Janetti Signorelli

Neuroligin-2 (Nlgn2) is a key synaptic adhesion protein at virtually all GABAergic synapses, which recruits GABA_ARs by promoting assembly of the postsynaptic gephyrin scaffold. Intriguingly, loss of Nlgn2 differentially affects subsets of GABAergic synapses, indicating that synapse-specific interactors and redundancies define its function, but the nature of these interactions remain poorly understood. Here we investigated how Nlgn2 function in hippocampal area CA1 is modulated by two proposed interaction partners, MDGA1 and MDGA2. We show that loss of MDGA1 expression, but not heterozygous deletion of MDGA2, ameliorates the abnormal cytosolic gephyrin aggregation, the reduction in inhibitory synaptic transmission and the exacerbated anxiety-related behaviour characterizing Nlgn2 knockout (KO) mice. Additionally, combined Nlgn2 and MDGA1 deletion causes an exacerbated layer-specific loss of gephyrin puncta. Given that both Nlgn2 and the MDGA1 have been correlated with many psychiatric disorders, our data support the notion that cytosolic gephyrin aggregation may represent an interesting target for novel therapeutic strategies.

Regulated systems for transgene expression are useful tools in basic research and a promising platform in biomedicine due to their regulated transgene expression by an inducer. The emergence of optogenetics expression systems enabled the construction of light-switchable systems, enhancing the spatial and temporal resolution of a transgene. The LightOn system is an optogenetic tool that regulates the expression of a gene of interest using blue light as an inducer. This system is based on a photosensitive protein (GAVPO), which dimerizes and binds to the UASG sequence in response to blue light, triggering the expression of a downstream transgene. Previously, we adapted the LightOn system to a dual lentiviral vector system for neurons. Here, we continue the optimization and assemble all components of the LightOn system into a single lentiviral plasmid, the OPTO-BLUE system. For functional validation, we used enhanced green fluorescent protein (EGFP) as an expression reporter (OPTO-BLUE-EGFP) and evaluated the efficiency of EGFP expression by transfection and transduction in HEK293-T cells exposed to continuous blue-light illumination. Altogether, these results prove that the optimized OPTO-BLUE system allows the light-controlled expression of a reporter protein according to a specific time and light intensity. Likewise, this system should provide an important molecular tool to modulate gene expression of any protein by blue light.

Autism spectrum disorder (ASD) is a heterogeneous neurodevelopment disorder resulting from different etiological factors, both genetic and/or environmental. These factors can lead to abnormal neuronal development on dendrite and synaptic function at the central nervous system. Recent studies have shown that a subset of ASD patients display increased circulation levels of the tyrosine metabolite, p‐cresol, related to chronic intestinal disorders because of dysbiosis of the intestinal microbiota. In particular, abnormal presence of intestinal Clostridium sp. has been linked to high levels of p‐cresol in ASD children younger than 8 years. However, the role of p‐cresol during development of the central nervous system is unknown. Here, we evaluated in vitro the effect of p‐cresol on neurite outgrowth in N2a and PC12 cell lines and dendritic morphology, synaptic density, neuronal activity, and calcium responses in primary rat hippocampal neurons. p‐cresol inhibits neural differentiation and neurites outgrowth in N2a and PC12 neuronal cell lines. In hippocampal neuronal cultures, Sholl's analysis shows a decrease in the dendritic arborization of neurons treated with p‐cresol. Synaptic density analyzed with the synaptic markers Piccolo and Shank2 is diminished in hippocampal neurons treated with p‐cresol. Electrically evoked intracellular calcium rise was drastically, but reversely, blocked by p‐cresol, whereas that spontaneous neuronal activity was severely affected by early addition of the metabolite. These findings show that p‐cresol alters dendrite development, synaptogenesis, and synapse function of neurons in culture, therefore, neuronal alterations occurring in ASD children may be related to this metabolite and dysbiosis of the intestinal microbiota. image

Fibronectin (Fn) enhances human sperm capacitation via the cAMP/PKA pathway, and the endocannabinoid system participates in this process. Moreover, Fn has been linked to endocannabinoid system components in different cellular models, even though no evidence of such interactions in human sperm is available. Normal semen samples were evaluated over a 4‐year period. Our findings suggest that (a) the capacitating effects of Fn were reversed by preincubating the sperm with a cannabinoid receptor 1 (CB1) or transient receptor potential cation channel subfamily V member 1 (TRPV1) antagonist ( p < 0.001 and p < 0.05, respectively); (b) cooperation between CB1 and TRPV1 may exist ( p < 0.01); (c) the activity of specific fatty acid amide hydroxylase (FAAH) decreased after 1 min ( p < 0.01) and increased after 60 min ( p < 0.01) of capacitation in the presence of Fn; (d) the effects of Fn on FAAH activity were prevented by preincubating spermatozoa with a protein kinase A (PKA) inhibitor ( p < 0.01); (e) Fn modulated both the cyclic adenosine monophosphate concentration and PKA activity ( p < 0.05) during early capacitation; and (f) FAAH was a PKA substrate modulated by phosphorylation. These findings indicate that Fn stimulates human sperm capacitation via the cAMP/PKA pathway through modulation of the endocannabinoid system. Understanding the functional competence of human spermatozoa is essential for facilitating clinical advances in infertility treatment and for developing novel contraceptive strategies.

Summary: In human spermatozoa, protein kinases have a role in the acrosome reaction (AR) induced by a variety of stimuli. However, there is disagreement or a lack of information regarding the role of protein kinases and phosphatases in the progesterone (P)‐induced increase in intracellular calcium concentration ([Ca²⁺]_i). In addition, there are no studies regarding the role of Ser/Thr and Tyr phosphatases and there are contradictory results regarding the role of Tyr kinases in the P‐induced acrosome reaction. Here, we performed a simultaneous evaluation of the involvement of protein kinases and phosphatases in the P‐induced acrosome reaction and in the P‐induced calcium influx. Motile spermatozoa were capacitated for 18 h and different aliquots were allocated to treated or control groups and then evaluated for their ability to undergo the acrosome reaction and to increase [Ca²⁺]_i in response to P. The acrosome reaction was evaluated using Pisum sativum agglutinin (PSA)‐FITC, and [Ca²⁺]_i was evaluated using fura 2AM. At all of the concentrations tested, PKA inhibitors significantly reduced the percentage of the P‐induced acrosome reaction (p < 0.001). However, only the highest concentrations of PKA inhibitors reduced the P‐induced calcium influx; lower concentrations of PKA inhibitors did not affect it. Similar results were apparent for PKC inhibitors and for tyrosine kinase inhibitors. None of the Ser/Thr phosphatase inhibitors affected the P‐induced acrosome reaction or the P‐induced calcium influx, except for the PP2B inhibitors that significantly reduced the P‐induced acrosome reaction without affecting calcium influx. Finally, the protein tyrosine phosphatase inhibitors significantly blocked the P‐induced acrosome reaction and reduced the amplitude of the P‐induced calcium transient (p < 0.001) as well as the amplitude of the plateau phase (p < 0.01). The data suggest that protein kinases and possibly PP2B have a role on the acrosome reaction at some point downstream of calcium entry and that Tyr phosphatases have a role on the acrosome reaction upstream of calcium entry.

Does fibronectin (Fn) stimulate the sperm capacitation process in humans? Fibronectin stimulates human sperm capacitation. Capacitation is a process that occurs in the oviduct. It has been suggested that some molecules present in the oviductal fluid and cells as well as proteins present in the cumulus oophorus could be involved in the modulation of sperm function and their acquisition of fertilizing capacity. Fibronectin is a glycoprotein that is present in the fluid and the oviduct epithelium, and its receptor (alpha 5 beta 1 integrin) is present in human sperm. When alpha 5 beta 1 (α₅β₁) integrin binds to fibronectin, intracellular signals similar to the process of sperm capacitation are activated. Human sperm were selected via a percoll gradient and were then incubated in non-capacitated medium (NCM) or reconstituted capacitated medium (RCM), in the presence or absence of fibronectin for different time periods. A total of 39 donors were used during the study, which lasted 3 years. Freshly ejaculated sperm from healthy volunteers were obtained by masturbation. All semen samples were normal according to the World Health Organization parameters. Six approaches were used to determine the effects of fibronectin on sperm capacitation: chlortetracycline (CTC) assay, heterologous co-culture of human sperm with bovine oviductal epithelial cells (BOEC), measurement of cyclic (c) AMP levels, activity of protein kinase A (PKA), phosphorylation of proteins in tyrosine (Tyr) residues, and induction of acrosome reaction with progesterone. When sperm were incubated in RCM in the presence of Fn, we observed differences with respect to sperm incubated in RCM without Fn (control): (i) a 10% increase in the percentage of sperm with the B pattern (capacitated sperm) of CTC fluorescence from the beginning of capacitation (P < 0.001); (ii) an effect on both the concentration of cAMP (P < 0.05) and PKA activity (P < 0.05) during early capacitation; (iii) an increase in the degree of phosphorylation of proteins on tyrosine residues after 60 min of capacitation (P < 0.01); (iv) an increase in the percentage of acrosome-reacted sperm in response to progesterone (P < 0.05); and (v) a decrease in the percentage of sperm attached to BOEC (P < 0.05). Moreover, we noted that the effect of Fn was specific and mediated by alpha 5 beta 1 integrin (P < 0.001). Fn by itself had no effect on sperm capacitation. This study was carried out with sperm from young adult men. Men with abnormal semen samples were excluded. The results cannot be directly extrapolated to other mammalian species. Currently, male subfertility has become a huge public health problem, which makes it imperative to develop new treatments. This is a novel discovery that extends our current knowledge concerning normal and pathological sperm physiology as well as events that regulate the process of fertilization. This study was supported by grants from FONDECYT (1130341, E.S.D. and 1120056, P.M.) and FONCYT (PIP 2011-0496, S.P.-M). The authors have no conflicts of interest.

There are few reports on the role of protein phosphatases during capacitation. Here, we report on the role of PP2B, PP1, and PP2A during human sperm capacitation. Motile sperm were resuspended in non-capacitating medium (NCM, Tyrode's medium, albumin- and bicarbonate-free) or in reconstituted medium (RCM, NCM plus 2.6% albumin/25 mM bicarbonate). The presence of the phosphatases was evaluated by western blotting and the subcellular localization by indirect immunofluorescence. The function of these phosphatases was analyzed by incubating the sperm with specific inhibitors: okadaic acid, I2, endothall, and deltamethrin. Different aliquots were incubated in the following media: 1) NCM; 2) NCM plus inhibitors; 3) RCM; and 4) RCM plus inhibitors. The percent capacitated sperm and phosphatase activities were evaluated using the chlortetracycline assay and a phosphatase assay kit, respectively. The results confirm the presence of PP2B and PP1 in human sperm. We also report the presence of PP2A, specifically, the catalytic subunit and the regulatory subunits PR65 and B. PP2B and PP2A were present in the tail, neck, and postacrosomal region, and PP1 was present in the postacrosomal region, neck, middle, and principal piece of human sperm. Treatment with phosphatase inhibitors rapidly (≤1 min) increased the percent of sperm depicting the pattern B, reaching a maximum of ∼40% that was maintained throughout incubation; after 3 h, the percent of capacitated sperm was similar to that of the control. The enzymatic activity of the phosphatases decreased during capacitation without changes in their expression. The pattern of phosphorylation on threonine residues showed a sharp increase upon treatment with the inhibitors. In conclusion, human sperm express PP1, PP2B, and PP2A, and the activity of these phosphatases decreases during capacitation. This decline in phosphatase activities and the subsequent increase in threonine phosphorylation may be an important requirement for the success of sperm capacitation.

Fertilization is the process by which male and female haploid gametes (sperm and egg) unite to produce a genetically distinct individual. In mammals, fertilization involves a number of sequential steps, including sperm migration through the female genital tract, sperm penetration through the cumulus mass, sperm adhesion and binding to the zona pellucida, acrosome exocytosis, sperm penetration through the zona and fusion of the sperm and egg plasma membranes. However, freshly ejaculated sperm are not capable of fertilizing an oocyte. They must first undergo a series of biochemical and physiological changes, collectively known as capacitation, before acquiring fertilizing capabilities. Several molecules are required for successful capacitation and in vitro fertilization; these include bicarbonate, serum albumin (normally bovine serum albumin, BSA) and Ca²⁺. Bicarbonate activates the sperm protein soluble adenylyl cyclase (SACY), which results in increased levels of cAMP and cAMP-dependent protein kinase (PKA) activation. The response to bicarbonate is fast and cAMP levels increase within 60 s followed by an increase in PKA activity. Several studies with an anti-phospho-PKA substrate antibody have demonstrated a rapid increase in protein phosphorylation in human, mouse and boar sperm. The target proteins of PKA are not known and the precise role of BSA during capacitation is unclear. Most of the studies provide support for the idea that BSA acts by removing cholesterol from the sperm. The loss of cholesterol has been suggested to affect the bilayer of the sperm plasma membrane making it more fusogenic. The relationship between cholesterol loss and the activation of the cAMP/PKA pathway is also unclear. During early stages of capacitation, Ca²⁺ might be involved in the stimulation of SACY, although definitive proof is lacking. Protein tyrosine phosphorylation is another landmark of capacitation but occurs during the late stages of capacitation on a different time-scale from cAMP/PKA activation. Additionally, the tyrosine kinases present in sperm are not well characterized. Although protein phosphorylation depends upon the balanced action of protein kinases and protein phosphatase, we have even less information regarding the role of protein phosphatases during sperm capacitation. Over the last few years, several reports have pointed out that the ubiquitin-proteasome system might play a role during sperm capacitation, acrosome reaction and/or sperm-egg fusion. In the present review, we summarize the information regarding the role of protein kinases, phosphatases and the proteasome during sperm capacitation. Where appropriate, we give examples of the way that these molecules interact and regulate each other’s activities.