Effect of Transcranial Pulse Stimulation for the Treatment of Alzheimer´s Disease and its Related Symptoms.

Alzheimer's disease (AD) is the most common cause of neurodegenerative cognitive impairment. Brain stimulation techniques based on the delivery of transcranial shockwaves are currently being studied for their increasing popularity as an approach to modulate the human brain in a focal and targeted manner making this therapy a promising line of action against AD. In the present manuscript, we review for further understanding whether transcranial pulse stimulation (TPS) is a beneficial treatment for AD patients. PubMed, Google Scholar, and Cochrane databases were accessed with the search criteria set from year 2001 to 2022 and the following keywords were used: "transcranial pulse stimulation", "focused ultrasound", "noninvasive treatment and Alzheimer" and "TPS". The search was focused on papers that provide evidence on the biological bases of the method, as well as its safety and tolerability. Even though more studies are needed with greater scientific rigor, such as a doubleblind and randomized study versus a placebo, TPS is an excellent and safe therapeutic option for AD. This novel approach accompanies currently available treatments and complements them, helping to maintain greater stability of the disease and slowing its progression. The biological effects and potential mechanisms of action of TPS for the improvement of cognitive function are further discussed.

Reproductive outcomes of oocyte donation in patients with uterine Müllerian anomalies.

OBJECTIVE: To evaluate live-birth rates per embryo transfer in patients with uterine Müllerian anomalies (UMAs). Secondary objectives were to compare reproductive outcomes between the normal uterus group, the different UMA types, and UMA subgroups with and without required surgery. DESIGN: This retrospective study compared two cohorts, one with UMAs and other with normal uteri of our oocyte donation program at 12 Instituto Valenciano De Infertilidad/Reproductive Medicine Associates University affiliated clinics from January 2000 to 2020. The oocyte donation reduces confounding because of differences in embryo quality. The primary outcome was the live-birth rate per embryo transfer. Secondary outcomes included the rates of implantation, clinical pregnancy, miscarriage, and ongoing pregnancy. We calculated odds ratios with 95% confidence intervals. PATIENTS: Infertile women undergoing oocyte donation with UMAs. INTERVENTION: None. MAIN OUTCOME MEASURES: The rates of implantation, clinical pregnancy, miscarriage, ongoing pregnancy, and live birth. RESULTS: We analyzed 58,337 cycles of oocyte donation: 57,869 patients had no uterine malformation, and 468 women had UMAs. Compared with patients with normal uteri, patients with UMAs had lower rates of live births (36.67% [32.84-40.65] vs. 38.1% [95% confidence intervals {CI}: 37.82-38.42]) and ongoing pregnancy (39.74% [35.93-43.66] vs. 41.5% [41.24-41.83]). The miscarriage rate was higher in patients with UMAs (19.5% [16.55-22.85] vs. 16.6% [16.47-16.92]). Specifically, patients with a unicornuate uterus (n=29) had lower rates of implantation (24.07% [13.49-37.64] vs. 42.85% [95% CI: 42.6-43.09]), pregnancy (41.86% [27.01-57.87] vs. 59.51% [59.22-59.81]), ongoing pregnancy (16.67% [6.97-31.36] vs. 41.54% [41.24-41.83]), and live births (16.67% [6.97-31.36] vs. 38.12% [37.83-38.42]). In addition, patients with a partial septate uterus (n=91) had a higher miscarriage rate (26.50% [18.44-34.89] vs. 16.7% [16.47-16.92]). Compared with the normal uterus group, the live-birth rates were lower in the UMA without surgery group (33.09% [27.59-38.96] vs. 38.12% [37.83-38.42]). CONCLUSION: Among patients who received embryos derived from donated oocytes, live birth and ongoing pregnancy rates were lower in patients with UMAs compared with patients with normal uteri. A higher miscarriage rate was found in patients with UMAs. Patients with a unicornuate uterus had worse reproductive outcomes. Our results show that the uterus is less competent in patients with UMAs. TRIAL REGISTRATION: This study was registered at clinicaltrial.gov (NCT04571671).

Transfer of H2O2 from Mitochondria to the endoplasmic reticulum via Aquaporin-11.

Some aquaporins (AQPs) can transport H2O2 across membranes, allowing redox signals to proceed in and between cells. Unlike other peroxiporins, human AQP11 is an endoplasmic reticulum (ER)-resident that can conduit H2O2 to the cytosol. Here, we show that silencing Ero1α, an ER flavoenzyme that generates abundant H2O2 during oxidative folding, causes a paradoxical increase in luminal H2O2 levels. The simultaneous AQP11 downregulation prevents this increase, implying that H2O2 reaches the ER from an external source(s). Pharmacological inhibition of the electron transport chain reveals that Ero1α downregulation activates superoxide production by complex III. In the intermembrane space, superoxide dismutase 1 generates H2O2 that enters the ER channeled by AQP11. Meanwhile, the number of ER-mitochondria contact sites increases as well, irrespective of AQP11 expression. Taken together, our findings identify a novel interorganellar redox response that is activated upon Ero1α downregulation and transfers H2O2 from mitochondria to the ER via AQP11.

Human aquaporin-11 guarantees efficient transport of H2O2 across the endoplasmic reticulum membrane.

Hydrogen peroxide (H2O2) is an essential second intracellular messenger. To reach its targets in the cytosol, H2O2 must cross a membrane, a feat that requires aquaporins (AQP) endowed with 'peroxiporin' activity (AQP3, AQP8, AQP9). Here, we exploit different organelle-targeted H2O2-sensitive probes to show that also AQP11 efficiently conduits H2O2. Unlike other peroxiporins, AQP11 is localized in the endoplasmic reticulum (ER), accumulating partly in mitochondrial-associated ER membranes (MAM). Its downregulation severely perturbs the flux of H2O2 through the ER, but not through the mitochondrial or plasma membranes. These properties make AQP11 a potential regulator of ER redox homeostasis and signaling.

Monitoring cytosolic H2O2 fluctuations arising from altered plasma membrane gradients or from mitochondrial activity.

Genetically encoded probes monitoring H2O2 fluctuations in living organisms are key to decipher redox signaling events. Here we use a new probe, roGFP2-Tpx1.C169S, to monitor pre-toxic fluctuations of peroxides in fission yeast, where the concentrations linked to signaling or to toxicity have been established. This probe is able to detect nanomolar fluctuations of intracellular H2O2 caused by extracellular peroxides; expression of human aquaporin 8 channels H2O2 entry into fission yeast decreasing membrane gradients. The probe also detects H2O2 bursts from mitochondria after addition of electron transport chain inhibitors, the extent of probe oxidation being proportional to the mitochondrial activity. The oxidation of this probe is an indicator of steady-state levels of H2O2 in different genetic backgrounds. Metabolic reprogramming during growth in low-glucose media causes probe reduction due to the activation of antioxidant cascades. We demonstrate how peroxiredoxin-based probes can be used to monitor physiological H2O2 fluctuations.

The Plasma Membrane: A Platform for Intra- and Intercellular Redox Signaling.

Membranes are of outmost importance to allow for specific signal transduction due to their ability to localize, amplify, and direct signals. However, due to the double-edged nature of reactive oxygen species (ROS)-toxic at high concentrations but essential signal molecules-subcellular localization of ROS-producing systems to the plasma membrane has been traditionally regarded as a protective strategy to defend cells from unwanted side-effects. Nevertheless, specialized regions, such as lipid rafts and caveolae, house and regulate the activated/inhibited states of important ROS-producing systems and concentrate redox targets, demonstrating that plasma membrane functions may go beyond acting as a securing lipid barrier. This is nicely evinced by nicotinamide adenine dinucleotide phosphate (NADPH)-oxidases (NOX), enzymes whose primary function is to generate ROS and which have been shown to reside in specific lipid compartments. In addition, membrane-inserted bidirectional H2O2-transporters modulate their conductance precisely during the passage of the molecules through the lipid bilayer, ensuring time-scaled delivery of the signal. This review aims to summarize current evidence supporting the role of the plasma membrane as an organizing center that serves as a platform for redox signal transmission, particularly NOX-driven, providing specificity at the same time that limits undesirable oxidative damage in case of malfunction. As an example of malfunction, we explore several pathological situations in which an inflammatory component is present, such as inflammatory bowel disease and neurodegenerative disorders, to illustrate how dysregulation of plasma-membrane-localized redox signaling impacts normal cell physiology.

A persulfidation-based mechanism controls aquaporin-8 conductance.

Upon engagement of tyrosine kinase receptors, nicotinamide adenine dinucleotide phosphate (NADPH)-oxidases release H2O2 in the extracellular space. We reported previously that aquaporin-8 (AQP8) transports H2O2 across the plasma membrane and is reversibly gated during cell stress, modulating signal strength and duration. We show that AQP8 gating is mediated by persulfidation of cysteine 53 (C53). Treatment with H2S is sufficient to block H2O2 entry in unstressed cells. Silencing cystathionine ß-synthase (CBS) prevents closure, suggesting that this enzyme is the main source of H2S. Molecular modeling indicates that C53 persulfidation displaces a nearby histidine located in the narrowest part of the channel. We propose that H2O2 molecules transported through AQP8 sulfenylate C53, making it susceptible to H2S produced by CBS. This mechanism tunes H2O2 transport and may control signaling and limit oxidative stress.

Restoring microenvironmental redox and pH homeostasis inhibits neoplastic cell growth and migration: therapeutic efficacy of esomeprazole plus sulfasalazine on 3-MCA-induced sarcoma.

Neoplastic cells live in a stressful context and survive thanks to their ability to overcome stress. Thus, tumor cell responses to stress are potential therapeutic targets. We selected two such responses in melanoma and sarcoma cells: the xc- antioxidant system, that opposes oxidative stress, and surface v-ATPases that counteract the low pHi by extruding protons, and targeted them with the xc- blocker sulfasalazine and the proton pump inhibitor esomeprazole. Sulfasalazine inhibited the cystine/cysteine redox cycle and esomeprazole decreased pHi while increasing pHe in tumor cell lines. Although the single treatment with either drug slightly inhibited cell proliferation and motility, the association of sulfasalazine and esomeprazole powerfully decreased sarcoma and melanoma growth and migration. In the 3-methylcholanthrene (3-MCA)-induced sarcoma model, the combined therapy strongly reduced the tumor burden and increased the survival time: notably, 22 % of double-treated mice recovered and survived off therapy. Tumor-associated macrophages (TAM) displaying M2 markers, that abundantly infiltrate sarcoma and melanoma, overexpress xc- and membrane v-ATPases and were drastically decreased in tumors from mice undergone the combined therapy. Thus, the double targeting of tumor cells and macrophages by sulfasalazine and esomeprazole has a double therapeutic effect, as decreasing TAM infiltration deprives tumor cells of a crucial allied. Sulfasalazine and esomeprazole may therefore display unexpected therapeutic values, especially in case of hard-to-treat cancers.

Stress Regulates Aquaporin-8 Permeability to Impact Cell Growth and Survival.

UNLABELLED: Aquaporin-8 (AQP8) allows the bidirectional transport of water and hydrogen peroxide across biological membranes. Depending on its concentration, H2O2 exerts opposite roles, amplifying growth factor signaling in physiological conditions, but causing severe cell damage when in excess. Thus, H2O2 permeability is likely to be tightly controlled in living cells. AIMS: In this study, we investigated whether and how the transport of H2O2 through plasma membrane AQP8 is regulated, particularly during cell stress. RESULTS: We show that diverse cellular stress conditions, including heat, hypoxia, and ER stress, reversibly inhibit the permeability of AQP8 to H2O2 and water. Preventing the accumulation of intracellular reactive oxygen species (ROS) during stress counteracts AQP8 blockade. Once inhibition is established, AQP8-dependent transport can be rescued by reducing agents. Neither H2O2 nor water transport is impaired in stressed cells expressing a mutant AQP8, in which cysteine 53 had been replaced by serine. Cells expressing this mutant are more resistant to stress-, drug-, and radiation-induced growth arrest and death. INNOVATION AND CONCLUSION: The control of AQP8-mediated H2O2 transport provides a novel mechanism to regulate cell signaling and survival during stress. Antioxid. Redox Signal. 24, 1031-1044.

Oocyte donation outcome after oncological treatment in cancer survivors.

OBJECTIVE: To study reproductive outcome in patients cured of cancer who required oocyte donation (OD) owing to iatrogenic ovarian dysfunction. DESIGN: Multicenter, unmatched, retrospective cohorts study. SETTING: Private, university-affiliated group of clinics. PATIENT(S): Women treated and cured of cancer (n = 142) who underwent 333 cycles of OD (exposed group) and women without a previous cancer diagnosis (n = 17,844) who underwent 29,778 cycles of OD (unexposed cohort) between January 2000 and January 2012. INTERVENTION(S): Retrospective chart review. MAIN OUTCOME MEASURE(S): Pregnancy, implantation, miscarriage, and ongoing pregnancy rates. RESULT(S): There were no differences in terms of pregnancy (55.7% vs. 54.7%), implantation (39.8% vs. 38.2%), miscarriage (29.5% vs. 26.9%), or delivery rates (39.3% vs. 39.9%) between the unexposed group and the patients previously diagnosed and cured of cancer, respectively. There was no correlation between OD outcome and cancer type. CONCLUSION(S): Endometrial receptivity in women treated and cured of cancer was comparable to that of general patients without previous malignancies who had received OD, based on the largest series available in the literature.

Different redox sensitivity of endoplasmic reticulum associated degradation clients suggests a novel role for disulphide bonds in secretory proteins.

To maintain proteostasis in the endoplasmic reticulum (ER), terminally misfolded secretory proteins must be recognized, partially unfolded, and dislocated to the cytosol for proteasomal destruction, in a complex process called ER-associated degradation (ERAD). Dislocation implies reduction of inter-chain disulphide bonds. When in its reduced form, protein disulphide isomerase (PDI) can act not only as a reductase but also as an unfoldase, preparing substrates for dislocation. PDI oxidation by Ero1 favours substrate release and transport across the ER membrane. Here we addressed the redox dependency of ERAD and found that DTT stimulates the dislocation of proteins with DTT-resistant disulphide bonds (i.e., orphan Ig-µ chains) but stabilizes a ribophorin mutant (Ri332) devoid of them. DTT promotes the association of Ri332, but not of Ig-µ, with PDI. This discrepancy may suggest that disulphide bonds in cargo proteins can be utilized to oxidize PDI, hence facilitating substrate detachment and degradation also in the absence of Ero1. Accordingly, Ero1 silencing retards Ri332 degradation, but has little if any effect on Ig-µ. Thus, some disulphides can increase the stability and simultaneously favour quality control of secretory proteins.

Treatment of luteal phase defects in assisted reproduction.

Abnormal luteal function is a common issue in assisted reproduction techniques associated with ovarian stimulation probably due to low levels of LH in the middle and in the late luteal phase. This defect seems to be associated with supraphysiological steroid levels at the end of follicular phase. The luteal phase insufficiency has not got a diagnostic test which has proven reliable in a clinical setting. Luteal phase after ovarian stimulation becomes shorter and insufficient, resulting in lower pregnancy rates. Luteal phase support with progesterone or hCG improves pregnancy outcomes and no differences are found among different routes of administration. However, hCG increases the risk of ovarian hyperstimulation syndrome. In relation to the length of luteal support, the day of starting it remains controversial and it does not seem necessary to continue once a pregnancy has been established. After GnRHa triggering ovulation, intensive luteal support or hCG bolus can overcome the defect in luteal phase, but more studies are needed to show the LH utility as support.

Tyrosine kinase signal modulation: a matter of H2O2 membrane permeability?

Abstract H2O2 produced by extracellular NADPH oxidases regulates tyrosine kinase signaling inhibiting phosphatases. How does it cross the membrane to reach its cytosolic targets? Silencing aquaporin-8 (AQP8), but not AQP3 or AQP4, inhibited H2O2 entry into HeLa cells. Re-expression of AQP8 with silencing-resistant vectors rescued H2O2 transport, whereas a C173A-AQP8 mutant failed to do so. Lowering AQP8 levels affected H2O2 entry into the endoplasmic reticulum, but not into mitochondria. AQP8 silencing also inhibited the H2O2 spikes and phosphorylation of downstream proteins induced by epidermal growth factor. These observations lead to the hypothesis that H2O2 does not freely diffuse across the plasma membrane and AQP8 and other H2O2 transporters are potential targets for manipulating key signaling pathways in cancer and degenerative diseases.

RIAM (Rap1-interacting adaptor molecule) regulates complement-dependent phagocytosis.

Phagocytosis mediated by the complement receptor CR3 (also known as integrin αMß2 or Mac-1) is regulated by the recruitment of talin to the cytoplasmic tail of the ß2 integrin subunit. Talin recruitment to this integrin is dependent on Rap1 activation. However, the mechanism by which Rap1 regulates this event and CR3-dependent phagocytosis remains largely unknown. In the present work, we examined the role of the Rap1 effector RIAM, a talin-binding protein, in the regulation of complement-mediated phagocytosis. Using the human myeloid cell lines HL-60 and THP-1, we determined that knockdown of RIAM impaired αMß2 integrin affinity changes induced by stimuli fMLP and LPS. Phagocytosis of complement-opsonized RBC particles, but not of IgG-opsonized RBC particles, was impaired in RIAM knockdown cells. Rap1 activation via EPAC induced by 8-pCPT-2'-O-Me-cAMP resulted in an increase of complement-mediated phagocytosis that was abrogated by knockdown of RIAM in HL-60 and THP-1 cell lines and in macrophages derived from primary monocytes. Furthermore, recruitment of talin to ß2 integrin during complement-mediated phagocytosis was reduced in RIAM knockdown cells. These results indicate that RIAM is a critical component of the phagocytosis machinery downstream of Rap1 and mediates its function by recruiting talin to the phagocytic complement receptors.

The role of LH in ovarian stimulation.

LH is a glycoprotein that plays a crucial role in folliculogenesis during the natural ovarian cycles. It has the same activity and shares receptors with hCG. However the use of LH in combination with FSH in controlled ovarian stimulation remains controversial. A practical approach concerning the usefulness of LH according to the endogenous level of LH is described herein. Specific groups of patients can benefit from ovarian stimulation with LH. New applications of LH/hCG activity are also discussed.

Rap1-GTP-interacting adaptor molecule (RIAM) protein controls invasion and growth of melanoma cells.

The Mig-10/RIAM/lamellipodin (MRL) family member Rap1-GTP-interacting adaptor molecule (RIAM) interacts with active Rap1, a small GTPase that is frequently activated in tumors such as melanoma and prostate cancer. We show here that RIAM is expressed in metastatic human melanoma cells and that both RIAM and Rap1 are required for BLM melanoma cell invasion. RIAM silencing in melanoma cells led to inhibition of tumor growth and to delayed metastasis in a severe combined immunodeficiency xenograft model. Defective invasion of RIAM-silenced melanoma cells arose from impairment in persistent cell migration directionality, which was associated with deficient activation of a Vav2-RhoA-ROCK-myosin light chain pathway. Expression of constitutively active Vav2 and RhoA in cells depleted for RIAM partially rescued their invasion, indicating that Vav2 and RhoA mediate RIAM function. These results suggest that inhibition of cell invasion in RIAM-silenced melanoma cells is likely based on altered cell contractility and cell polarization. Furthermore, we show that RIAM depletion reduces ß1 integrin-dependent melanoma cell adhesion, which correlates with decreased activation of both Erk1/2 MAPK and phosphatidylinositol 3-kinase, two central molecules controlling cell growth and cell survival. In addition to causing inhibition of cell proliferation, RIAM silencing led to higher susceptibility to cell apoptosis. Together, these data suggest that defective activation of these kinases in RIAM-silenced cells could account for inhibition of melanoma cell growth and that RIAM might contribute to the dissemination of melanoma cells.

Mechanism suppressing glycogen synthesis in neurons and its demise in progressive myoclonus epilepsy.

Glycogen synthesis is normally absent in neurons. However, inclusion bodies resembling abnormal glycogen accumulate in several neurological diseases, particularly in progressive myoclonus epilepsy or Lafora disease. We show here that mouse neurons have the enzymatic machinery for synthesizing glycogen, but that it is suppressed by retention of muscle glycogen synthase (MGS) in the phosphorylated, inactive state. This suppression was further ensured by a complex of laforin and malin, which are the two proteins whose mutations cause Lafora disease. The laforin-malin complex caused proteasome-dependent degradation both of the adaptor protein targeting to glycogen, PTG, which brings protein phosphatase 1 to MGS for activation, and of MGS itself. Enforced expression of PTG led to glycogen deposition in neurons and caused apoptosis. Therefore, the malin-laforin complex ensures a blockade of neuronal glycogen synthesis even under intense glycogenic conditions. Here we explain the formation of polyglucosan inclusions in Lafora disease by demonstrating a crucial role for laforin and malin in glycogen synthesis.

Laforin, the dual-phosphatase responsible for Lafora disease, interacts with R5 (PTG), a regulatory subunit of protein phosphatase-1 that enhances glycogen accumulation.

Progressive myoclonus epilepsy of Lafora type (LD, MIM 254780) is a fatal autosomal recessive disorder characterized by the presence of progressive neurological deterioration, myoclonus, epilepsy and polyglucosan intracellular inclusion bodies, called Lafora bodies. Lafora bodies resemble glycogen with reduced branching, suggesting an alteration in glycogen metabolism. Linkage analysis and homozygosity mapping localized EPM2A, a major gene for LD, to chromosome 6q24. EPM2A encodes a protein of 331 amino acids (named laforin) with two domains, a dual-specificity phosphatase domain and a carbohydrate binding domain. Here we show that, in addition, laforin interacts with itself and with the glycogen targeting regulatory subunit R5 of protein phosphatase 1 (PP1). R5 is the human homolog of the murine Protein Targeting to Glycogen, a protein that also acts as a molecular scaffold assembling PP1 with its substrate, glycogen synthase, at the intracellular glycogen particles. The laforin-R5 interaction was confirmed by pull-down and co-localization experiments. Full-length laforin is required for the interaction. However, a minimal central region of R5 (amino acids 116-238), including the binding sites for glycogen and for glycogen synthase, is sufficient to interact with laforin. Point-mutagenesis of the glycogen synthase-binding site completely blocked the interaction with laforin. The majority of the EPM2A missense mutations found in LD patients result in lack of phosphatase activity, absence of binding to glycogen and lack of interaction with R5. Interestingly, we have found that the LD-associated EPM2A missense mutation G240S has no effect on the phosphatase or glycogen binding activities of laforin but disrupts the interaction with R5, suggesting that binding to R5 is critical for the laforin function. These results place laforin in the context of a multiprotein complex associated with intracellular glycogen particles, reinforcing the concept that laforin is involved in the regulation of glycogen metabolism.