prefibrillar oligomeric intermediates are believed to be the most toxic species, able to induce cellular damages directly by membrane damage. We present a neutron-scattering study on the interaction of large unilamellar vesicles (LUV), as cell membrane models, with both freshly dissolved Aß and early toxic prefibrillar oligomers, intermediate states in the amyloid pathway. In addition, we explore the effect of coincubating the Aß-peptide with the chaperonin Hsp60, which is known to strongly interact with it in its aggregation pattern. In fact, the interaction of the LUV with coincubated Aß/Hsp60, right after mixing and after following the aggregation protocol leading to the toxic intermediates in the absence of Hsp60, is studied. Neutron spin echo experiments show that the interaction with both freshly dissolved and aggregate Aß species brings about an increase in membrane stiffness, whereas the presence of even very low amounts of Hsp60 (ratio Aß/Hsp60 = 25:1) maintains unaltered the elastic properties of the membrane bilayer. A coherent interpretation of these results, related to previous literature, can be based on the ability of the chaperonin to interfere with Aß aggregation, by the specific recognition of the Aß-reactive transient species. In this framework, our results strongly suggest that early in a freshly dissolved Aß solution are present some species able to modify the bilayer dynamics, and the chaperonin plays the role of an assistant in such stochastic "misfolding events", avoiding the insult on the membrane as well as the onset of the aggregation cascade.

Biophysical characterization of asolectin-squalene liposomes.

Liposomes are shell nanoparticles able to embed hydrophobic molecules into their lipid layers to be released to cells. In pharmaceutical sciences, liposomes remain the delivery system with the highest biocompatibility, stability, loading characteristics, tunable physicochemical properties. Squalene is a natural, water insoluble, lipid, abundant in olive oil and shark liver. Studies in vitro and in animal models suggest protective and inhibitory effects of squalene against cancer. To study its effect on cells, and to overcome its insolubility in water, we have designed and produced large unilamellar liposomes containing different quantities of this terpene (0%, 2.8%, 5% w/w). Liposomes have been characterized by different biophysical techniques. Size-exclusion and affinity chromatography showed a unimodal size distribution and confirmed the squalene loaded dose. Laurdan fluorescence evidenced the changes in the hydration of the external layer of liposomes as a function of squalene concentration. Dynamic light scattering and small angle X-ray scattering revealed squalene induced structural differences in the hydrodynamic radius distribution and in the bilayer thickness respectively. Finally, preliminary experiments on the effects of liposome-delivered squalene on tumor and non-tumor cell lines showed time- and dose-dependent cytotoxic effects on LAN5 tumor cells and no effect on NIH-3T3 normal cells.

The effects of pressure on the energy landscape of proteins.

Protein dynamics is characterized by fluctuations among different conformational substates, i.e. the different minima of their energy landscape. At temperatures above ~200 K, these fluctuations lead to a steep increase in the thermal dependence of all dynamical properties, phenomenon known as Protein Dynamical Transition. In spite of the intense studies, little is known about the effects of pressure on these processes, investigated mostly near room temperature. We studied by neutron scattering the dynamics of myoglobin in a wide temperature and pressure range. Our results show that high pressure reduces protein motions, but does not affect the onset temperature for the Protein Dynamical Transition, indicating that the energy differences and barriers among conformational substates do not change with pressure. Instead, high pressure values strongly reduce the average structural differences between the accessible conformational substates, thus increasing the roughness of the free energy landscape of the system.

Investigation on different chemical stability of mitochondrial Hsp60 and its precursor.

In the large class of molecules that maintain protein homeostasis, called molecular chaperones, chaperonins constitute a subclass that specifically assist the correct folding of newly synthesized proteins. Among them, Hsp60 is composed of a double heptameric ring structure with a large central cavity where the unfolded protein binds via hydrophobic interactions and is supported, in this function, by the co-chaperonin Hsp10. Hsp60 is typically located in the mitochondria, but in some pathological situations, such as cancers and chronic inflammatory diseases, Hsp60 accumulates in the cytoplasm. In these cases, cytoplasmatic Hsp60 is a mixture of mitochondrial Hsp60 secreted from mitochondria upon stress, and its precursor, called naïve Hsp60, never entered into the organella. The difference between the naïve and mitochondrial Hsp60s resides in the absence of the mitochondrial import signal (MIS) in the mitochondrial form, but information on their different structure and stability is still lacking. We present here a study on the stability against a chemical denaturant, of the different cytoplasmic Hsp60 species. By combining Circular Dichroism and Small Angle X-ray Scattering as experimental biophysical techniques to investigate Hsp60, we find that naïve and mitochondrial Hsp60 (mtHsp60) forms differ in their stability. Furthermore, specific responses from the two forms are discussed in terms of the biological environment they are working in, thus opening new questions on their biological function.

Biological and biophysics aspects of metformin-induced effects: cortex mitochondrial dysfunction and promotion of toxic amyloid pre-fibrillar aggregates.

The onset of Alzheimer disease (AD) is influenced by several risk factors comprising diabetes. Within this context, antidiabetic drugs, including metformin, are investigated for their effect on AD. We report that in the C57B6/J mice, metformin is delivered to the brain where activates AMP-activated kinase (AMPK), its molecular target. This drug affects the levels of ß-secretase (BACE1) and ß-amyloid precursor protein (APP), promoting processing and aggregation of ß-amyloid (Aß), mainly in the cortex region. Moreover, metformin induces mitochondrial dysfunction and cell death by affecting the level and conformation of Translocase of the Outer Membrane 40 (TOM40), voltage-dependent anion-selective channels 1 (VDAC1) and hexokinase I (HKI), proteins involved in mitochondrial transport of molecules, including Aß. By using biophysical techniques we found that metformin is able to directly interact with Aß influencing its aggregation kinetics and features. These findings indicate that metformin induces different adverse effects, leading to an overall increase of the risk of AD onset.

Hsp60, amateur chaperone in amyloid-beta fibrillogenesis.

BACKGROUND: Molecular chaperones are a very special class of proteins that play essential roles in many cellular processes like folding, targeting and transport of proteins. Moreover, recent evidence indicates that chaperones can act as potentially strong suppressor agents in Alzheimer's disease (AD). Indeed, in vitro experiments demonstrate that several chaperones are able to significantly slow down or suppress aggregation of Aß peptide and in vivo studies reveal that treatment with specific chaperones or their overexpression can ameliorate some distinct pathological signs characterizing AD. METHODS: Here we investigate using a biophysical approach (fluorescence, circular dichroism (CD), transmission electron (TEM) and atomic force (AFM) microscopy, size exclusion chromatography (SEC)) the effect of the human chaperonin Hsp60 on Aß fibrillogenesis. RESULTS: We found that Hsp60 powerfully inhibits Aß amyloid aggregation, by closing molecular pathways leading to peptide fibrillogenesis. CONCLUSIONS: We observe that Hsp60 inhibits Aß aggregation through a more complex mechanism than a simple folding chaperone action. The action is specifically directed toward the early oligomeric species behaving as aggregation seeds for on-pathway amyloid fibrillogenesis. GENERAL SIGNIFICANCE: Understanding the specificity of the molecular interactions of Hsp60 with amyloid Aß peptide allowed us to emphasize the important aspects to be taken into consideration when considering the recent promising therapeutic strategies for neurodegeneration.

Amyloid ß-peptide insertion in liposomes containing GM1-cholesterol domains.

Neuronal membrane damage is related to the early impairments appearing in Alzheimer's disease due to the interaction of the amyloid ß-peptide (Aß) with the phospholipid bilayer. In particular, the ganglioside GM1, present with cholesterol in lipid rafts, seems to be able to initiate Aß aggregation on membrane. We studied the thermodynamic and structural effects of the presence of GM1 on the interaction between Aß and liposomes, a good membrane model system. Isothermal Titration Calorimetry highlighted the importance of the presence of GM1 in recruiting monomeric Aß toward the lipid bilayer. Light and Small Angle X-ray Scattering revealed a different pattern for GM1 containing liposomes, both before and after interaction with Aß. The results suggest that the interaction with GM1 brings to insertion of Aß in the bilayer, producing a structural perturbation down to the internal layers of the liposome, as demonstrated by the obtained electron density profiles.

Stability and disassembly properties of human naïve Hsp60 and bacterial GroEL chaperonins.

Human Hsp60 chaperonin and its bacterial homolog GroEL, in association with the corresponding co-chaperonins Hsp10 and GroES, constitute important chaperone systems promoting the proper folding of several mitochondrial proteins. Hsp60 is also currently described as a ubiquitous molecule with multiple roles both in health conditions and in several diseases. Naïve Hsp60 bearing the mitochondrial import signal has been recently demonstrated to present different oligomeric organizations with respect to GroEL, suggesting new possible physiological functions. Here we present a combined investigation with circular dichroism and small-angle X-ray scattering of structure, self-organization, and stability of naïve Hsp60 in solution in comparison with bacterial GroEL. Experiments have been performed in different concentrations of guanidine hydrochloride, monitoring the dissociation of tetradecamers into heptamers and monomers, until unfolding. GroEL is proved to be more stable with respect to Hsp60, and the unfolding free energy as well as its dependence on denaturant concentration is obtained.

Human Hsp60 with its mitochondrial import signal occurs in solution as heptamers and tetradecamers remarkably stable over a wide range of concentrations.

It has been established that Hsp60 can accumulate in the cytosol in various pathological conditions, including cancer and chronic inflammatory diseases. Part or all of the cytosolic Hsp60 could be naïve, namely, bear the mitochondrial import signal (MIS), but neither the structure nor the in solution oligomeric organization of this cytosolic molecule has still been elucidated. Here we present a detailed study of the structure and self-organization of naïve cytosolic Hsp60 in solution. Results were obtained by different biophysical methods (light and X ray scattering, single molecule spectroscopy and hydrodynamics) that all together allowed us to assay a wide range of concentrations of Hsp60. We found that Naïve Hsp60 in aqueous solution is assembled in very stable heptamers and tetradecamers at all concentrations assayed, without any trace of monomer presence.

α-Casein Inhibits Insulin Amyloid Formation by Preventing the Onset of Secondary Nucleation Processes.

α-Casein is known to inhibit the aggregation of several proteins, including the amyloid ß-peptide, by mechanisms that are not yet completely clear. We studied its effects on insulin, a system extensively used to investigate the properties of amyloids, many of which are common to all proteins and peptides. In particular, as for other proteins, insulin aggregation is affected by secondary nucleation pathways. We found that α-casein strongly delays insulin amyloid formation, even at extremely low doses, when the aggregation process is characterized by secondary nucleation. At difference, it has a vanishing inhibitory effect on the initial oligomer formation, which is observed at high concentration and does not involve any secondary nucleation pathway. These results indicate that an efficient inhibition of amyloid formation can be achieved by chaperone-like systems, by sequestering the early aggregates, before they can trigger the exponential proliferation brought about by secondary nucleation mechanisms.