Most recent advances and applications of extracellular vesicles in tackling neurological challenges.

Over the past few decades, there has been a notable increase in the global burden of central nervous system (CNS) diseases. Despite advances in technology and therapeutic options, neurological and neurodegenerative disorders persist as significant challenges in treatment and cure. Recently, there has been a remarkable surge of interest in extracellular vesicles (EVs) as pivotal mediators of intercellular communication. As carriers of molecular cargo, EVs demonstrate the ability to traverse the blood-brain barrier, enabling bidirectional communication. As a result, they have garnered attention as potential biomarkers and therapeutic agents, whether in their natural form or after being engineered for use in the CNS. This review article aims to provide a comprehensive introduction to EVs, encompassing various aspects such as their diverse isolation methods, characterization, handling, storage, and different routes for EV administration. Additionally, it underscores the recent advances in their potential applications in neurodegenerative disorder therapeutics. By exploring their unique capabilities, this study sheds light on the promising future of EVs in clinical research. It considers the inherent challenges and limitations of these emerging applications while incorporating the most recent updates in the field.

Extreme Biocatalysts: From Basic Research to Biotechnological Applications.

Biocatalysis, a cornerstone of modern biotechnology, is poised to revolutionize industrial processes across diverse sectors [...].

Thermophilic Hemicellulases Secreted by Microbial Consortia Selected from an Anaerobic Digester.

The rise of agro-industrial activities over recent decades has exponentially increased lignocellulose biomasses (LCB) production. LCB serves as a cost-effective source for fermentable sugars and other renewable chemicals. This study explores the use of microbial consortia, particularly thermophilic consortia, for LCB deconstruction. Thermophiles produce stable enzymes that retain activity under industrial conditions, presenting a promising approach for LCB conversion. This research focused on two microbial consortia (i.e., microbiomes) that were analyzed for enzyme production using a cheap medium, i.e., a mixture of spent mushroom substrate (SMS) and digestate. The secreted xylanolytic enzymes were characterized in terms of temperature and pH optima, thermal stability, and hydrolysis products from LCB-derived polysaccharides. These enzymes showed optimal activity aligning with common biorefinery conditions and outperformed a formulated enzyme mixture in thermostability tests in the digestate. Phylogenetic and genomic analyses highlighted the genetic diversity and metabolic potential of these microbiomes. Bacillus licheniformis was identified as a key species, with two distinct strains contributing to enzyme production. The presence of specific glycoside hydrolases involved in the cellulose and hemicellulose degradation underscores these consortia's capacity for efficient LCB conversion. These findings highlight the potential of thermophilic microbiomes, isolated from an industrial environment, as a robust source of robust enzymes, paving the way for more sustainable and cost-effective bioconversion processes in biofuel and biochemical production and other biotechnological applications.

Lignocellulolytic Potential of Microbial Consortia Isolated from a Local Biogas Plant: The Case of Thermostable Xylanases Secreted by Mesophilic Bacteria.

Lignocellulose biomasses (LCB), including spent mushroom substrate (SMS), pose environmental challenges if not properly managed. At the same time, these renewable resources hold immense potential for biofuel and chemicals production. With the mushroom market growth expected to amplify SMS quantities, repurposing or disposal strategies are critical. This study explores the use of SMS for cultivating microbial communities to produce carbohydrate-active enzymes (CAZymes). Addressing a research gap in using anaerobic digesters for enriching microbiomes feeding on SMS, this study investigates microbial diversity and secreted CAZymes under varied temperatures (37 °C, 50 °C, and 70 °C) and substrates (SMS as well as pure carboxymethylcellulose, and xylan). Enriched microbiomes demonstrated temperature-dependent preferences for cellulose, hemicellulose, and lignin degradation, supported by thermal and elemental analyses. Enzyme assays confirmed lignocellulolytic enzyme secretion correlating with substrate degradation trends. Notably, thermogravimetric analysis (TGA), coupled with differential scanning calorimetry (TGA-DSC), emerged as a rapid approach for saccharification potential determination of LCB. Microbiomes isolated at mesophilic temperature secreted thermophilic hemicellulases exhibiting robust stability and superior enzymatic activity compared to commercial enzymes, aligning with biorefinery conditions. PCR-DGGE and metagenomic analyses showcased dynamic shifts in microbiome composition and functional potential based on environmental conditions, impacting CAZyme abundance and diversity. The meta-functional analysis emphasised the role of CAZymes in biomass transformation, indicating microbial strategies for lignocellulose degradation. Temperature and substrate specificity influenced the degradative potential, highlighting the complexity of environmental-microbial interactions. This study demonstrates a temperature-driven microbial selection for lignocellulose degradation, unveiling thermophilic xylanases with industrial promise. Insights gained contribute to optimizing enzyme production and formulating efficient biomass conversion strategies. Understanding microbial consortia responses to temperature and substrate variations elucidates bioconversion dynamics, emphasizing tailored strategies for harnessing their biotechnological potential.

Neural Stem Cell-Derived Extracellular Vesicles Counteract Insulin Resistance-Induced Senescence of Neurogenic Niche.

Neural stem and progenitor cell (NSPC) depletion may play a crucial role in the cognitive impairment observed in many age-related non-communicable diseases. Insulin resistance affects brain functions through a plethora of mechanisms that remain poorly understood. In an experimental model of insulin resistant NSPCs, we identified a novel molecular circuit relying on insulin receptor substrate-1 (IRS-1)/ Forkhead box O (FoxO) signaling cascade and inhibiting the recruitment of transcription factors FoxO1 and FoxO3a on the promoters of genes regulating proliferation and self-renewal. Insulin resistance also epigenetically increased the expression of cyclin-dependent kinase inhibitor 1 (p21) and accelerated NSPC senescence. Of note, we found that stimulation of NSPCs with NSPC-derived exosomes (exo-NSPC) rescued IRS-1/FoxO activation and counteracted both the reduced proliferation and senescence of stem cells. Accordingly, intranasal administration of exo-NSPC counteracted the high-fat diet-dependent impairment of adult hippocampal neurogenesis in mice by restoring the balance between proliferating and senescent NSPCs in the hippocampus. Our findings suggest a novel mechanism underlying the metabolic control of NSPC fate potentially involved in the detrimental effects of metabolic disorders on brain plasticity. In addition, our data highlight the role of extracellular vesicle-mediated signals in the regulation of cell fate within the adult neurogenic niche.

Chronic mild stress alters synaptic plasticity in the nucleus accumbens through GSK3ß-dependent modulation of Kv4.2 channels.

Although major depressive disorder (MDD) is highly prevalent, its pathophysiology is poorly understood. Recent evidence suggests that glycogen-synthase kinase 3ß (GSK3ß) plays a key role in memory formation, yet its role in mood regulation remains controversial. Here, we investigated whether GSK3ß activity in the nucleus accumbens (NAc) is associated with depression-like behaviors and synaptic plasticity. We performed whole-cell patch-clamp recordings of medium spiny neurons (MSNs) in the NAc and determined the role of GSK3ß in spike timing-dependent long-term potentiation (tLTP) in the chronic unpredictable mild stress (CUMS) mouse model of depression. To assess the specific role of GSK3ß in tLTP, we used in vivo genetic silencing by an adeno-associated viral vector (AAV2) short hairpin RNA against GSK3ß. In addition, we examined the role of the voltage-gated potassium Kv4.2 subunit, a molecular determinant of A-type K+ currents, as a potential downstream target of GSK3ß. We found increased levels of active GSK3ß and augmented tLTP in CUMS mice, a phenotype that was prevented by selective GSK3ß knockdown. Furthermore, knockdown of GSK3ß in the NAc ameliorated depressive-like behavior in CUMS mice. Electrophysiological, immunohistochemical, biochemical, and pharmacological experiments revealed that inhibition of the Kv4.2 channel through direct phosphorylation at Ser-616 mediated the GSK3ß-dependent tLTP changes in CUMS mice. Our results identify GSK3ß regulation of Kv4.2 channels as a molecular mechanism of MSN maladaptive plasticity underlying depression-like behaviors and suggest that the GSK3ß-Kv4.2 axis may be an attractive therapeutic target for MDD.

High-Fat Diet Leads to Reduced Protein O-GlcNAcylation and Mitochondrial Defects Promoting the Development of Alzheimer's Disease Signatures.

The disturbance of protein O-GlcNAcylation is emerging as a possible link between altered brain metabolism and the progression of neurodegeneration. As observed in brains with Alzheimer's disease (AD), flaws of the cerebral glucose uptake translate into reduced protein O-GlcNAcylation, which promote the formation of pathological hallmarks. A high-fat diet (HFD) is known to foster metabolic dysregulation and insulin resistance in the brain and such effects have been associated with the reduction of cognitive performances. Remarkably, a significant role in HFD-related cognitive decline might be played by aberrant protein O-GlcNAcylation by triggering the development of AD signature and mitochondrial impairment. Our data support the impairment of total protein O-GlcNAcylation profile both in the brain of mice subjected to a 6-week high-fat-diet (HFD) and in our in vitro transposition on SH-SY5Y cells. The reduction of protein O-GlcNAcylation was associated with the development of insulin resistance, induced by overfeeding (i.e., defective insulin signaling and reduced mitochondrial activity), which promoted the dysregulation of the hexosamine biosynthetic pathway (HBP) flux, through the AMPK-driven reduction of GFAT1 activation. Further, we observed that a HFD induced the selective impairment of O-GlcNAcylated-tau and of O-GlcNAcylated-Complex I subunit NDUFB8, thus resulting in tau toxicity and reduced respiratory chain functionality respectively, highlighting the involvement of this posttranslational modification in the neurodegenerative process.

GSK3ß Modulates Timing-Dependent Long-Term Depression Through Direct Phosphorylation of Kv4.2 Channels.

Spike timing-dependent plasticity (STDP) is a form of activity-dependent remodeling of synaptic strength that underlies memory formation. Despite its key role in dictating learning rules in the brain circuits, the molecular mechanisms mediating STDP are still poorly understood. Here, we show that spike timing-dependent long-term depression (tLTD) and A-type K+ currents are modulated by pharmacological agents affecting the levels of active glycogen-synthase kinase 3 (GSK3) and by GSK3ß knockdown in layer 2/3 of the mouse somatosensory cortex. Moreover, the blockade of A-type K+ currents mimics the effects of GSK3 up-regulation on tLTD and occludes further changes in synaptic strength. Pharmacological, immunohistochemical and biochemical experiments revealed that GSK3ß influence over tLTD induction is mediated by direct phosphorylation at Ser-616 of the Kv4.2 subunit, a molecular determinant of A-type K+ currents. Collectively, these results identify the functional interaction between GSK3ß and Kv4.2 channel as a novel mechanism for tLTD modulation providing exciting insight into the understanding of GSK3ß role in synaptic plasticity.

Neural Stem Cell-Derived Exosomes Revert HFD-Dependent Memory Impairment via CREB-BDNF Signalling.

Overnutrition and metabolic disorders impair cognitive functions through molecular mechanisms still poorly understood. In mice fed with a high fat diet (HFD) we analysed the expression of synaptic plasticity-related genes and the activation of cAMP response element-binding protein (CREB)-brain-derived neurotrophic factor (BDNF)-tropomyosin receptor kinase B (TrkB) signalling. We found that a HFD inhibited both CREB phosphorylation and the expression of a set of CREB target genes in the hippocampus. The intranasal administration of neural stem cell (NSC)-derived exosomes (exo-NSC) epigenetically restored the transcription of Bdnf, nNOS, Sirt1, Egr3, and RelA genes by inducing the recruitment of CREB on their regulatory sequences. Finally, exo-NSC administration rescued both BDNF signalling and memory in HFD mice. Collectively, our findings highlight novel mechanisms underlying HFD-related memory impairment and provide evidence of the potential therapeutic effect of exo-NSC against metabolic disease-related cognitive decline.

Galactomannan degradation by thermophilic enzymes: a hot topic for biotechnological applications.

Extremophilic microorganisms are valuable sources of enzymes for various industrial applications. In fact, given their optimal catalytic activity and operational stability under harsh physical and chemical conditions, they represent a suitable alternative to their mesophilic counterparts. For instance, extremophilic enzymes are important to foster the switch from fossil-based to lignocellulose-based industrial processes. Indeed, more stable enzymes are needed, because the conversion of the lignocellulosic biomass to a wide palette of value-added products requires extreme chemo-physical pre-treatments. Galactomannans are part of the hemicellulose fraction in lignocellulosic biomass. They are heteropolymers constituted by a ß-1,4-linked mannan backbone substituted with side chains of α-1,6-linked galactose residues. Therefore, the joint action of different hydrolytic enzymes (i.e. ß-mannanase, ß-mannosidase and α-galactosidase) is needed to accomplish their complete hydrolysis. So far, numerous galactomannan-degrading enzymes have been isolated and characterized from extremophilic microorganisms. Besides applications in biorefinery, these biocatalysts are also useful to improve the quality (i.e. digestibility and prebiotic properties) of food and feed as well as in paper industries to aid the pulp bleaching process. In this review, an overview about the structure, function and applications of galactomannans is provided. Moreover, a survey of (hyper)-thermophilic galactomannans-degrading enzymes, mainly characterized in the last decade, has been carried out. These extremozymes are described in the light of their biotechnological application in industrial processes requiring harsh conditions.

Nutrient-Dependent Changes of Protein Palmitoylation: Impact on Nuclear Enzymes and Regulation of Gene Expression.

Diet is the main environmental stimulus chronically impinging on the organism throughout the entire life. Nutrients impact cells via a plethora of mechanisms including the regulation of both protein post-translational modifications and gene expression. Palmitoylation is the most-studied protein lipidation, which consists of the attachment of a molecule of palmitic acid to residues of proteins. S-palmitoylation is a reversible cysteine modification finely regulated by palmitoyl-transferases and acyl-thioesterases that is involved in the regulation of protein trafficking and activity. Recently, several studies have demonstrated that diet-dependent molecules such as insulin and fatty acids may affect protein palmitoylation. Here, we examine the role of protein palmitoylation on the regulation of gene expression focusing on the impact of this modification on the activity of chromatin remodeler enzymes, transcription factors, and nuclear proteins. We also discuss how this physiological phenomenon may represent a pivotal mechanism underlying the impact of diet and nutrient-dependent signals on human diseases.

Insights into the anticancer properties of the first antimicrobial peptide from Archaea.

BACKGROUND: The peptide VLL-28, identified in the sequence of an archaeal protein, the transcription factor Stf76 from Sulfolobus islandicus, was previously identified and characterized as an antimicrobial peptide, possessing a broad-spectrum antibacterial activity. METHODS: Through a combined approach of NMR and Circular Dichroism spectroscopy, Dynamic Light Scattering, confocal microscopy and cell viability assays, the interaction of VLL-28 with the membranes of both parental and malignant cell lines has been characterized and peptide mechanism of action has been studied. RESULTS: It is here demonstrated that VLL-28 selectively exerts cytotoxic activity against murine and human tumor cells. By means of structural methodologies, VLL-28 interaction with the membranes has been proven and the binding residues have been identified. Confocal microscopy data show that VLL-28 is internalized only into tumor cells. Finally, it is shown that cell death is mainly caused by a time-dependent activation of apoptotic pathways. CONCLUSIONS: VLL-28, deriving from the archaeal kingdom, is here found to be endowed with selective cytotoxic activity towards both murine and human cancer cells and consequently can be classified as an ACP. GENERAL SIGNIFICANCE: VLL-28 represents the first ACP identified in an archaeal microorganism, exerting a trans-kingdom activity.

Thermus thermophilus as source of thermozymes for biotechnological applications: homologous expression and biochemical characterization of an α-galactosidase.

BACKGROUND: The genus Thermus, which has been considered for a long time as a fruitful source of biotechnological relevant enzymes, has emerged more recently as suitable host to overproduce thermozymes. Among these, α-galactosidases are widely used in several industrial bioprocesses that require high working temperatures and for which thermostable variants offer considerable advantages over their thermolabile counterparts. RESULTS: Thermus thermophilus HB27 strain was used for the homologous expression of the TTP0072 gene encoding for an α-galactosidase (TtGalA). Interestingly, a soluble and active histidine-tagged enzyme was produced in larger amounts (5 mg/L) in this thermophilic host than in Escherichia coli (0.5 mg/L). The purified recombinant enzyme showed an optimal activity at 90 °C and retained more than 40% of activity over a broad range of pH (from 5 to 8). CONCLUSIONS: TtGalA is among the most thermoactive and thermostable α-galactosidases discovered so far, thus pointing to T. thermophilus as cell factory for the recombinant production of biocatalysts active at temperature values over 90 °C.

Unravelling the Role of the F55 Regulator in the Transition from Lysogeny to UV Induction of Sulfolobus Spindle-Shaped Virus 1.

UNLABELLED: Sulfolobus spindle-shaped virus 1 represents a model for studying virus-host interaction in harsh environments, and it is so far the only member of the family Fuselloviridae that shows a UV-inducible life cycle. Although the virus has been extensively studied, mechanisms underpinning the maintenance of lysogeny as well as those regulating the UV induction have received little attention. Recently, a novel SSV1 transcription factor, F55, was identified. This factor was able to bind in vitro to several sequences derived from the early and UV-inducible promoters of the SSV1 genome. The location of these binding sites together with the differential affinity of F55 for these sequences led to the hypothesis that this protein might be involved in the maintenance of the SSV1 lysogeny. Here, we report an in vivo survey of the molecular events occurring at the UV-inducible region of the SSV1 genome, with a focus on the binding profile of F55 before and after the UV irradiation. The binding of F55 to the target promoters correlates with transcription repression, whereas its dissociation is paralleled by transcription activation. Therefore, we propose that F55 acts as a molecular switch for the transcriptional regulation of the early viral genes. IMPORTANCE: Functional genomic studies of SSV1 proteins have been hindered by the lack of similarity with other characterized proteins. As a result, few insights into their in vivo roles have been gained throughout the last 3 decades. Here, we report the first in vivo investigation of an SSV1 transcription regulator, F55, that plays a key role in the transition from the lysogenic to the induced state of SSV1. We show that F55 regulates the expression of the UV-inducible as well as the early genes. Moreover, the differential affinity of this transcription factor for these targets allows a fine-tuned and temporal coordinated regulation of transcription of viral genes.

Structural and functional studies of Stf76 from the Sulfolobus islandicus plasmid-virus pSSVx: a novel peculiar member of the winged helix-turn-helix transcription factor family.

The hybrid plasmid-virus pSSVx from Sulfolobus islandicus presents an open reading frame encoding a 76 amino acid protein, namely Stf76, that does not show significant sequence homology with any protein with known 3D structure. The recombinant protein recognizes specifically two DNA-binding sites located in its own promoter, thus suggesting an auto-regulated role of its expression. Circular dichroism, spectrofluorimetric, light scattering and isothermal titration calorimetry experiments indicated a 2:1 molar ratio (protein:DNA) upon binding to the DNA target containing a single site. Furthermore, the solution structure of Stf76, determined by nuclear magnetic resonance (NMR) using chemical shift Rosetta software, has shown that the protein assumes a winged helix-turn-helix fold. NMR chemical shift perturbation analysis has been performed for the identification of the residues responsible for DNA interaction. In addition, a model of the Stf76-DNA complex has been built using as template a structurally related homolog.

Monitoring the Response of Hyperbilirubinemia in the Mouse Brain by In Vivo Bioluminescence Imaging.

Increased levels of unconjugated bilirubin are neurotoxic, but the mechanism leading to neurological damage has not been completely elucidated. Innovative strategies of investigation are needed to more precisely define this pathological process. By longitudinal in vivo bioluminescence imaging, we noninvasively visualized the brain response to hyperbilirubinemia in the MITO-Luc mouse, in which light emission is restricted to the regions of active cell proliferation. We assessed that acute hyperbilirubinemia promotes bioluminescence in the brain region, indicating an increment in the cell proliferation rate. Immunohistochemical detection in brain sections of cells positive for both luciferase and the microglial marker allograft inflammatory factor 1 suggests proliferation of microglial cells. In addition, we demonstrated that brain induction of bioluminescence was altered by pharmacological displacement of bilirubin from its albumin binding sites and by modulation of the blood-brain barrier permeability, all pivotal factors in the development of bilirubin-induced neurologic dysfunction. We also determined that treatment with minocycline, an antibiotic with anti-inflammatory and neuroprotective properties, or administration of bevacizumab, an anti-vascular endothelial growth factor antibody, blunts bilirubin-induced bioluminescence. Overall the study supports the use of the MITO-Luc mouse as a valuable tool for the rapid response monitoring of drugs aiming at preventing acute bilirubin-induced neurological dysfunction.

A standardized protocol for the UV induction of Sulfolobus spindle-shaped virus 1.

The Fuselloviridae prototype member Sulfolobus spindle-shaped virus 1 is a model of UV-inducible viruses infecting Crenarchaeota. Previous works on SSV1 UV induction were bases on empirically determined parameters that have not yet been standardized. Thus, in many peer reviewed literature, it is not clear how the fluence and irradiance have been determined. Here, we describe a protocol for the UV induction of SSV1 replication, which is based on the combination of the following instrumentally monitored parameters: (1) the fluence; (2) the irradiance; (3) the exposure time, and (4) the exposure distance. With the aim of finding a good balance between the viral replication induction and the host cells viability, UV-irradiated cultures were monitored for their ability to recover in the aftermath of the UV exposure. This UV irradiation procedure has been set up using the well-characterized Sulfolobus solfataricus P2 strain as model system to study host-virus interaction.

The identification of a novel Sulfolobus islandicus CAMP-like peptide points to archaeal microorganisms as cell factories for the production of antimicrobial molecules.

BACKGROUND: Pathogenic bacteria easily develop resistance to c onventional antibiotics so that even relatively new molecules are quickly losing efficacy. This strongly encourages the quest of new antimicrobials especially for the treatment of chronic infections. Cationic antimicrobial peptides (CAMPs) are small positively charged peptides with an amphipathic structure, active against Gram-positive and Gram-negative bacteria, fungi, as well as protozoa. RESULTS: A novel (CAMP)-like peptide (VLL-28) was identified in the primary structure of a transcription factor, Stf76, encoded by pSSVx, a hybrid plasmid-virus from the archaeon Sulfolobus islandicus. VLL-28 displays chemical, physical and functional properties typical of CAMPs. Indeed, it has a broad-spectrum antibacterial activity and acquires a defined structure in the presence of membrane mimetics. Furthermore, it exhibits selective leakage and fusogenic capability on vesicles with a lipid composition similar to that of bacterial membranes. VLL-28 localizes not only on the cell membrane but also in the cytoplasm of Escherichia coli and retains the ability to bind nucleic acids. These findings suggest that this CAMP-like peptide could exert its antimicrobial activity both on membrane and intra cellular targets. CONCLUSIONS: VLL-28 is the first CAMP-like peptide identified in the archaeal kingdom, thus pointing to archaeal microorganisms as cell factories to produce antimicrobial molecules of biotechnological interest. Furthermore, results from this work show that DNA/RNA-binding proteins could be used as sources of CAMPs.

A role for neuronal cAMP responsive-element binding (CREB)-1 in brain responses to calorie restriction.

Calorie restriction delays brain senescence and prevents neurodegeneration, but critical regulators of these beneficial responses other than the NAD(+)-dependent histone deacetylase Sirtuin-1 (Sirt-1) are unknown. We report that effects of calorie restriction on neuronal plasticity, memory and social behavior are abolished in mice lacking cAMP responsive-element binding (CREB)-1 in the forebrain. Moreover, CREB deficiency drastically reduces the expression of Sirt-1 and the induction of genes relevant to neuronal metabolism and survival in the cortex and hippocampus of dietary-restricted animals. Biochemical studies reveal a complex interplay between CREB and Sirt-1: CREB directly regulates the transcription of the sirtuin in neuronal cells by binding to Sirt-1 chromatin; Sirt-1, in turn, is recruited by CREB to DNA and promotes CREB-dependent expression of target gene peroxisome proliferator-activated receptor-γ coactivator-1α and neuronal NO Synthase. Accordingly, expression of these CREB targets is markedly reduced in the brain of Sirt KO mice that are, like CREB-deficient mice, poorly responsive to calorie restriction. Thus, the above circuitry, modulated by nutrient availability, links energy metabolism with neurotrophin signaling, participates in brain adaptation to nutrient restriction, and is potentially relevant to accelerated brain aging by overnutrition and diabetes.