Upregulation of S100A8 in peripheral blood mononuclear cells from patients with depression treated with SSRIs: a pilot study.

BACKGROUND: Major depressive disorder (MDD) affects more than 350 million people worldwide, and there is currently no laboratory test to diagnose it. This pilot study aimed to identify potential biomarkers in peripheral blood mononuclear cells (PBMCs) from MDD patients. METHODS: We used tandem mass tagging coupled to synchronous precursor selection (mass spectrometry) to obtain the differential proteomic profile from a pool of PBMCs from MDD patients and healthy subjects, and quantitative PCR to assess gene expression of differentially expressed proteins (DEPs) of our interest. RESULTS: We identified 247 proteins, of which 133 had a fold change ≥ 2.0 compared to healthy volunteers. Using pathway enrichment analysis, we found that some processes, such as platelet degranulation, coagulation, and the inflammatory response, are perturbed in MDD patients. The gene-disease association analysis showed that molecular alterations in PBMCs from MDD patients are associated with cerebral ischemia, vascular disease, thrombosis, acute coronary syndrome, and myocardial ischemia, in addition to other conditions such as inflammation and diabetic retinopathy. CONCLUSIONS: We confirmed by qRT-PCR that S100A8 is upregulated in PBMCs from MDD patients and thus could be an emerging biomarker of this disorder. This report lays the groundwork for future studies in a broader and more diverse population and contributes to a deeper characterization of MDD.

Thinking outside the black box: are the brain endothelial cells the new main target in Alzheimer's disease?

The blood-brain barrier is the interface through which the brain interacts with the milieu and consists mainly of a sophisticated network of brain endothelial cells that forms blood vessels and selectively moves molecules inside and outside the brain through multiple mechanisms of transport. Although brain endothelial cell function is crucial for brain homeostasis, their role in neurodegenerative diseases has historically not been considered with the same importance as other brain cells such as microglia, astroglia, neurons, or even molecules such as amyloid beta, Tau, or alpha-synuclein. Alzheimer's disease is the most common neurodegenerative disease, and brain endothelial cell dysfunction has been reported by several groups. However, its impairment has barely been considered as a potential therapeutic target. Here we review the most recent advances in the relationship between Alzheimer's disease and brain endothelial cells commitment and analyze the possible mechanisms through which their alterations contribute to this neurodegenerative disease, highlighting their inflammatory phenotype and the possibility of an impaired secretory pattern of brain endothelial cells that could contribute to the progression of this ailment. Finally, we discuss why shall brain endothelial cells be appreciated as a therapeutic target instead of solely an obstacle for delivering treatments to the injured brain in Alzheimer's disease.

Sucrose Consumption during Late Adolescence Impairs Adult Neurogenesis of the Ventral Dentate Gyrus without Inducing an Anxiety-like Behavior.

Sucrose consumption impairs behavioral and cognitive functions that correlate with decreased neurogenesis in animal models. When consumed during early adolescence, this disaccharide promotes anxious and depressive behaviors, along with a reduction in the generation of new neurons in the dentate gyrus of the hippocampus. Data concerning sucrose consumption during late adolescence are lacking, and the effect of sucrose intake on the ventral dentate gyrus of the hippocampus (which modulates anxiety and depression) remains elusive. Here, we tested whether sucrose intake during late adolescence causes anxiety or impaired neurogenesis in the ventral dentate gyrus. Rats did not display anxiety-like behaviors neither at the light−dark box test nor at the open field exploration. However, there was a significant increase in proliferative cells in the subgranular zone of the ventral dentate gyrus in rats exposed to sucrose (p < 0.05). This increased proliferation corresponded to neural stem cells (Radial Type 1 cells) in the group exposed to sucrose until adulthood but was not present in rats exposed to sucrose only during late adolescence. Remarkably, the phosphorylation of ERK1/2 kinases was increased in the hippocampi of rats exposed to sucrose only during late adolescence, suggesting that the increased proliferation in this group could be mediated by the MAPK pathway. On the other hand, although no differences were found in the number of immature granular neurons, we observed more immature granular neurons with impaired dendritic orientation in both groups exposed to sucrose. Finally, GAD65/67 and BCL2 levels did not change between groups, suggesting an unaltered hippocampal GABAergic system and similar apoptosis, respectively. This information provides the first piece of evidence of how sucrose intake, starting in late adolescence, impacts ventral dentate gyrus neurogenesis and contributes to a better understanding of the effects of this carbohydrate on the brain at postnatal stages.

Diabetes Induces Permanent Deleterious Effects in the Olfactory Bulb Associated with Increased Tyrosine Hydroxylase Expression and ERK1/2 Phosphorylation.

Diabetes mellitus type 2 (T2D) complications include brain damage which increases the risk of neurodegenerative diseases and dementia. An early manifestation of neurodegeneration is olfactory dysfunction (OD), which is also presented in diabetic patients. Previously, we demonstrated that OD correlates with IL-1ß and miR-146a overexpression in the olfactory bulb (OB) on a T2D rodent model, suggesting the participation of inflammation on OD. Here, we found that OD persists on a long-term T2D condition after the downregulation of IL-1ß. Remarkably, OD was associated with the increased expression of the dopaminergic neuronal marker tyrosine hydroxylase, ERK1/2 phosphorylation, and reduced neuronal activation on the OB of diabetic rats, suggesting the participation of the dopaminergic tone on the OD derived from T2D. Dopaminergic neurons are susceptible in neurodegenerative diseases such as Parkinson's disease; therefore further studies must be performed to completely elucidate the participation of these neurons and ERK1/2 signaling on olfactory impairment.

The Impairment of Blood-Brain Barrier in Alzheimer's Disease: Challenges and Opportunities with Stem Cells.

Alzheimer's disease (AD) is the most common neurodegenerative disorder and its prevalence is increasing. Nowadays, very few drugs effectively reduce AD symptoms and thus, a better understanding of its pathophysiology is vital to design new effective schemes. Presymptomatic neuronal damage caused by the accumulation of Amyloid ß peptide and Tau protein abnormalities remains a challenge, despite recent efforts in drug development. Importantly, therapeutic targets, biomarkers, and diagnostic techniques have emerged to detect and treat AD. Of note, the compromised blood-brain barrier (BBB) and peripheral inflammation in AD are becoming more evident, being harmful factors that contribute to the development of the disease. Perspectives from different pre-clinical and clinical studies link peripheral inflammation with the onset and progression of AD. This review aims to analyze the main factors and the contribution of impaired BBB in AD development. Additionally, we describe the potential therapeutic strategies using stem cells for AD treatment.

Cryopreservation of Gametes and Embryos and Their Molecular Changes.

The process of freezing cells or tissues and depositing them in liquid nitrogen at -196 °C is called cryopreservation. Sub-zero temperature is not a physiological condition for cells and water ice crystals represent the main problem since they induce cell death, principally in large cells like oocytes, which have a meiotic spindle that degenerates during this process. Significantly, cryopreservation represents an option for fertility preservation in patients who develop gonadal failure for any condition and those who want to freeze their germ cells for later use. The possibility of freezing sperm, oocytes, and embryos has been available for a long time, and in 1983 the first birth with thawed oocytes was achieved. From the mid-2000s forward, the use of egg vitrification through intracytoplasmic sperm injection has improved pregnancy rates. Births using assisted reproductive technologies (ART) have some adverse conditions and events. These risks could be associated with ART procedures or related to infertility. Cryopreservation generates changes in the epigenome of gametes and embryos, given that ART occurs when the epigenome is most vulnerable. Furthermore, cryoprotective agents induce alterations in the integrity of germ cells and embryos. Notably, cryopreservation extensively affects cell viability, generates proteomic profile changes, compromises crucial cellular functions, and alters sperm motility. This technique has been widely employed since the 1980s and there is a lack of knowledge about molecular changes. The emerging view is that molecular changes are associated with cryopreservation, affecting metabolism, cytoarchitecture, calcium homeostasis, epigenetic state, and cell survival, which compromise the fertilization in ART.

Dynamic landscape of chromatin accessibility and transcriptomic changes during differentiation of human embryonic stem cells into dopaminergic neurons.

Chromatin architecture influences transcription by modulating the physical access of regulatory factors to DNA, playing fundamental roles in cell identity. Studies on dopaminergic differentiation have identified coding genes, but the relationship with non-coding genes or chromatin accessibility remains elusive. Using RNA-Seq and ATAC-Seq we profiled differentially expressed transcripts and open chromatin regions during early dopaminergic neuron differentiation. Hierarchical clustering of differentially expressed genes, resulted in 6 groups with unique characteristics. Surprisingly, the abundance of long non-coding RNAs (lncRNAs) was high in the most downregulated transcripts, and depicted positive correlations with target mRNAs. We observed that open chromatin regions decrease upon differentiation. Enrichment analyses of accessibility depict an association between open chromatin regions and specific functional pathways and gene-sets. A bioinformatic search for motifs allowed us to identify transcription factors and structural nuclear proteins that potentially regulate dopaminergic differentiation. Interestingly, we also found changes in protein and mRNA abundance of the CCCTC-binding factor, CTCF, which participates in genome organization and gene expression. Furthermore, assays demonstrated co-localization of CTCF with Polycomb-repressed chromatin marked by H3K27me3 in pluripotent cells, progressively decreasing in neural precursor cells and differentiated neurons. Our work provides a unique resource of transcription factors and regulatory elements, potentially involved in the acquisition of human dopaminergic neuron cell identity.

Mouse Embryonic Stem Cells Expressing GDNF Show Enhanced Dopaminergic Differentiation and Promote Behavioral Recovery After Grafting in Parkinsonian Rats.

Parkinson's disease (PD) is characterized by the progressive loss of midbrain dopaminergic neurons (DaNs) of the substantia nigra pars compacta and the decrease of dopamine in the brain. Grafting DaN differentiated from embryonic stem cells (ESCs) has been proposed as an alternative therapy for current pharmacological treatments. Intrastriatal grafting of such DaNs differentiated from mouse or human ESCs improves motor performance, restores DA release, and suppresses dopamine receptor super-sensitivity. However, a low percentage of grafted neurons survive in the brain. Glial cell line-derived neurotrophic factor (GDNF) is a strong survival factor for DaNs. GDNF has proved to be neurotrophic for DaNs in vitro and in vivo, and induces axonal sprouting and maturation. Here, we engineered mouse ESCs to constitutively produce human GDNF, to analyze DaN differentiation and the possible neuroprotection by transgenic GDNF after toxic challenges in vitro, or after grafting differentiated DaNs into the striatum of Parkinsonian rats. GDNF overexpression throughout in vitro differentiation of mouse ESCs increases the proportion of midbrain DaNs. These transgenic cells were less sensitive than control cells to 6-hydroxydopamine in vitro. After grafting control or GDNF transgenic DaNs in hemi-Parkinsonian rats, we observed significant recoveries in both pharmacological and non-pharmacological behavioral tests, as well as increased striatal DA release, indicating that DaNs are functional in the brain. The graft volume, the number of surviving neurons, the number of DaNs present in the striatum, and the proportion of DaNs in the grafts were significantly higher in rats transplanted with GDNF-expressing cells, when compared to control cells. Interestingly, no morphological alterations in the brain of rats were found after grafting of GDNF-expressing cells. This approach is novel, because previous works have use co-grafting of DaNs with other cell types that express GDNF, or viral transduction in the host tissue before or after grafting of DaNs. In conclusion, GDNF production by mouse ESCs contributes to enhanced midbrain differentiation and permits a higher number of surviving DaNs after a 6-hydroxydopamine challenge in vitro, as well as post-grafting in the lesioned striatum. These GDNF-expressing ESCs can be useful to improve neuronal survival after transplantation.

Human Keratinocytes Adopt Neuronal Fates After In Utero Transplantation in the Developing Rat Brain.

Human skin contains keratinocytes in the epidermis. Such cells share their ectodermal origin with the central nervous system (CNS). Recent studies have demonstrated that terminally differentiated somatic cells can adopt a pluripotent state, or can directly convert its phenotype to neurons, after ectopic expression of transcription factors. In this article we tested the hypothesis that human keratinocytes can adopt neural fates after culturing them in suspension with a neural medium. Initially, keratinocytes expressed Keratins and Vimentin. After neural induction, transcriptional upregulation of NESTIN, SOX2, VIMENTIN, SOX1, and MUSASHI1 was observed, concomitant with significant increases in NESTIN detected by immunostaining. However, in vitro differentiation did not yield the expression of neuronal or astrocytic markers. We tested the differentiation potential of control and neural-induced keratinocytes by grafting them in the developing CNS of rats, through ultrasound-guided injection. For this purpose, keratinocytes were transduced with lentivirus that contained the coding sequence of green fluorescent protein. Cell sorting was employed to select cells with high fluorescence. Unexpectedly, 4 days after grafting these cells in the ventricles, both control and neural-induced cells expressed green fluorescent protein together with the neuronal proteins ßIII-Tubulin and Microtubule-Associated Protein 2. These results support the notion that in vivo environment provides appropriate signals to evaluate the neuronal differentiation potential of keratinocytes or other non-neural cell populations.

Olfactory Dysfunction in Diabetic Rats is Associated with miR-146a Overexpression and Inflammation.

Type 2 diabetes (T2D) is associated with cognitive decline and dementia. Both neurodegenerative conditions are characterized by olfactory dysfunction (OD) which is also observed in diabetic patients. Diabetes and neurodegeneration display altered miRNAs expression; therefore, the study of miRNAs in the diabetic olfactory system is important in order to know the mechanisms involved in neurodegeneration induced by T2D. In this work we evaluated the expression of miRs206, 451, 146a and 34a in the olfactory bulb (OB) of T2D rats and its association with OD. T2D induction was performed by administering streptozotocin to neonatal rats. The olfactory function was evaluated after reaching the adulthood by employing the buried pellet and social recognition tests. After 18 weeks, animals were sacrificed to determinate miRNAs and protein expression in the OB. T2D animals showed a significant increase in the latency to find the odor stimulus in the buried pellet test and a significant reduction in the interest to investigate the novel juvenile subjects in the social recognition test, indicating OD. In miRNAs analysis we observed a significant increase of miR-146a expression in the OB of T2D rats when compared to controls. This increase in miR-146a correlated with the overexpression of IL-1ß in the OB of T2D rats. The present results showed that OD in T2D rats is associated with IL-1ß mediated-inflammation and miR-146a overexpression, suggesting that high levels of IL-1ß could trigger miR-146a upregulation as a negative feedback of the inflammatory response in the OB of T2D rats.

Aryl Hydrocarbon Receptor in Post-Mortem Hippocampus and in Serum from Young, Elder, and Alzheimer's Patients.

One of the characteristics of the cerebral aging process is the presence of chronic inflammation through glial cells, which is particularly significant in neurodegeneration. On the other hand, it has been demonstrated that the aryl hydrocarbon receptor (AHR) participates in the inflammatory response. Currently, evidence in animal models shows that the hallmarks of aging are associated with changes in the AHR levels. However, there is no information concerning the behavior and participation of AHR in the human aging brain or in Alzheimer's disease (AD). We evaluated the expression of AHR in human hippocampal post-mortem tissue and its association with reactive astrocytes by immunohistochemistry. Besides this, we analyzed through ELISA the AHR levels in blood serum from young and elder participants, and from AD patients. The levels of AHR and glial fibrillar acid protein were higher in elder than in young post-mortem brain samples. AHR was localized mainly in the cytosol of astrocytes and displayed a pattern that resembles extracellular vesicles; this latter feature was more conspicuous in AD subjects. We found higher serum levels of AHR in AD patients than in the other participants. These results suggest that AHR participates in the aging process, and probably in the development of neurodegenerative diseases like AD.

Annexin A1, Annexin A2, and Dyrk 1B are upregulated during GAS1-induced cell cycle arrest.

GAS1 is a pleiotropic protein that has been investigated because of its ability to induce cell proliferation, cell arrest, and apoptosis, depending on the cellular or the physiological context in which it is expressed. At this point, we have information about the molecular mechanisms by which GAS1 induces proliferation and apoptosis; but very few studies have been focused on elucidating the mechanisms by which GAS1 induces cell arrest. With the aim of expanding our knowledge on this subject, we first focused our research on finding proteins that were preferentially expressed in cells arrested by serum deprivation. By using a proteomics approach and mass spectrometry analysis, we identified 17 proteins in the 2-DE protein profile of serum deprived NIH3T3 cells. Among them, Annexin A1 (Anxa1), Annexin A2 (Anxa2), dual specificity tyrosine-phosphorylation-regulated kinase 1B (Dyrk1B), and Eukaryotic translation initiation factor 3, F (eIf3f) were upregulated at transcriptional the level in proliferative NIH3T3 cells. Moreover, we demonstrated that Anxa1, Anxa2, and Dyrk1b are upregulated at both the transcriptional and translational levels by the overexpression of GAS1. Thus, our results suggest that the upregulation of Anxa1, Anxa2, and Dyrk1b could be related to the ability of GAS1 to induce cell arrest and maintain cell viability. Finally, we provided further evidence showing that GAS1 through Dyrk 1B leads not only to the arrest of NIH3T3 cells but also maintains cell viability.

GAS1 is present in the cerebrospinal fluid and is expressed in the choroid plexus of the adult rat.

Growth arrest specific 1 (GAS1) is a GPI-anchored protein that inhibits proliferation when overexpressed in tumors but during development it promotes proliferation and survival of different organs and tissues. This dual ability is caused by its capacity to interact both by inhibiting the signaling induced by the glial cell line-derived neurotrophic factor and by facilitating the activity of the sonic hedgehog pathway. GAS1 is expressed as membrane bound in different organs and as a secreted form by glomerular mesangial cells. In the developing central nervous system, GAS1 is found in neural progenitors; however, it continues to be expressed in the adult brain. Here, we demonstrate that soluble GAS1 is present in the cerebrospinal fluid (CSF) and it is expressed in the choroid plexus (CP) of the adult rat, the main producer of CSF. Additionally, we confirm the presence of GAS1 in blood plasma and liver of the adult rat, the principal source of blood plasma proteins. The pattern of expression of GAS1 is perivascular in both the CP and the liver. In vitro studies show that the fibroblast cell line NIH/3T3 expresses one form of GAS1 and releases two soluble forms into the supernatant. Briefly, in the present work, we show the presence of GAS1 in adult rat body fluids focusing in the CSF and the CP, and suggest that secreted GAS1 exists as two different isoforms.

A soluble form of GAS1 inhibits tumor growth and angiogenesis in a triple negative breast cancer model.

We previously demonstrated the capacity of GAS1 (Growth Arrest Specific 1) to inhibit the growth of gliomas by blocking the GDNF-RET signaling pathway. Here, we show that a soluble form of GAS1 (tGAS1), decreases the number of viable MDA MB 231 human breast cancer cells, acting in both autocrine and paracrine manners when secreted from producing cells. Moreover, tGAS1 inhibits the growth of tumors implanted in female nu/nu mice through a RET-independent mechanism which involves interfering with the Artemin (ARTN)-GFRα3-(GDNF Family Receptor alpha 3) mediated intracellular signaling and the activation of ERK. In addition, we observed that the presence of tGAS1 reduces the vascularization of implanted tumors, by preventing the migration of endothelial cells. The present results support a potential adjuvant role for tGAS1 in the treatment of breast cancer, by detaining tumor growth and inhibiting angiogenesis.