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Background: Cerebral small vessel disease is the most common cause of lacunar strokes (LS). Understanding LS pathogenesis is vital for predicting disease severity, prognosis, and developing therapies. Objectives: To research molecular profiles that differentiate LS in deep brain structures from those in subcortical white matter. Design: Prospective case–control study involving 120 patients with imaging-confirmed LS and a 120 control group. Methods: We examined the relationship between Alzheimer’s disease biomarkers [amyloid beta (Aβ 1–40 , Aβ 1–42 )], serum inflammatory marker (interleukin-6, IL-6), and endothelial dysfunction markers [soluble tumor necrosis factor-like weak inducer of apoptosis, and pentraxin-3 (sTWEAK, PTX3)] with respect to LS occurring in deep brain structures and subcortical white matter. In addition, we investigated links between LS, leukoaraiosis presence (white matter hyperintensities, WMHs), and functional outcomes at 3 months. Poor outcome was defined as a modified Rankin scale >2 at 3 months. Results: Significant differences were observed in levels of IL-6, PTX3, and sTWEAK between patients with deep lacunar infarcts and those with recent small subcortical infarcts (20.8 versus 15.6 pg/mL, p

Background: Monogenic Alzheimer’s disease (AD) has severe health and socioeconomic repercussions. Its rarest cause is presenilin 2 (PSEN2) gene mutations. We present two new cases with presumed PSEN2-AD with unusual clinical and neuroimaging findings in order to provide more information on the pathophysiology and semiology of these patients. Methods: Women aged 69 and 62 years at clinical onset, marked by prominent behavioral and language dysfunction, progressing to severe dementia within three years were included. The complete study is depicted. In addition, a systematic review of the PSEN2-AD was performed. Results: Neuroimaging revealed pronounced frontal white matter hyperintensities (WMH) and frontotemporal atrophy/hypometabolism. The genetic study unveiled PSEN2 variants: c.772G>A (p.Ala258Thr) and c.1073-2_1073-1del. Both cerebrospinal fluid (CSF) and experimental blood biomarkers shouldered AD etiology. Conclusions: Prominent behavioral and language dysfunction suggesting frontotemporal dementia (FTD) may be underestimated in the literature as a clinical picture in PSEN2 mutations. Thus, it may be reasonable to include PSEN2 in genetic panels when suspecting FTDL. PSEN2 mutations may cause striking WMH, arguably related to myelin disruption induced by amyloid accumulation.

Glutamate grabbers, such as glutamate oxaloacetate transaminase (GOT), have been proposed to prevent excitotoxicity secondary to high glutamate levels in stroke patients. However, the efficacy of blood glutamate grabbing by GOT could be dependent on the extent and severity of the disruption of the blood–brain barrier (BBB). Our purpose was to analyze the relationship between GOT and glutamate concentration with the patient’s functional status differentially according to BBB serum markers (soluble tumor necrosis factor-like weak inducer of apoptosis (sTWEAK) and leukoaraiosis based on neuroimaging). This retrospective observational study includes 906 ischemic stroke patients. We studied the presence of leukoaraiosis and the serum levels of glutamate, GOT, and sTWEAK in blood samples. Functional outcome was assessed using the modified Rankin Scale (mRS) at 3 months. A significant negative correlation between GOT and glutamate levels at admission was shown in those patients with sTWEAK levels > 2900 pg/mL (Pearson’s correlation coefficient: −0.249; p < 0.0001). This correlation was also observed in patients with and without leukoaraiosis (Pearson’s correlation coefficients: −0.299; p < 0.001 vs. −0.116; p = 0.024). The logistic regression model confirmed the association of higher levels of GOT with lower odds of poor outcome at 3 months when sTWEAK levels were >2900 pg/mL (OR: 0.41; CI 95%: 0.28–0.68; p < 0.0001) or with leukoaraiosis (OR: 0.75; CI 95%: 0.69–0.82; p < 0.0001). GOT levels are associated with glutamate levels and functional outcomes at 3 months, but only in those patients with leukoaraiosis and elevated sTWEAK levels. Consequently, therapies targeting glutamate grabbing might be more effective in patients with BBB dysfunction.

Abstract Background Alzheimer's disease (AD) exhibits mitochondrial dysfunctions associated with dysregulated metabolism, brain inflammation, synaptic loss, and neuronal cell death. As a key protein serving as the mitochondrial gatekeeper, the voltage-dependent anion channel-1 (VDAC1) that controls metabolism and Ca2+ homeostasis is positioned at a convergence point for various cell survival and death signals. Here, we targeted VDAC1 with VBIT-4, a newly developed inhibitor of VDAC1 that prevents its pro-apoptotic activity, and mitochondria dysfunction. Methods To address the multiple pathways involved in AD, neuronal cultures and a 5 × FAD mouse model of AD were treated with VBIT-4. We addressed multiple topics related to the disease and its molecular mechanisms using immunoblotting, immunofluorescence, q-RT-PCR, 3-D structural analysis and several behavioral tests. Results In neuronal cultures, amyloid-beta (Aβ)-induced VDAC1 and p53 overexpression and apoptotic cell death were prevented by VBIT-4. Using an AD-like 5 × FAD mouse model, we showed that VDAC1 was overexpressed in neurons surrounding Aβ plaques, but not in astrocytes and microglia, and this was associated with neuronal cell death. VBIT-4 prevented the associated pathophysiological changes including neuronal cell death, neuroinflammation, and neuro-metabolic dysfunctions. VBIT-4 also switched astrocytes and microglia from being pro-inflammatory/neurotoxic to neuroprotective phenotype. Moreover, VBIT-4 prevented cognitive decline in the 5 × FAD mice as evaluated using several behavioral assessments of cognitive function. Interestingly, VBIT-4 protected against AD pathology, with no significant change in phosphorylated Tau and only a slight decrease in Aβ-plaque load. Conclusions The study suggests that mitochondrial dysfunction with its gatekeeper VDAC1 is a promising target for AD therapeutic intervention, and VBIT-4 is a promising drug candidate for AD treatment.

IntroductionDynamic susceptibility-weighted contrast-enhanced (DSC) perfusion studies in magnetic resonance imaging (MRI) provide valuable data for studying vascular cerebral pathophysiology in different rodent models of brain diseases (stroke, tumor grading, and neurodegenerative models). The extraction of these hemodynamic parameters via DSC-MRI is based on tracer kinetic modeling, which can be solved using deconvolution-based methods, among others. Most of the post-processing software used in preclinical studies is home-built and custom-designed. Its use being, in most cases, limited to the institution responsible for the development. In this study, we designed a tool that performs the hemodynamic quantification process quickly and in a reliable way for research purposes.MethodsThe DSC-MRI quantification tool, developed as a Python project, performs the basic mathematical steps to generate the parametric maps: cerebral blood flow (CBF), cerebral blood volume (CBV), mean transit time (MTT), signal recovery (SR), and percentage signal recovery (PSR). For the validation process, a data set composed of MRI rat brain scans was evaluated: i) healthy animals, ii) temporal blood–brain barrier (BBB) dysfunction, iii) cerebral chronic hypoperfusion (CCH), iv) ischemic stroke, and v) glioblastoma multiforme (GBM) models. The resulting perfusion parameters were then compared with data retrieved from the literature.ResultsA total of 30 animals were evaluated with our DSC-MRI quantification tool. In all the models, the hemodynamic parameters reported from the literature are reproduced and they are in the same range as our results. The Bland–Altman plot used to describe the agreement between our perfusion quantitative analyses and literature data regarding healthy rats, stroke, and GBM models, determined that the agreement for CBV and MTT is higher than for CBF.ConclusionAn open-source, Python-based DSC post-processing software package that performs key quantitative perfusion parameters has been developed. Regarding the different animal models used, the results obtained are consistent and in good agreement with the physiological patterns and values reported in the literature. Our development has been built in a modular framework to allow code customization or the addition of alternative algorithms not yet implemented.

Background: One of the main biological mechanisms behind the link between periodontitis and atherosclerotic vascular diseases is vascular endothelial dysfunction. Particularly, circulating endothelial progenitor cells (EPCs) have been considered a biomarker of altered vascular endothelial function. Objectives: The aim of this study was to investigate relationship between periodontal inflammation and increased number of circulating EPCs. Design: This is retrospective cohort study. Methods: In this study, 85 elderly patients with a previous history of hypertension were followed up to 12 months. A baseline full-mouth periodontal assessment was carried out, and the amount of periodontal tissue inflamed per subject was calculated as a proxy of periodontal inflammation [periodontal inflamed surface area (PISA)]. The number of circulating EPCs (CD34 + /CD133 + /KDR + ) was determined by flow cytometry from peripheral blood samples collected at baseline and 12 months. Results: Mean concentrations of CD34 + /CD133 + /KDR + progenitor cells were higher in periodontitis patients than in those without periodontitis at baseline [55.4, 95% confidence interval (CI) = 20.8 to 90.0 versus 27.2, 95% CI = 13.6 to 40.8, p = 0.008] and 12 months (114.6, 95% CI = 53.5 to 175.7 versus 19.1, 95% CI = 10.8 to 27.4, p = 0.003). A significant increase over the follow-up was noticed in the group of subjects with periodontitis ( p = 0.049) but not in the nonperiodontitis group ( p = 0.819). PISA was independently associated with CD34 + /CD133 + /KDR + EPCs at baseline ( B coefficient = 0.031, 95% CI = 0.005 to 0.058; p = 0.021). The relationship between PISA and CD34 + /CD133 + /KDR + EPCs at 12 months was confounded by increased baseline body mass index ( B coefficient = 0.064, 95% CI = −0.005 to 0.132; p = 0.066). Conclusion: Periodontal inflammation is associated with high number of CD34 + /CD133 + /KDR + EPCs, thus supporting a potential link between periodontitis and endothelial dysfunction.

Ischemic stroke is an important cause of mortality and disability worldwide. Given that current treatments do not allow a remarkably better outcome in patients after stroke, it is mandatory to seek new approaches to preventing stroke and/or complementing the current treatments or ameliorating the ischemic insult. Multiple preclinical and clinical studies highlighted the potential beneficial roles of exercise and a Mediterranean diet following a stroke. Here, we investigated the effects of a pre-stroke Mediterranean-like diet supplemented with hydroxytyrosol and with/without physical exercise on male rats undergoing transient middle cerebral artery occlusion (tMCAO). We also assessed a potential synergistic effect with physical exercise. Our findings indicated that the diet reduced infarct and edema volumes, modulated acute immune response by altering cytokine and chemokine levels, decreased oxidative stress, and improved acute functional recovery post-ischemic injury. Interestingly, while physical exercise alone improved certain outcomes compared to control animals, it did not enhance, and in some aspects even impaired, the positive effects of the Mediterranean-like diet in the short term. Overall, these data provide the first preclinical evidence that a preemptive enriched Mediterranean diet modulates cytokines/chemokines levels downwards which eventually has an important role during the acute phase following ischemic damage, likely mediating neuroprotection.

Abstract Background Ischemic stroke is the most common cerebrovascular disease and is caused by interruption of blood supply to the brain. To date, recombinant tissue plasminogen activator (rtPA) has been the main pharmacological treatment in the acute phase. However, this treatment has some drawbacks, such as a short half-life, low reperfusion rate, risk of hemorrhagic transformations, and neurotoxic effects. To overcome the limitations of rtPA and improve its effectiveness, we recently designed sonosensitive sub-micrometric capsules (SCs) loaded with rtPA with a size of approximately 600 nm, synthesized using the layer-by-layer (LbL) technique, and coated with gelatine for clot targeting. In this study, we evaluated the rtPA release of ultrasound (US)-responsive SCs in healthy mice and the therapeutic effect in a thromboembolic stroke model. Results In healthy mice, SCs loaded with rtPA 1 mg/kg responded properly to external US exposure, extending the half-life of the drug in the blood stream more than the group treated with free rtPA solution. The gelatine coating also contributed to stabilizing the encapsulation and maintaining the response to US. When the same particles were administered in the stroke model, these SCs appeared to aggregate in the ischemic brain region, probably generating secondary embolisms and limiting the thrombolytic effect of rtPA. Despite the promising results of these thrombolytic particles, at least under the dose and size conditions used in this study, the administration of these capsules represents a risk factor for stroke. Conclusions This is the first study to report the aggregation risk of a drug carrier in neurological pathologies such as stroke. Biocompatibility analysis related to the use of nano-and microparticles should be deeply studied to anticipate the limitations and orientate the design of new nanoparticles for translation to humans. Graphical Abstract

Ischemic stroke is becoming one of the most common causes of death and disability in developed countries. Since current therapeutic options are quite limited, focused on acute reperfusion therapies that are hampered by a very narrow therapeutic time window, it is essential to discover novel treatments that not only stop the progression of the ischemic cascade during the acute phase, but also improve the recovery of stroke patients during the sub-acute or chronic phase. In this regard, several studies have shown that endothelial progenitor cells (EPCs) can repair damaged vessels as well as generate new ones following cerebrovascular damage. EPCs are circulating cells with characteristics of both endothelial cells and adult stem cells presenting the ability to differentiate into mature endothelial cells and self-renew, respectively. Moreover, EPCs have the advantage of being already present in healthy conditions as circulating cells that participate in the maintenance of the endothelium in a direct and paracrine way. In this scenario, EPCs appear as a promising target to tackle stroke by self-promoting re-endothelization, angiogenesis and vasculogenesis. Based on clinical data showing a better neurological and functional outcome in ischemic stroke patients with higher levels of circulating EPCs, novel and promising therapeutic approaches would be pharmacological treatment promoting EPCs-generation as well as EPCs-based therapies. Here, we will review the latest advances in preclinical as well as clinical research on EPCs application following stroke, not only as a single treatment but also in combination with new therapeutic approaches.

Abstract The real-time live fluorescent monitoring of surface AMPA receptors (AMPARs) could open new opportunities for drug discovery and phenotypic screening concerning neuropsychiatric disorders. We have developed FORTIS, a tool based on pH sensitivity capable of detecting subtle changes in surface AMPARs at a neuronal population level. The expression of SEP-GluA1 or pHuji-GluA1 recombinant AMPAR subunits in mammalian neurons cultured in 96-well plates enables surface AMPARs to be monitored with a microplate reader. Thus, FORTIS can register rapid changes in surface AMPARs induced by drugs or genetic modifications without having to rely on conventional electrophysiology or imaging. By combining FORTIS with pharmacological manipulations, basal surface AMPARs, and plasticity-like changes can be monitored. We expect that employing FORTIS to screen for changes in surface AMPARs will accelerate both neuroscience research and drug discovery.

Alzheimer’s disease (AD) is the most common degenerative disorder in the elderly in developed countries. Currently, growing evidence is pointing at endothelial dysfunction as a key player in the cognitive decline course of AD. As a main component of the blood–brain barrier (BBB), the dysfunction of endothelial cells driven by vascular risk factors associated with AD allows the passage of toxic substances to the cerebral parenchyma, producing chronic hypoperfusion that eventually causes an inflammatory and neurotoxic response. In this process, the levels of several biomarkers are disrupted, such as an increase in adhesion molecules that allow the passage of leukocytes to the cerebral parenchyma, increasing the permeability of the BBB; moreover, other vascular players, including endothelin-1, also mediate artery inflammation. As a consequence of the disruption of the BBB, a progressive neuroinflammatory response is produced that, added to the astrogliosis, eventually triggers neuronal degeneration (possibly responsible for cognitive deterioration). Recently, new molecules have been proposed as early biomarkers for endothelial dysfunction that can constitute new therapeutic targets as well as early diagnostic and prognostic markers for AD.

Ischemic stroke, caused by the interruption of blood flow to the brain and subsequent neuronal death, represents one of the main causes of disability in worldwide. Although reperfusion therapies have shown efficacy in a limited number of patients with acute ischemic stroke, neuroprotective drugs and recovery strategies have been widely assessed, but none of them have been successful in clinical practice. Therefore, the search for new therapeutic approaches is still necessary. Sphingolipids consist of a family of lipidic molecules with both structural and cell signaling functions. Regulation of sphingolipid metabolism is crucial for cell fate and homeostasis in the body. Different works have emphasized the implication of its metabolism in different pathologies, such as diabetes, cancer, neurodegeneration, or atherosclerosis. Other studies have shown its implication in the risk of suffering a stroke and its progression. This review will highlight the implications of sphingolipid metabolism enzymes in acute ischemic stroke.

Combined hormone drugs are the basis for orally administered contraception. However, they are associated with severe side effects that are even more impactful for women in developing countries, where resources are limited. The risk of side effects may be reduced by non-hormonal small molecules which specifically target proteins involved in fertilization. In this study, we present a virtual docking experiment directed to discover molecules that target the crucial fertilization interactions of JUNO (oocyte) and IZUMO1 (sperm). We docked 913,000 molecules to two crystal structures of JUNO and ranked them on the basis of energy-related criteria. Of the 32 tested candidates, two molecules (i.e., Z786028994 and Z1290281203) demonstrated fertilization inhibitory effect in both an in vitro fertilization (IVF) assay in mice and an in vitro penetration of human sperm into hamster oocytes. Despite this clear effect on fertilization, these two molecules did not show JUNO–IZUMO1 interaction blocking activity as assessed by AVidity-based EXtracellular Interaction Screening (AVEXIS). Therefore, further research is required to determine the mechanism of action of these two fertilization inhibitors.

Alzheimer’s disease (AD) is a neurodegenerative disease representing the most common type of dementia worldwide. The early diagnosis of AD is very difficult to achieve due to its complexity and the practically unknown etiology. Therefore, this is one of the greatest challenges in the field in order to develop an accurate therapy. Within the different etiological hypotheses proposed for AD, we will focus on the two-hit vascular hypothesis and vascular alterations occurring in the disease. According to this hypothesis, the accumulation of β-amyloid protein in the brain starts as a consequence of damage in the cerebral vasculature. Given that there are several vascular and angiogenic alterations in AD, and that endothelial progenitor cells (EPCs) play a key role in endothelial repair processes, the study of EPCs in AD may be relevant to the disease etiology and perhaps a biomarker and/or therapeutic target. This review focuses on the involvement of endothelial dysfunction in the onset and progression of AD with special emphasis on EPCs as a biomarker and potential therapeutic target.

Blood-brain barrier (BBB) integrity is essential to maintaining brain health. Aging-related alterations could lead to chronic progressive leakiness of the BBB, which is directly correlated with cerebrovascular diseases. Indeed, the BBB breakdown during acute ischemic stroke is critical. It remains unclear, however, whether BBB dysfunction is one of the first events that leads to brain disease or a down-stream consequence. This review will focus on the BBB dysfunction associated with cerebrovascular disease. An added difficulty is its association with the deleterious or reparative effect, which depends on the stroke phase. We will first outline the BBB structure and function. Then, we will focus on the spatiotemporal chronic, slow, and progressive BBB alteration related to ischemic stroke. Finally, we will propose a new perspective on preventive therapeutic strategies associated with brain aging based on targeting specific components of the BBB. Understanding BBB age-evolutions will be beneficial for new drug development and the identification of the best performance window times. This could have a direct impact on clinical translation and personalised medicine.

Alzheimer’s disease (AD) is considered the most prevalent neurodegenerative disease and the leading cause of dementia worldwide. Sphingolipids, such as ceramide or sphingosine 1-phosphate, are bioactive molecules implicated in structural and signaling functions. Metabolic dysfunction in the highly conserved pathways to produce sphingolipids may lead to or be a consequence of an underlying disease. Recent studies on transcriptomics and sphingolipidomics have observed alterations in sphingolipid metabolism of both enzymes and metabolites involved in their synthesis in several neurodegenerative diseases, including AD. In this review, we highlight the most relevant findings related to ceramide and neurodegeneration, with a special focus on AD.

Ischemic stroke is a leading cause of death and disability worldwide. Following an ischemic insult, cells undergo endoplasmic reticulum (ER) stress, which increases the ER’s protein-folding and degradative capacities and blocks the global synthesis of proteins by phosphorylating the eukaryotic translation initiation factor 2-alpha (eIF2α). Phosphorylation of eIF2α is directly related to the dynamics of stress granules (SGs), which are membraneless organelles composed of RNA-binding proteins and mRNA. SGs play a critical role in mRNA metabolism and translational control. Other translation factors are also linked to cellular pathways, including SG dynamics following a stroke. Because the formation of SGs is closely connected to mRNA translation, it is interesting to study the relationship between SG dynamics and cellular outcome in cases of ischemic damage. Therefore, in this review, we focus on the role of SG dynamics during cerebral ischemia.

Alzheimer’s Disease (AD) is one of the main neurodegenerative diseases worldwide. Unfortunately, AD shares many similarities with other dementias at early stages, which impedes an accurate premortem diagnosis. Therefore, it is urgent to find biomarkers to allow for early diagnosis of the disease. There is increasing scientific evidence highlighting the similarities between the eye and other structures of the CNS, suggesting that knowledge acquired in eye research could be useful for research and diagnosis of AD. For example, the retina and optic nerve are considered part of the central nervous system, and their damage can result in retrograde and anterograde axon degeneration, as well as abnormal protein aggregation. In the anterior eye segment, the aqueous humor and tear film may be comparable to the cerebrospinal fluid. Both fluids are enriched with molecules that can be potential neurodegenerative biomarkers. Indeed, the pathophysiology of AD, characterized by cerebral deposits of amyloid-beta (Aβ) and tau protein, is also present in the eyes of AD patients, besides numerous structural and functional changes observed in the structure of the eyes. Therefore, all this evidence suggests that ocular changes have the potential to be used as either predictive values for AD assessment or as diagnostic tools.

In 1959, E. G. Gray described two different types of synapses in the brain for the first time: symmetric and asymmetric. Later on, symmetric synapses were associated with inhibitory terminals, and asymmetric synapses to excitatory signaling. The balance between these two systems is critical to maintain a correct brain function. Likewise, the modulation of both types of synapses is also important to maintain a healthy equilibrium. Cerebral circuitry responds differently depending on the type of damage and the timeline of the injury. For example, promoting symmetric signaling following ischemic damage is beneficial only during the acute phase; afterwards, it further increases the initial damage. Synapses can be also altered by players not directly related to them; the chronic and long-term neurodegeneration mediated by tau proteins primarily targets asymmetric synapses by decreasing neuronal plasticity and functionality. Dopamine represents the main modulating system within the central nervous system. Indeed, the death of midbrain dopaminergic neurons impairs locomotion, underlying the devastating Parkinson’s disease. Herein, we will review studies on symmetric and asymmetric synapses plasticity after three different stressors: symmetric signaling under acute damage—ischemic stroke; asymmetric signaling under chronic and long-term neurodegeneration—Alzheimer’s disease; symmetric and asymmetric synapses without modulation—Parkinson’s disease.

In the last few decades, great advances have been made in the detection and treatment of cancer, thus increasing the survival rate [...]

Sphingolipids are both structural molecules that are essential for cell architecture and second messengers that are involved in numerous cell functions. Ceramide is the central hub of sphingolipid metabolism. In addition to being the precursor of complex sphingolipids, ceramides induce cell cycle arrest and promote cell death and inflammation. At least some of the enzymes involved in the regulation of sphingolipid metabolism are altered in carcinogenesis, and some are targets for anticancer drugs. A number of scientific reports have shown how alterations in sphingolipid pools can affect cell proliferation, survival and migration. Determination of sphingolipid levels and the regulation of the enzymes that are implicated in their metabolism is a key factor for developing novel therapeutic strategies or improving conventional therapies. The present review highlights the importance of bioactive sphingolipids and their regulatory enzymes as targets for therapeutic interventions with especial emphasis in carcinogenesis and cancer dissemination.

Ceramide is a bioactive sphingolipid involved in numerous cellular processes. In addition to being the precursor of complex sphingolipids, ceramides can act as second messengers, especially when they are generated at the plasma membrane of cells. Its metabolic dysfunction may lead to or be a consequence of an underlying disease. Recent reports on transcriptomics and electrospray ionization mass spectrometry analysis have demonstrated the variation of specific levels of sphingolipids and enzymes involved in their metabolism in different neurodegenerative diseases. In the present review, we highlight the most relevant discoveries related to ceramide and neurodegeneration, with a special focus on Parkinson’s disease.

Phosphatidic acid (PA) is a bioactive phospholipid capable of regulating key biological functions, including neutrophil respiratory burst, chemotaxis, or cell growth and differentiation. However, the mechanisms whereby PA exerts these actions are not completely understood. In this work, we show that PA stimulates myoblast proliferation, as determined by measuring the incorporation of [3H]thymidine into DNA and by staining the cells with crystal violet. PA induced the rapid phosphorylation of Akt and ERK1/2, and pretreatment of the cells with specific small interferin RNA (siRNA) to silence the genes encoding these kinases, or with selective pharmacologic inhibitors, blocked PA-stimulated myoblast proliferation. The mitogenic effects of PA were abolished by the preincubation of the myoblasts with pertussis toxin, a Gi protein inhibitor, suggesting the implication of Gi protein-coupled receptors in this action. Although some of the effects of PA have been associated with its possible conversion to lysoPA (LPA), treatment of the myoblasts with PA for up to 60 min did not produce any significant amount of LPA in these cells. Of interest, pharmacological blockade of the LPA receptors 1 and 2, or specific siRNA to silence the genes encoding these receptors, abolished PA-stimulated myoblast proliferation. Moreover, PA was able to compete with LPA for binding to LPA receptors, suggesting that PA can act as a ligand of LPA receptors. It can be concluded that PA stimulates myoblast proliferation through interaction with LPA1 and LPA2 receptors and the subsequent activation of the PI3K/Akt and MEK/ERK1-2 pathways, independently of LPA formation.

Sphingolipids are not only crucial for membrane architecture but act as critical regulators of cell functions. The bioactive sphingolipid ceramide 1-phosphate (C1P), generated by the action of ceramide kinase, has been reported to stimulate cell proliferation, cell migration and to regulate inflammatory responses via activation of different signaling pathways. We have previously shown that skeletal muscle is a tissue target for C1P since the phosphosphingolipid plays a positive role in myoblast proliferation implying a role in muscle regeneration. Skeletal muscle displays strong capacity of regeneration thanks to the presence of quiescent adult stem cells called satellite cells that upon trauma enter into the cell cycle and start proliferating. However, at present, the exact molecular mechanism by which C1P triggers its mitogenic effect in myoblasts is lacking. Here, we report for the first time that C1P stimulates C2C12 myoblast proliferation via lysophosphatidic acid (LPA) signaling axis. Indeed, C1P subsequently to phospholipase A2 activation leads to LPA1 and LPA3 engagement, which in turn drive Akt (protein kinase B) and ERK1/2 (extracellular signal-regulated kinases 1/2) activation, thus stimulating DNA synthesis. The present findings shed new light on the key role of bioactive sphingolipids in skeletal muscle and provide further support to the notion that these pleiotropic molecules might be useful therapeutic targets for skeletal muscle regeneration.