ALS-linked protein disulfide isomerase variants cause motor dysfunction.

Disturbance of endoplasmic reticulum (ER) proteostasis is a common feature of amyotrophic lateral sclerosis (ALS). Protein disulfide isomerases (PDIs) areERfoldases identified as possibleALSbiomarkers, as well as neuroprotective factors. However, no functional studies have addressed their impact on the disease process. Here, we functionally characterized fourALS-linked mutations recently identified in two majorPDIgenes,PDIA1 andPDIA3/ERp57. Phenotypic screening in zebrafish revealed that the expression of thesePDIvariants induce motor defects associated with a disruption of motoneuron connectivity. Similarly, the expression of mutantPDIs impaired dendritic outgrowth in motoneuron cell culture models. Cellular and biochemical studies identified distinct molecular defects underlying the pathogenicity of thesePDImutants. Finally, targetingERp57 in the nervous system led to severe motor dysfunction in mice associated with a loss of neuromuscular synapses. This study identifiesERproteostasis imbalance as a risk factor forALS, driving initial stages of the disease.

Association between depressive symptoms in mothers and metabolic control in adolescents with type 1 diabetes. / Asociación entre síntomas depresivos de las madres y control metabólico en adolescentes con Diabetes Mellitus tipo 1.

INTRODUCTION: Poor metabolic control in patients with Type 1 Diabetes Mellitus (T1DM) is associated with short- and long-term complications. Adolescents with T1DM present poorer metabolic control than pa tients of other age groups. Few studies have shown an association between mothers with depressive symptoms and the metabolic control of their adolescent children. OBJECTIVE: To evaluate the associa tion between maternal depressive symptoms and metabolic control of their adolescents with T1DM. SUBJECTS AND METHOD: Cross-sectional observational study carried out with adolescents aged between 10 and 18 years, with T1DM diagnosis of at least 1 year ago and their mothers. The Beck Depression Inventory-II and the SALUFAM questionnaire were applied, and sociodemographic data were co llected. Glycosylated hemoglobin from capillary blood was used as a marker of metabolic control. RESULTS: 86 couples (mother-adolescent children) were studied. The average age of the adolescents was 14.04 years and the average evolution time of T1DM was 5.95 years. 27.325.6% of mothers had depressive symptoms, which was associated with worse metabolic control of their children (HbA1c of 7.66% and 8.91%, p-value <0.001). 17.9% of adolescents had depressive symptoms, which was not associated with maternal depressive symptoms or worse metabolic control. Maternal depressive symptoms were also associated with lower maternal and paternal educational levels, high number of children in the family, presence of other siblings with chronic illnesses, and high health vulnera bility (SALUFAM). CONCLUSIONS: The mother's depressive symptoms can be associated with worst metabolic control in T1MD adolescents. It is fundamental a multidisciplinary family approach to get better metabolic controls in T1DM adolescents.

The secretory phenotype of senescent astrocytes isolated from Wistar newborn rats changes with anti-inflammatory drugs, but does not have a short-term effect on neuronal mitochondrial potential.

In the central nervous system (CNS), senescent astrocytes have been associated with neurodegeneration. Senescent cells secrete a complex mixture of pro-inflammatory factors, which are collectively called Senescence Associated Secretory Phenotype (SASP). The SASP components can vary depending on the cell type, senescence inducer and time. The SASP has been mainly studied in fibroblasts and epithelial cells, but little is known in the context of the CNS. Here, the SASP profile in senescent astrocytes isolated from Wistar newborn rats induced to senescence by oxidative stress or by proteasome inhibition was analyzed. Senescent astrocytes secreted predominantly chemokines and IL-1α, but no IL-6. The effect of the anti-inflammatory drugs, sulforaphane (SFN) and dehydroepiandrosterone (DHEA), on the SASP profile was evaluated. Our results showed that SFN and DHEA decreased IL-1α secretion while increasing IL-10, thus modifying the SASP to a less anti-inflammatory profile. Primary neurons were subjected to the conditioned media obtained from drug-treated senescent astrocytes, and their mitochondrial membrane potential was evaluated.

Cyclophilin D over-expression increases mitochondrial complex III activity and accelerates supercomplex formation.

Cyclophilin D (CyPD), a mitochondrial matrix protein, has been widely studied for its role in mitochondrial-mediated cell death. Unexpectedly, we previously discovered that overexpression of CyPD in a stable cell line, increased mitochondrial membrane potentials and enhanced cell survival under conditions of oxidative stress. Here, we investigated the underlying mechanisms responsible for these findings. Spectrophotometric measurements in isolated mitochondria revealed that overexpression of CyPD in HEK293 cells increased respiratory chain activity, but only for Complex III (CIII). Acute treatment of mitochondria with the immumosupressant cyclosporine A did not affect CIII activity. Expression levels of the CIII subunits cytochrome b and Rieske-FeS were elevated in HEK293 cells overexpressing CyPD. However, CIII activity was still significantly higher compared to control mitochondria, even when normalized by protein expression. Blue native gel electrophoresis and Western blot assays revealed a molecular interaction of CyPD with CIII and increased levels of supercomplexes in mitochondrial protein extracts. Radiolabeled protein synthesis in mitochondria showed that CIII assembly and formation of supercomplexes containing CIII were significantly faster when CyPD was overexpressed. Taken together, these data indicate that CyPD regulates mitochondrial metabolism, and likely cell survival, by promoting more efficient electrons flow through the respiratory chain via increased supercomplex formation.

Long-lived species have improved proteostasis compared to phylogenetically-related shorter-lived species.

Our previous studies have shown that the liver from Naked Mole Rats (NMRs), a long-lived rodent, has increased proteasome activity and lower levels of protein ubiquitination compared to mice. This suggests that protein quality control might play a role in assuring species longevity. To determine whether enhanced proteostasis is a common mechanism in the evolution of other long-lived species, here we evaluated the major players in protein quality control including autophagy, proteasome activity, and heat shock proteins (HSPs), using skin fibroblasts from three phylogenetically-distinct pairs of short- and long-lived mammals: rodents, marsupials, and bats. Our results indicate that in all cases, macroautophagy was significantly enhanced in the longer-lived species, both at basal level and after induction by serum starvation. Similarly, basal levels of most HSPs were elevated in all the longer-lived species. Proteasome activity was found to be increased in the long-lived rodent and marsupial but not in bats. These observations suggest that long-lived species may have superior mechanisms to ensure protein quality, and support the idea that protein homeostasis might play an important role in promoting longevity.

Cell proliferation arrest and redox state status as part of different stages during senescence establishment in mouse fibroblasts.

Senescence phenotype can be achieved by multiple pathways. Most of them involve the activation of negative cell cycle regulators as well as a shift to an oxidative status. However, the exact participation of these events in senescence establishment and maintenance is not completely understood. In this study we investigated the content of three final cell cycle regulators, as well as the redox state in some critical points during the pre-senescent and the full-senescent states. Our results highlight the existence of a critical pre-phase in senescent phenotype establishment, in which cell proliferation stops with the participation of the cell cycle inhibitors, and a second maintenance stage where the exacerbated pro-oxidant state inside the cell induces the physiological decline characteristic in senescent cells.

Repeated dose toxicity study of Vibrio cholerae-loaded gastro-resistant microparticles.

CONTEXT: Microencapsulation of antigens has been extensively studied over the last decades aiming at improving the immunogenicity of vaccine candidates. OBJECTIVE: Addressing microparticles (MPs) toxicity in rats. MATERIAL AND METHODS: Spray-dried Eudragit® L 30 D-55 MPs and Eudragit® L 30 D-55 alginate MPs were elaborated and characterized. MPs obtained were administered to rats, three groups were defined: G1, control group; G2, administered with Vibrio cholerae (VC)-loaded MPs; G3, receiving VC-loaded alginate MPs. Animals received three vaccine doses. Body weight, food and water intake were controlled during the study. Haematological parameters, vibriocidal titres, organ weight and histology in necropsy were also analyzed. RESULTS: All animals grew healthy. Body weight gain, food and water intake and haematological parameters remained within physiological values, showing no treatment-related differences. Moreover, organ weight changes were not detected and animals developed protective vibriocidal titres. CONCLUSION: VC-loaded MPs and VC-loaded alginate MPs have proved to be safe and effective in the assessed conditions.

The coupling between healthspan and lifespan in Caenorhabditis depends on complex interactions between compound intervention and genetic background.

Aging is characterized by declining health that results in decreased cellular resilience and neuromuscular function. The relationship between lifespan and health, and the influence of genetic background on that relationship, has important implications in the development of pharmacological anti-aging interventions. Here we assessed swimming performance as well as survival under thermal and oxidative stress across a nematode genetic diversity test panel to evaluate health effects for three compounds previously studied in the Caenorhabditis Intervention Testing Program and thought to promote longevity in different ways - NP1 (nitrophenyl piperazine-containing compound 1), propyl gallate, and resveratrol. Overall, we find the relationships among median lifespan, oxidative stress resistance, thermotolerance, and mobility vigor to be complex. We show that oxidative stress resistance and thermotolerance vary with compound intervention, genetic background, and age. The effects of tested compounds on swimming locomotion, in contrast, are largely species-specific. In this study, thermotolerance, but not oxidative stress or swimming ability, correlates with lifespan. Notably, some compounds exert strong impact on some health measures without an equally strong impact on lifespan. Our results demonstrate the importance of assessing health and lifespan across genetic backgrounds in the effort to identify reproducible anti-aging interventions, with data underscoring how personalized treatments might be required to optimize health benefits.

Phage display of functional αß single-chain T-cell receptor molecules specific for CD1b:Ac2SGL complexes from Mycobacterium tuberculosis-infected cells.

The development of molecules specific for M. tuberculosis-infected cells has important implications, as these tools may facilitate understanding of the mechanisms regulating host pathogen interactions in vivo. In addition, development of new tools capable to targeting M. tuberculosis-infected cells may have potential applications to diagnosis, treatment, and prevention of tuberculosis (TB). Due to the lack of CD1b polymorphism, M. tuberculosis lipid-CD1b complexes could be considered as universal tuberculosis infection markers. The aim of the present study was to display on the PIII surface protein of m13 phage, a human αß single-chain T-cell receptor molecule specific for CD1b:2-stearoyl-3-hydroxyphthioceranoyl-2´-sulfate-α-α´-D-trehalose (Ac2SGL) which is a complex presented by human cells infected with M. tuberculosis. The results showed the pIII fusion particle was successfully displayed on the phage surface. The study of the recognition of the recombinant phage in ELISA and immunohistochemistry showed the recognition of CD1b:Ac2SGL complexes and cells in human lung tissue from a tuberculosis patient respectively, suggesting the specific recognition of the lipid-CD1b complex.

Effect of Nrf2 loss on senescence and cognition of tau-based P301S mice.

Cellular senescence may contribute to chronic inflammation involved in the progression of age-related diseases such as Alzheimer's disease (AD), and its removal prevents cognitive impairment in a model of tauopathy. Nrf2, the major transcription factor for damage response pathways and regulators of inflammation, declines with age. Our previous work showed that silencing Nrf2 gives rise to premature senescence in cells and mice. Others have shown that Nrf2 ablation can exacerbate cognitive phenotypes of some AD models. In this study, we aimed to understand the relationship between Nrf2 elimination, senescence, and cognitive impairment in AD, by generating a mouse model expressing a mutant human tau transgene in an Nrf2 knockout (Nrf2KO) background. We assessed senescent cell burden and cognitive decline of P301S mice in the presence and absence of Nrf2. Lastly, we administered 4.5-month-long treatments with two senotherapeutic drugs to analyze their potential to prevent senescent cell burden and cognitive decline: the senolytic drugs dasatinib and quercetin (DQ) and the senomorphic drug rapamycin. Nrf2 loss accelerated the onset of hind-limb paralysis in P301S mice. At 8.5 months of age, P301S mice did not exhibit memory deficits, while P301S mice without Nrf2 were significantly impaired. However, markers of senescence were not elevated by Nrf2 ablation in any of tissues that we examined. Neither drug treatment improved cognitive performance, nor did it reduce expression of senescence markers in brains of P301S mice. Contrarily, rapamycin treatment at the doses used delayed spatial learning and led to a modest decrease in spatial memory. Taken together, our data suggests that the emergence of senescence may be causally associated with onset of cognitive decline in the P301S model, indicate that Nrf2 protects brain function in a model of AD through mechanisms that may include, but do not require the inhibition of senescence, and suggest possible limitations for DQ and rapamycin as therapies for AD.

[Prevalence of recurrent aphthous stomatitis in a family medical office, Manzanillo, Cuban. A cross-sectional study]. / Prevalencia de estomatitis aftosa recurrente en un consultorio médico de familia, Manzanillo, Cuba. Un estudio transversal.

Introduction: Recurrent Aphthous Stomatitis, also known as aphthous ulcers or simply aphthous, is considered the most common of oral mucosal lesions. Objective: To describe the prevalence of recurrent aphthous stomatitis. Methods: Descriptive, cross-sectional and prospective study. 847 patients who attended the Family Medical Office No. 28, San Francisco comunity, Manzanillo, from July 1, 2021 to June 30, 2022, Cuba, were evaluated. A calibrated and trained assistant investigator evaluated the following variables: Clinical classification of recurrent aphthous stomatitis (minor aphthosis, major aphthosis, or aphthosis herpetiformis), lesion pain intensity, lesion location, and risk factors (viral infection), bacterial infection, immunological alterations, psychosomatic alterations, oral trauma, gastrointestinal alterations, endocrine factors, allergic conditions, heredity, blood and nutritional deficiencies, smoking), age group, sex, race, and duration of the lesion. Results: Aphthous stomatitis occurred in 30.46%, with greater frequency in the age group 30 - 39 years (24.42%). Minor aphthosis was the most frequent with 91.09%. The duration of the lesion of 10 to 12 days predominated with 37.60%, the most frequent location corresponded to the edge and tip of the tongue with 32.94% and the most representative pain intensity was mild with a total of 63.18%. The highest frequency among the risk factors corresponded to psychosomatic alterations with 100%. Conclusions: Recurrent Aphthous Stomatitis had a prevalence greater than 30% with a predominance of the female sex and young adults. Minor Aphtosis and a stay time of more than 10 days were the most frequent. The most common location is the tongue and bottom of the vestibular sulcus with the possible existence of a relationship between the mobile parts of the mouth. Stress, the main risk factor, exacerbated by Covid-19.

Soluble pathogenic tau enters brain vascular endothelial cells and drives cellular senescence and brain microvascular dysfunction in a mouse model of tauopathy.

Vascular mechanisms of Alzheimer's disease (AD) may constitute a therapeutically addressable biological pathway underlying dementia. We previously demonstrated that soluble pathogenic forms of tau (tau oligomers) accumulate in brain microvasculature of AD and other tauopathies, including prominently in microvascular endothelial cells. Here we show that soluble pathogenic tau accumulates in brain microvascular endothelial cells of P301S(PS19) mice modeling tauopathy and drives AD-like brain microvascular deficits. Microvascular impairments in P301S(PS19) mice were partially negated by selective removal of pathogenic soluble tau aggregates from brain. We found that similar to trans-neuronal transmission of pathogenic forms of tau, soluble tau aggregates are internalized by brain microvascular endothelial cells in a heparin-sensitive manner and induce microtubule destabilization, block endothelial nitric oxide synthase (eNOS) activation, and potently induce endothelial cell senescence that was recapitulated in vivo in microvasculature of P301S(PS19) mice. Our studies suggest that soluble pathogenic tau aggregates mediate AD-like brain microvascular deficits in a mouse model of tauopathy, which may arise from endothelial cell senescence and eNOS dysfunction triggered by internalization of soluble tau aggregates.

The Neurospora crassa mutant NcΔEgt-1 identifies an ergothioneine biosynthetic gene and demonstrates that ergothioneine enhances conidial survival and protects against peroxide toxicity during conidial germination.

Ergothioneine (EGT) is a histidine derivative with sulfur on the imidazole ring and a trimethylated amine; it is postulated to have an antioxidant function. Although EGT apparently is only produced by fungi and some prokaryotes, it is acquired by animals and plants from the environment, and is concentrated in animal tissues in cells with an EGT transporter. Monobromobimane derivatives of EGT allowed conclusive identification of EGT by LC/MS and the quantification of EGT in Colletotrichum graminicola and Neurospora crassa conidia and mycelia. EGT concentrations were significantly (α=0.05) higher in conidia than in mycelia, with approximately 17X and 5X more in C. graminicola and N. crassa, respectively. The first EGT biosynthetic gene in a fungus was identified by quantifying EGT in N. crassa wild type and knockouts in putative homologs of actinomycete EGT biosynthetic genes. NcΔEgt-1, a strain with a knockout in gene NCU04343, does not produce EGT, in contrast to the wild type. To determine the effects of EGT in vivo, we compared NcΔEgt-1 to the wild type. NcΔEgt-1 is not pleiotropically affected in rate of hyphal elongation in Vogel's medium either with or without ammonium nitrate and in the rate of germination of macroconidia on Vogel's medium. The superoxide-producer menadione had indistinguishable effects on conidial germination between the two strains. Cupric sulfate also had indistinguishable effects on conidial germination and on hyphal growth between the two strains. In contrast, germination of NcΔEgt-1 conidia was significantly more sensitive to tert-butyl hydroperoxide than the wild type; germination of 50% (GI(50)) of the NcΔEgt-1 conidia was prevented at 2.7 mM tert-butyl hydroperoxide whereas the GI(50) for the wild type was 4.7 mM tert-butyl hydroperoxide, or at a 1.7X greater concentration. In the presence of tert-butyl hydroperoxide and the fluorescent reactive oxygen species indicator 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate, significantly (P=0.0002) more NcΔEgt-1 conidia fluoresced than wild type conidia, indicating that EGT quenched peroxides in vivo. While five to 21-day-old conidia of both strains germinated 100%, NcΔEgt-1 conidia had significantly (P<0.001) diminished longevity. Linear regression analysis indicates that germination of the wild type declined to 50% in 35 days, in comparison to 25 days for the NcΔEgt-1, which is equivalent to a 29% reduction in conidial life span in the NcEgt-1 deletion strain. Consequently, the data indicate that endogenous EGT helps protect conidia during the quiescent period between conidiogenesis and germination, and that EGT helps protect conidia during the germination process from the toxicity of peroxide but not from superoxide or Cu(2+). Based on an in silico analysis, we postulate that NcEgt-1 was acquired early in the mycota lineage as a fusion of two adjacent prokaryotic genes, that was then lost in the Saccharomycotina, and that NcEgt-1 catalyzes the first two steps of EGT biosynthesis from histidine to hercynine to hercynylcysteine sulfoxide.

Protein stability and resistance to oxidative stress are determinants of longevity in the longest-living rodent, the naked mole-rat.

The widely accepted oxidative stress theory of aging postulates that aging results from accumulation of oxidative damage. Surprisingly, data from the longest-living rodent known, naked mole-rats [MRs; mass 35 g; maximum lifespan (MLSP) > 28.3 years], when compared with mice (MLSP 3.5 years) exhibit higher levels of lipid peroxidation, protein carbonylation, and DNA oxidative damage even at a young age. We hypothesize that age-related changes in protein structural stability, oxidation, and degradation are abrogated over the lifespan of the MR. We performed a comprehensive study of oxidation states of protein cysteines [both reversible (sulfenic, disulfide) and indirectly irreversible (sulfinic/sulfonic acids)] in liver from young and old C57BL/6 mice (6 and 28 months) and MRs (2 and >24 years). Furthermore, we compared interspecific differences in urea-induced protein unfolding and ubiquitination and proteasomal activity. Compared with data from young mice, young MRs have 1.6 times as much free protein thiol groups and similar amounts of reversible oxidative damage to cysteine. In addition, they show less urea-induced protein unfolding, less protein ubiquitination, and higher proteasome activity. Mice show a significant age-related increase in cysteine oxidation and higher levels of ubiquitination. In contrast, none of these parameters were significantly altered over 2 decades in MRs. Clearly MRs have markedly attenuated age-related accrual of oxidation damage to thiol groups and age-associated up-regulation of homeostatic proteolytic activity. These pivotal mechanistic interspecies differences may contribute to the divergent aging profiles and strongly implicate maintenance of protein stability and integrity in successful aging.

Brain cellular senescence in mouse models of Alzheimer's disease.

The accumulation of senescent cells contributes to aging pathologies, including neurodegenerative diseases, and its selective removal improves physiological and cognitive function in wild-type mice as well as in Alzheimer's disease (AD) models. AD models recapitulate some, but not all components of disease and do so at different rates. Whether brain cellular senescence is recapitulated in some or all AD models and whether the emergence of cellular senescence in AD mouse models occurs before or after the expected onset of AD-like cognitive deficits in these models are not yet known. The goal of this study was to identify mouse models of AD and AD-related dementias that develop measurable markers of cellular senescence in brain and thus may be useful to study the role of cellular senescence in these conditions. We measured the levels of cellular senescence markers in the brains of P301S(PS19), P301L, hTau, and 3xTg-AD mice that model amyloidopathy and/or tauopathy in AD and related dementias and in wild-type, age-matched control mice for each strain. Expression of cellular senescence markers in brains of transgenic P301L and 3xTg-AD mice was largely indistinguishable from that in WT control age-matched mice. In contrast, markers of cellular senescence were differentially increased in brains of transgenic hTau and P301S(PS19) mice as compared to WT control mice before the onset of AD-like cognitive deficits. Taken together, our data suggest that P301S(PS19) and hTau mice may be useful models for the study of brain cellular senescence in tauopathies including, but not limited to, AD.

Mitochondrial Dysfunction and the Glycolytic Switch Induced by Caveolin-1 Phosphorylation Promote Cancer Cell Migration, Invasion, and Metastasis.

Cancer cells often display impaired mitochondrial function, reduced oxidative phosphorylation, and augmented aerobic glycolysis (Warburg effect) to fulfill their bioenergetic and biosynthetic needs. Caveolin-1 (CAV1) is a scaffolding protein that promotes cancer cell migration, invasion, and metastasis in a manner dependent on CAV1 phosphorylation on tyrosine-14 (pY14). Here, we show that CAV1 expression increased glycolysis rates, while mitochondrial respiration was reduced by inhibition of the mitochondrial complex IV. These effects correlated with increased reactive oxygen species (ROS) levels that favored CAV1-induced migration and invasion. Interestingly, pY14-CAV1 promoted the metabolic switch associated with increased migration/invasion and augmented ROS-inhibited PTP1B, a phosphatase that controls pY14 levels. Finally, the glycolysis inhibitor 2-deoxy-D-glucose reduced CAV1-enhanced migration in vitro and metastasis in vivo of murine melanoma cells. In conclusion, CAV1 promotes the Warburg effect and ROS production, which inhibits PTP1B to augment CAV1 phosphorylation on tyrosine-14, thereby increasing the metastatic potential of cancer cells.

Genetic diversity estimates for the Caenorhabditis Intervention Testing Program screening panel.

The Caenorhabditis Intervention Testing Program (CITP) was founded on the principle that compounds with positive effects across a genetically diverse test-set should have an increased probability of engaging conserved biochemical pathways with mammalian translational potential. To fulfill its mandate, the CITP uses a genetic diversity panel of Caenorhabditis strains for assaying longevity effects of candidate compounds. The panel comprises 22 strains from three different species, collected globally, to achieve inter-population genetic diversity. The three represented species, C. elegans, C. briggsae, and C. tropicalis, are all sequential hermaphrodites, which simplifies experimental procedures while maximizing intra-population homogeneity. Here, we present estimates of the genetic diversity encapsulated by the constituent strains in the panel based on their most recently published and publicly available whole-genome sequences, as well as two newly generated genomic data sets. We observed average genome-wide nucleotide diversity (π) within the C. elegans (1.2e-3), C. briggsae (7.5e-3), and C. tropicalis strains (2.6e-3) greater than estimates for human populations, and comparable to that found in mouse populations. Our analysis supports the assumption that the CITP screening panel encompasses broad genetic diversity, suggesting that lifespan-extending chemicals with efficacy across the panel should be enriched for interventions that function on conserved processes that are shared across genetic backgrounds. While the diversity panel was established by the CITP for studying longevity interventions, the panel may prove useful for the broader research community when seeking broadly efficacious interventions for any phenotype with potential genetic background effects.

Transport and Secretion of the Wnt3 Ligand by Motor Neuron-like Cells and Developing Motor Neurons.

The vertebrate neuromuscular junction (NMJ) is formed by a presynaptic motor nerve terminal and a postsynaptic muscle specialization. Cumulative evidence reveals that Wnt ligands secreted by the nerve terminal control crucial steps of NMJ synaptogenesis. For instance, the Wnt3 ligand is expressed by motor neurons at the time of NMJ formation and induces postsynaptic differentiation in recently formed muscle fibers. However, the behavior of presynaptic-derived Wnt ligands at the vertebrate NMJ has not been deeply analyzed. Here, we conducted overexpression experiments to study the expression, distribution, secretion, and function of Wnt3 by transfection of the motor neuron-like NSC-34 cell line and by in ovo electroporation of chick motor neurons. Our findings reveal that Wnt3 is transported along motor axons in vivo following a vesicular-like pattern and reaches the NMJ area. In vitro, we found that endogenous Wnt3 expression increases as the differentiation of NSC-34 cells proceeds. Although NSC-34 cells overexpressing Wnt3 do not modify their morphological differentiation towards a neuronal phenotype, they effectively induce acetylcholine receptor clustering on co-cultured myotubes. These findings support the notion that presynaptic Wnt3 is transported and secreted by motor neurons to induce postsynaptic differentiation in nascent NMJs.

Rapamycin impairs bone accrual in young adult mice independent of Nrf2.

Advanced age is the strongest risk factor for osteoporosis. The immunomodulator drug rapamycin extends lifespan in numerous experimental model organisms and is being investigated as a potential therapeutic to slow human aging, but little is known about the effects of rapamycin on bone. We evaluated the impact of rapamycin treatment on bone mass, architecture, and indices of bone turnover in healthy adult (16-20 weeks old at treatment initiation) female wild-type (ICR) and Nrf2-/- mice, a mouse model of oxidative damage and aging-related disease vulnerability. Rapamycin (4 mg/kg bodyweight) was administered by intraperitoneal injection every other day for 12 weeks. Mice treated with rapamycin exhibited lower femur bone mineral content, bone mineral density, and bone volume compared to vehicle-treated mice. In midshaft femur diaphysis (cortical bone), rapamycin-treated mice had lower cortical volume and thickness, and in the distal femur metaphysis (cancellous bone), rapamycin-treated mice had higher trabecular spacing and lower connectivity density. Mice treated with rapamycin exhibited lower bone volume, bone volume fraction, and trabecular thickness in the 5th lumbar vertebra. Rapamycin-treated mice had lower levels of bone formation in the distal femur metaphysis compared to vehicle-treated mice which occurred co-incidentally with increased serum CTX-1, a marker of global bone resorption. Rapamycin had no impact on tibia inflammatory cytokine gene expression, and we found no independent effects of Nrf2 knockout on bone, nor did we find any interactions between genotype and treatment. These data show that rapamycin may have a negative impact on the skeleton of adult mice that should not be overlooked in the clinical context of its usage as a therapy to retard aging and reduce the incidence of age-related pathologies.