Truncated tau interferes with the autophagy and endolysosomal pathway and results in lipid accumulation.

The autophagy-lysosomal pathway plays a critical role in the clearance of tau protein aggregates that deposit in the brain in tauopathies, and defects in this system are associated with disease pathogenesis. Here, we report that expression of Tau35, a tauopathy-associated carboxy-terminal fragment of tau, leads to lipid accumulation in cell lines and primary cortical neurons. Our findings suggest that this is likely due to a deleterious block of autophagic clearance and lysosomal degradative capacity by Tau35. Notably, upon induction of autophagy by Torin 1, Tau35 inhibited nuclear translocation of transcription factor EB (TFEB), a key regulator of lysosomal biogenesis. Both cell lines and primary cortical neurons expressing Tau35 also exhibited changes in endosomal protein expression. These findings implicate autophagic and endolysosomal dysfunction as key pathological mechanisms through which disease-associated tau fragments could lead to the development and progression of tauopathy.

'I want to be a different kind of father': a qualitative analysis of adolescent fatherhood in Perú.

Adolescent parents and their offspring experience worse health outcomes throughout the life course. While over 90% of adolescent births occur in low- and middle-income countries, data from many such countries are lacking, particularly from fathers. This qualitative study conducted in Lima, Peru characterises the experience of adolescent fathers and identifies potential intervention targets. Interviews with young fathers and the mothers of their children were coded and analysed using thematic analysis and a grounded theory approach. Factors impacting their experience included family support, changes in their relationship with their partner, gender dynamics, and financial pressure. The study identified family and couple conflict, gendered expectations, and the father's personal development as potential intervention targets. Further research is needed to develop interventions that effectively engage adolescent fathers in low- and middle-income countries such as Peru, and support their transition to fatherhood.

Proteomics of the astrocyte secretome reveals changes in their response to soluble oligomeric Aß.

Astrocytes associate with amyloid plaques in Alzheimer's disease (AD). Astrocytes react to changes in the brain environment, including increasing concentrations of amyloid-ß (Aß). However, the precise response of astrocytes to soluble small Aß oligomers at concentrations similar to those present in the human brain has not been addressed. In this study, we exposed astrocytes to media from neurons that express the human amyloid precursor protein (APP) transgene with the double Swedish mutation (APPSwe), and which contains APP-derived fragments, including soluble human Aß oligomers. We then used proteomics to investigate changes in the astrocyte secretome. Our data show dysregulated secretion of astrocytic proteins involved in the extracellular matrix and cytoskeletal organization and increase secretion of proteins involved in oxidative stress responses and those with chaperone activity. Several of these proteins have been identified in previous transcriptomic and proteomic studies using brain tissue from human AD and cerebrospinal fluid (CSF). Our work highlights the relevance of studying astrocyte secretion to understand the brain response to AD pathology and the potential use of these proteins as biomarkers for the disease.

P2X7R influences tau aggregate burden in human tauopathies and shows distinct signalling in microglia and astrocytes.

The purinoceptor P2X7R is a promising therapeutic target for tauopathies, including Alzheimer's disease (AD). Pharmacological inhibition or genetic knockdown of P2X7R ameliorates cognitive deficits and reduces pathological tau burden in mice that model aspects of tauopathy, including mice expressing mutant human frontotemporal dementia (FTD)-causing forms of tau. However, disagreements remain over which glial cell types express P2X7R and therefore the mechanism of action is unresolved. Here, we show that P2X7R protein levels increase in human AD post-mortem brain, in agreement with an upregulation of P2RX7 mRNA observed in transcriptome profiles from the AMP-AD consortium. P2X7R protein increases mirror advancing Braak stage and coincide with synapse loss. Using RNAScope we detect P2RX7 mRNA in microglia and astrocytes in human AD brain, including in the vicinity of senile plaques. In cultured microglia, P2X7R activation modulates the NLRP3 inflammasome pathway by promoting the formation of active complexes and release of IL-1ß. In astrocytes, P2X7R activates NFκB signalling and increases production of the cytokines CCL2, CXCL1 and IL-6 together with the acute phase protein Lcn2. To further explore the role of P2X7R in a disease-relevant context, we expressed wild-type or FTD-causing mutant forms of tau in mouse organotypic brain slice cultures. Inhibition of P2X7R reduces insoluble tau levels without altering soluble tau phosphorylation or synaptic localisation, suggesting a non-cell autonomous role of glial P2X7R on pathological tau aggregation. These findings support further investigations into the cell-type specific effects of P2X7R-targeting therapies in tauopathies.

Astrocytic C-X-C motif chemokine ligand-1 mediates ß-amyloid-induced synaptotoxicity.

BACKGROUND: Pathological interactions between ß-amyloid (Aß) and tau drive synapse loss and cognitive decline in Alzheimer's disease (AD). Reactive astrocytes, displaying altered functions, are also a prominent feature of AD brain. This large and heterogeneous population of cells are increasingly recognised as contributing to early phases of disease. However, the contribution of astrocytes to Aß-induced synaptotoxicity in AD is not well understood. METHODS: We stimulated mouse and human astrocytes with conditioned medium containing concentrations and species of human Aß that mimic those in human AD brain. Medium from stimulated astrocytes was collected and immunodepleted of Aß before being added to naïve rodent or human neuron cultures. A cytokine, identified in unbiased screens of stimulated astrocyte media and in postmortem human AD brain lysates was also applied to neurons, including those pre-treated with a chemokine receptor antagonist. Tau mislocalisation, synaptic markers and dendritic spine numbers were measured in cultured neurons and organotypic brain slice cultures. RESULTS: We found that conditioned medium from stimulated astrocytes induces exaggerated synaptotoxicity that is recapitulated following spiking of neuron culture medium with recombinant C-X-C motif chemokine ligand-1 (CXCL1), a chemokine upregulated in AD brain. Antagonism of neuronal C-X-C motif chemokine receptor 2 (CXCR2) prevented synaptotoxicity in response to CXCL1 and Aß-stimulated astrocyte secretions. CONCLUSIONS: Our data indicate that astrocytes exacerbate the synaptotoxic effects of Aß via interactions of astrocytic CXCL1 and neuronal CXCR2 receptors, highlighting this chemokine-receptor pair as a novel target for therapeutic intervention in AD.

Regulation of mammalian autophagy in physiology and pathophysiology.

(Macro)autophagy is a bulk degradation process that mediates the clearance of long-lived proteins and organelles. Autophagy is initiated by double-membraned structures, which engulf portions of cytoplasm. The resulting autophagosomes ultimately fuse with lysosomes, where their contents are degraded. Although the term autophagy was first used in 1963, the field has witnessed dramatic growth in the last 5 years, partly as a consequence of the discovery of key components of its cellular machinery. In this review we focus on mammalian autophagy, and we give an overview of the understanding of its machinery and the signaling cascades that regulate it. As recent studies have also shown that autophagy is critical in a range of normal human physiological processes, and defective autophagy is associated with diverse diseases, including neurodegeneration, lysosomal storage diseases, cancers, and Crohn's disease, we discuss the roles of autophagy in health and disease, while trying to critically evaluate if the coincidence between autophagy and these conditions is causal or an epiphenomenon. Finally, we consider the possibility of autophagy upregulation as a therapeutic approach for various conditions.

siRNA screen identifies QPCT as a druggable target for Huntington's disease.

Huntington's disease (HD) is a currently incurable neurodegenerative condition caused by an abnormally expanded polyglutamine tract in huntingtin (HTT). We identified new modifiers of mutant HTT toxicity by performing a large-scale 'druggable genome' siRNA screen in human cultured cells, followed by hit validation in Drosophila. We focused on glutaminyl cyclase (QPCT), which had one of the strongest effects on mutant HTT-induced toxicity and aggregation in the cell-based siRNA screen and also rescued these phenotypes in Drosophila. We found that QPCT inhibition induced the levels of the molecular chaperone αB-crystallin and reduced the aggregation of diverse proteins. We generated new QPCT inhibitors using in silico methods followed by in vitro screening, which rescued the HD-related phenotypes in cell, Drosophila and zebrafish HD models. Our data reveal a new HD druggable target affecting mutant HTT aggregation and provide proof of principle for a discovery pipeline from druggable genome screen to drug development.

Phosphoproteomic analysis of protein kinase C signaling in Saccharomyces cerevisiae reveals Slt2 mitogen-activated protein kinase (MAPK)-dependent phosphorylation of eisosome core components.

The cell wall integrity (CWI) pathway of the model organism Saccharomyces cerevisiae has been thoroughly studied as a paradigm of the mitogen-activated protein kinase (MAPK) pathway. It consists of a classic MAPK module comprising the Bck1 MAPK kinase kinase, two redundant MAPK kinases (Mkk1 and Mkk2), and the Slt2 MAPK. This module is activated under a variety of stimuli related to cell wall homeostasis by Pkc1, the only member of the protein kinase C family in budding yeast. Quantitative phosphoproteomics based on stable isotope labeling of amino acids in cell culture is a powerful tool for globally studying protein phosphorylation. Here we report an analysis of the yeast phosphoproteome upon overexpression of a PKC1 hyperactive allele that specifically activates CWI MAPK signaling in the absence of external stimuli. We found 82 phosphopeptides originating from 43 proteins that showed enhanced phosphorylation in these conditions. The MAPK S/T-P target motif was significantly overrepresented in these phosphopeptides. Hyperphosphorylated proteins provide putative novel targets of the Pkc1-cell wall integrity pathway involved in diverse functions such as the control of gene expression, protein synthesis, cytoskeleton maintenance, DNA repair, and metabolism. Remarkably, five components of the plasma-membrane-associated protein complex known as eisosomes were found among the up-regulated proteins. We show here that Pkc1-induced phosphorylation of the eisosome core components Pil1 and Lsp1 was not exerted directly by Pkc1, but involved signaling through the Slt2 MAPK module.

Reactive astrocytes secrete the chaperone HSPB1 to mediate neuroprotection.

Molecular chaperones are protective in neurodegenerative diseases by preventing protein misfolding and aggregation, such as extracellular amyloid plaques and intracellular tau neurofibrillary tangles in Alzheimer's disease (AD). In addition, AD is characterized by an increase in astrocyte reactivity. The chaperone HSPB1 has been proposed as a marker for reactive astrocytes; however, its astrocytic functions in neurodegeneration remain to be elucidated. Here, we identify that HSPB1 is secreted from astrocytes to exert non-cell-autonomous protective functions. We show that in human AD brain, HSPB1 levels increase in astrocytes that cluster around amyloid plaques, as well as in the adjacent extracellular space. Moreover, in conditions that mimic an inflammatory reactive response, astrocytes increase HSPB1 secretion. Concomitantly, astrocytes and neurons can uptake astrocyte-secreted HSPB1, which is accompanied by an attenuation of the inflammatory response in reactive astrocytes and reduced pathological tau inclusions. Our findings highlight a protective mechanism in disease conditions that encompasses the secretion of a chaperone typically regarded as intracellular.

Microglial cytokines poison neuronal autophagy via CCR5, a druggable target.

In the prodromal phase of neurodegenerative diseases, microglia switch to an activated state resulting in increased secretion of pro-inflammatory factors. We reported that C - C chemokine ligand 3 (CCL3), C - C chemokine ligand 4 (CCL4) and C - C chemokine ligand 5 (CCL5) contained in the secretome of activated microglia inhibit neuronal autophagy via a non-cell autonomous mechanism. These chemokines bind and activate neuronal C - C chemokine receptor type 5 (CCR5), which, in turn, promotes phosphoinositide 3-kinase (PI3K) - protein kinase B (PKB, or AKT) - mammalian target of rapamycin complex 1 (mTORC1) pathway activation, which inhibits autophagy, thus causing the accumulation of aggregate-prone proteins in the cytoplasm of neurons. The levels of CCR5 and its chemokine ligands are increased in the brains of pre-manifesting Huntington disease (HD) and tauopathy mouse models. CCR5 accumulation might be due to a self-amplifying mechanism, since CCR5 is a substrate of autophagy and CCL5-CCR5-mediated autophagy inhibition impairs CCR5 degradation. Furthermore, pharmacological, or genetic inhibition of CCR5 rescues mTORC1-autophagy dysfunction and improves neurodegeneration in HD and tauopathy mouse models, suggesting that CCR5 hyperactivation is a pathogenic signal driving the progression of these diseases.

Astrocyte adaptation in Alzheimer's disease: a focus on astrocytic P2X7R.

Astrocytes are key homeostatic and defensive cells of the central nervous system (CNS). They undertake numerous functions during development and in adulthood to support and protect the brain through finely regulated communication with other cellular elements of the nervous tissue. In Alzheimer's disease (AD), astrocytes undergo heterogeneous morphological, molecular and functional alterations represented by reactive remodelling, asthenia and loss of function. Reactive astrocytes closely associate with amyloid ß (Aß) plaques and neurofibrillary tangles in advanced AD. The specific contribution of astrocytes to AD could potentially evolve along the disease process and includes alterations in their signalling, interactions with pathological protein aggregates, metabolic and synaptic impairments. In this review, we focus on the purinergic receptor, P2X7R, and discuss the evidence that P2X7R activation contributes to altered astrocyte functions in AD. Expression of P2X7R is increased in AD brain relative to non-demented controls, and animal studies have shown that P2X7R antagonism improves cognitive and synaptic impairments in models of amyloidosis and tauopathy. While P2X7R activation can induce inflammatory signalling pathways, particularly in microglia, we focus here specifically on the contributions of astrocytic P2X7R to synaptic changes and protein aggregate clearance in AD, highlighting cell-specific roles of this purinoceptor activation that could be targeted to slow disease progression.

Microglial-to-neuronal CCR5 signaling regulates autophagy in neurodegeneration.

In neurodegenerative diseases, microglia switch to an activated state, which results in excessive secretion of pro-inflammatory factors. Our work aims to investigate how this paracrine signaling affects neuronal function. Here, we show that activated microglia mediate non-cell-autonomous inhibition of neuronal autophagy, a degradative pathway critical for the removal of toxic, aggregate-prone proteins accumulating in neurodegenerative diseases. We found that the microglial-derived CCL-3/-4/-5 bind and activate neuronal CCR5, which in turn promotes mTORC1 activation and disrupts autophagy and aggregate-prone protein clearance. CCR5 and its cognate chemokines are upregulated in the brains of pre-manifesting mouse models for Huntington's disease (HD) and tauopathy, suggesting a pathological role of this microglia-neuronal axis in the early phases of these diseases. CCR5 upregulation is self-sustaining, as CCL5-CCR5 autophagy inhibition impairs CCR5 degradation itself. Finally, pharmacological or genetic inhibition of CCR5 rescues mTORC1 hyperactivation and autophagy dysfunction, which ameliorates HD and tau pathologies in mouse models.

Chemical inducers of autophagy that enhance the clearance of mutant proteins in neurodegenerative diseases.

Many of the neurodegenerative diseases that afflict people are caused by intracytoplasmic aggregate-prone proteins. These include Parkinson disease, tauopathies, and polyglutamine expansion diseases such as Huntington disease. In Mendelian forms of these diseases, the mutations generally confer toxic novel functions on the relevant proteins. Thus, one potential strategy for dealing with these mutant proteins is to enhance their degradation. This can be achieved by up-regulating macroautophagy, which we will henceforth call autophagy. In this minireview, we will consider the reasons why autophagy up-regulation may be a powerful strategy for these diseases. In addition, we will consider some of the drugs and associated signaling pathways that can be used to induce autophagy with these therapeutic aims in mind.

Influence of Coronal Preflaring on the Accuracy of Electronic Working Length Determination: Systematic Review and Meta-Analysis.

AIM: To conduct a systematic review and meta-analysis according to the following PICO question: in extracted human permanent teeth, does preflaring, compared with unflared canals, influence the accuracy of WL determination with EAL? MATERIAL AND METHODS: A systematic review was conducted according to the PRISMA checklist, using the following databases: PubMed, Science Direct, Scopus, and Web of Science. Studies related to WL determination using EAL both in preflared and unflared root canals of extracted human teeth were included. The outcome of interest was the accuracy of the electronic WL determination. A quality assessment of the included studies was performed, determining the risk of bias. The meta-analyses were calculated with the 5.4 RevMan software using the inverse variance method with random effects. PROSPERO registration: CRD42021243412. RESULTS: Ten experimental studies fulfilled the inclusion criteria, and most of them found that preflaring increases the accuracy of the EALs in WL determination. The calculated OR was 1.98 (95% CI = 1.65-2.37; p < 0.00001; I2 = 10%), indicating that the determination of WL by EALs is almost twice as accurate in preflared canals. The accuracy of Root ZX in WL determination increases more than three times (OR = 3.25; p < 0.00001). Preflaring with Protaper files significantly increases the accuracy of EALs (OR = 1.76; p < 0.00001). The total risk of bias of the included studies was low. No obvious publication bias was observed. CONCLUSIONS: The results indicate a significant increase in the accuracy of WL determination with EAL after preflaring, doubling the percentage of exact measurements. Preflaring should be recommended as an important step during mechanical enlargement of the root canal, not only because it improves the access of the files to the canal, but also because it allows one to obtain more accurate electronic determinations of WL.

Antibiotics Prescription by Spanish General Practitioners in Primary Dental Care.

The aim of this study was to analyze the antibiotics prescription habits, both prophylactically and therapeutically, of Spanish general dental practitioners in the management of endodontic infections in primary care. Two hundred Spanish general dental practitioners were asked to respond to a survey on indications for antibiotics prescription in the treatment of endodontic infections, being 190 general dentists (95%) included in the study. Data were analyzed using descriptive statistics and the chi-square test. The average duration of antibiotics therapy was 6.5 ± 1.0 days. In patients without medical allergies, most of them (97%) selected amoxicillin as the antibiotic of the first choice, alone (51.1%) or associated with clavulanic acid (45.8%); in patients with penicillin allergies, the drug of choice was clindamycin 300 mg (70%). For cases of symptomatic irreversible pulpitis, 44% of the respondents prescribed antibiotics, in the scenario of prophylactic antibiotic prescription, up to 27% of the general dentists prescribe according to non-current guidelines (1 g 1 h before or 1 g 1 h before and 1 g 1 h after) in non-indicated cases (16% in patients taking oral bisphosphonates). It is necessary to improve the antibiotic prescription habits of Spanish general dentists in endodontics.

Biomaterials in periapical regeneration after microsurgical endodontics: A narrative review.

BACKGROUND: The objective of this narrative review was to analyze the available scientific evidence regarding the application of biomaterials in endodontic microsurgery and its influence in post-surgical tissue repair. MATERIAL AND METHODS: The review question was Do biomaterials used in endodontic microsurgery influence post-surgical tissue repair and regeneration? Systematic MEDLINE/PubMed review was used to evaluate and present the results. RESULTS: The search yielded 131 references, 82 of which were selected for full text review after reading the abstracts. After a manual search in the references of the articles selected, 52 references were eliminated. Finally, 30 articles were selected. CONCLUSIONS: Bone grafts, membranes and bioceramics, especially MTA, are biomaterials with the ability to stimulate periapical tissue regeneration. This is one of many reason why bioceramics are the best choice as retrograde sealing materials. However, microsurgically treated periapical lesions can heal completely without the need to use bone grafts or membranes. Those techniques are indicated in endodontic microsurgery when additional stimulation of tissue regeneration is required, or when bone collapse needs to be prevented. Key words:Bioactive endodontic cements, endodontic surgery, periapical repair.

Preventing Functional Urinary Incontinence in Hip-Fractured Older Adults Through Patient Education: A Randomized Controlled Trial.

The purpose of this study was to evaluate whether an educational intervention would reduce the incidence of functional urinary incontinence (UI) in older adults with a fall-related hip fracture. The project was conducted as a multicenter randomized controlled trial (RCT). A total of 109 patients that had been admitted to six hospitals in Castilla-La Mancha (Spain) for acute treatment of hip fracture, previously continent and without cognitive impairment, were enrolled and randomly assigned to the experimental group (EG) or the control group (CG). Intervention (on EG): urinary habit training (Nursing Interventions Classifications taxonomy) was performed during hospital stay (second to fourth postoperative day), with a telephonic reinforcement 10 days after discharge. The CG received routine care. Primary outcome measure: incidence of UI. Follow-up: telephone assessment 3 and 6 months after discharge (blinded evaluation). The incidence of UI at 6 months was 49% (CG) versus 25.5% (EG) (relative risk = 0.52, 95% confidence interval [0.3, 0.9]; number necessary to treat = 4). The mean of UI episodes was 0.54 (EG) versus 1.8 (CG), p = .007. The educational intervention prevents the development of UI and decreases the number of episodes in case of appearance, in a statistically significant way.

Autophagy in Astrocytes and its Implications in Neurodegeneration.

Autophagy is a major degradation pathway where double-membrane vesicles called autophagosomes deliver cytoplasmic content to the lysosome. Increasing evidence suggests that autophagy dysfunction contributes to the pathogenesis of neurodegenerative diseases. In addition, misfolded proteins that accumulate in these diseases and constitute a common pathological hallmark are substrates for autophagic degradation. Astrocytes, a major type of glial cells, are emerging as a critical component in most neurodegenerative diseases. This review will summarize the recent efforts to investigate the role that autophagy plays in astrocytes in the context of neurodegenerative diseases. While the field has mostly focused on the implications of autophagy in neurons, autophagy may also be involved in the clearance of disease-related proteins in astrocytes as well as in maintaining astrocyte function, which could impact the cell autonomous and non-cell autonomous contribution of astrocytes to neurodegeneration.

A Microstructure Insight of MTA Repair HP of Rapid Setting Capacity and Bioactive Response.

Mineral trioxide aggregate (MTA) is considered a bioactive endodontic material, which promotes natural mineralization at the material-tooth tissue interface. MTA Repair HP stands out because of the short setting time and the quick and effective bioactive response in vitro. The bioactivity, depens on material composition and microstructure. This work is devoted to analyze MTA Repair HP microstructural features, of both the powder precursor and set material, to get insights into the material physicochemical parameters-functionality performance relationships. Transmission electron microscopy (TEM), and field emission gun scanning electron microscopy (FEG-SEM) coupled with energy-dispersive X-ray (EDX) analyses were performed. X-ray diffraction (XRD) measurements were carried out at different times to investigate setting process. Bioactivity evaluation in vitro was carried out by soaking the processed cement disk in simulated body fluid (SBF). The presented results point out those MTA Repair HP precursor material characteristics of tricalcium silicate particles of nanometric size and high aspect ratio, which provide an elevated surface area and maximized components dispersion of calcium silicate and very reactive calcium aluminate. The MTA Repair HP precursor powder nanostructure and formulation, allows a hydration process comprising silicate hydrate structures, which are very effective to achieve both fast setting and efficient bioactive response.