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Background: Vitamin B12 is essential for neurological function, red blood cell formation, and DNA synthesis. Deficiency can lead to diverse health conditions, including megaloblastic anemia and neurological issues. Oral supplementation is a standard treatment for B12 deficiency. The Sucrosomial ® carrier system offers an innovative approach that enhances supplemental nutrient absorption and bioavailability., Objectives: This study aimed to compare the effectiveness of oral Sucrosomial ® vitamin B12 formulation vs various conventional B12 supplements, randomly selected from local pharmacies, in increasing and maintaining circulatory B12 levels in healthy deficient adults (200-300 pg/mL)., Methods: A randomized, double-blind clinical trial was conducted across three centers in Pakistan from April to July 2024. At KEMU, participants received either Sucrosomial ® vitamin B12 or Mecogen SL B12; at LRH, Sucrosomial ® B12 or B-SUB B12; and at LUMHS, Sucrosomial ® B12, Evermin B12, or Neuromax B12. Participants took a daily single dose of 1,000 μg of the assigned B12 formulation for 7 days. Serum B12 levels were measured at baseline (day 0) and on days 1, 3, 5, and 7., Results: Sucrosomial ® B12 was significantly more effective than conventional B12 formulations in increasing and maintaining higher serum B12 levels across all time points. At KEMU, it reached a peak concentration of 454 ± 3.9 pg/mL by day 5, compared to 274 ± 11.1 pg/mL with Mecogen SL B12. At LRH, it peaked at 496 ± 34.4 pg/mL by day 5 versus 304 ± 49.4 pg/mL for B-SUB B12. At LUMHS, it reached 592.7 ± 74.3 pg/mL by day 7, compared to 407.24 ± 41.6 pg/mL for Evermin B12 and 263.82 ± 23.8 pg/mL for Neuromax B12. Sucrosomial ® B12 was the only formulation to surpass the deficiency-borderline threshold (200-300 pg/mL) within 24 h of the first dose and was well tolerated with no reported side effects., Conclusion: Sucrosomial ® vitamin B12 demonstrated superior efficacy in rapidly and consistently elevating and maintaining higher circulatory B12 levels compared to conventional supplements. Its characteristic absorption mode and proven efficacy suggest it could effectively address B12 deficiency in a broad range of populations, including those with gastrointestinal conditions and pernicious anemia, thereby supporting overall health., Clinical Trial Registration: clinicaltrials.gov, NCT06376591., Competing Interests: EB and GT were employed by PharmaNutra S.p.A, Pisa, Italy. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Memon, Conti, Brilli, Tarantino, Chaudhry, Baloch, Shafiq, Mumtaz, Qaisar, Iqtadar, Abrar, Kanwal, Akhtar, Latif, Rabbani, Ujjan, Turroni and Khan.)

Background: Vitamin D (Vit D) deficiency (VDD), associated with diverse health conditions, is commonly treated with Vit D3 supplements. However, the gastrointestinal (GI) absorption of Vit D3 in different formulations has not been well studied., Objective: We aimed to compare the absorption of an innovative phospholipids-sucrester matrix biodelivery vehicle-based (sucrosomial ® ) orodispersible Vit D3 preparation against a reference chewable tablet and soft gel capsule (SGC) Vit D3 formulations in Vit D-deficient healthy adults., Methods: In study 1, 25 subjects were randomized to receive a weekly single dose of 200,000 IU of sucrosomial ® Vit D3 ( n = 12) or chewable tablet Vit D3 ( n = 13) for 3 weeks. In study 2, 20 subjects were randomized to receive a single dose of 200,000 IU every other week of sucrosomial ® Vit D3 ( n = 10) or SGC Vit D3 ( n = 10) for 6 weeks. Circulatory 25-hydroxyvitamin D3 [25(OH)D] levels were reassessed after 2, 3, and 6 weeks in study 1 and after 4 and 6 weeks in study 2., Results: In study 1, after 2 weeks, circulatory 25(OH)D levels increased significantly in both Vit D3 treatment groups ( p < 0.0001) but improved markedly in the sucrosomial ® Vit D3 group, with no further considerable change after 3 and 6 weeks in both groups. Overall, at all three follow-ups, sucrosomial ® Vit D3 treatment achieved significantly higher and sustained 25(OH)D levels ( p < 0.001). In study 2, after 4 weeks, both Vit D3 treatment groups showed significant improvement in circulatory 25(OH)D levels ( p < 0.0001) but substantially higher in the sucrosomial ® group with statistically significant differences between the two treatment groups ( p = 0.02). At the 6-week follow-up, only subjects in the sucrosomial ® Vit D3 group showed a further increase in circulatory 25(OH)D levels ( p = 0.049), but no further significant changes in the levels of the SGC Vit D3 group ( p = 0.062), showing a statistically significant difference between the two treatment groups ( p = 0.002). The Vit D3 treatment was well tolerated by all participants, and no treatment-emergent effects or serious adverse events were reported., Conclusion: Our results suggest that the sucrosomial ® Vit D3 preparation absorbs efficiently in the GI system, achieving adequately higher and sustained circulatory Vit D levels in VDD, and thus can effectively contribute to the body protection against VDD-associated health conditions., Clinical Trial Registration: clinicaltrials.gov, identifier: NCT05706259., Competing Interests: EB and GT are employees of the PharmaNutra S.p.A., Pisa, Italy. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Bano, Abrar, Brilli, Tarantino, Bugti, Fabbrini, Conti, Turroni, Bugti, Afridi, Mureed, Zada, Din Ujjan, Ashraf, Ghafoor, Khan and Khan.)

Iron deficiency (ID) and iron deficiency anemia (IDA) are highly prevalent worldwide. Oral iron salts, especially ferrous sulfate, are commonly used for the treatment of iron deficiency (ID). However, its use is associated with gastrointestinal side effects, thus compromising treatment compliance. Intravenous iron administration is a more costly and logistically complex alternative and is not risk-free, as infusion and hypersensitivity reactions may occur. Sucrosomial ® iron is an oral formulation consisting of ferric pyrophosphate conveyed by a phospholipid and sucrester matrix (sucrosome ® ). Intestinal Sucrosomial ® iron absorption is mediated by enterocytes and M cells, through the paracellular and transcellular routes, and occurs mostly as intact particles. These pharmacokinetic properties of Sucrosomial ® iron result in higher iron intestinal absorption and excellent gastrointestinal tolerance compared to oral iron salts. The evidence derived from clinical studies supports the use of Sucrosomial ® iron as a valid first option for the treatment of ID and IDA, especially for subjects who are intolerant or refractory to conventional iron salts. Newer evidence also demonstrates the effectiveness of Sucrosomial ® iron, with a lower cost and fewer side effects, in certain conditions usually treated with IV iron in current clinical practice.

Background : Berberine is a natural alkaloid with hypoglycemic properties. However, its therapeutic use is limited by a very low oral bioavailability. Here we developed a new oral formulation of berberine based on Sucrosomial ® technology and tested its effect on insulin resistance. Methods : Sucrosomial ® berberine was first tested in vitro in the hepatoma cell line Huh7 to assess its effect on proteins involved in glucose homeostasis and insulin resistance. The pharmacokinetics and efficacy on insulin resistance were then studied in C57BL/6 mice fed with standard (SD) and high-fat diet (HFD) for 16 weeks and treated daily during the last 8 weeks with oral gavage of Sucrosomial ® berberine or berberine. Results : Sucrosomial ® berberine did not affect Huh7 cell viability at concentrations up to 40 µM. Incubation of Huh7 with 20 µM of Sucrosomial ® and control berberine induced glucokinase (GK) and the phosphorylation of 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK), both known targets for the control of insulin resistance. In vivo, we observed an 8-fold higher plasma concentration after 3 weeks of oral administration of 50 mg/kg/day of Sucrosomial ® formulation compared to berberine. HFD, compared to SD, induced insulin resistance in mice as determined by oral glucose tolerance test (OGTT). The treatment with a 6.25 mg/kg/daily dose of Sucrosomial ® berberine significantly reduced the area under the curve (AUC) of OGTT (73,103 ± 8645 vs. 58,830 ± 5597 mg/dL × min), while control berberine produced the same effects at 50 mg/Kg/day (51518 ± 1984 mg/dL × min). Under these conditions, the two formulations resulted in similar berberine plasma concentration in mice. Nevertheless, a different tissue distribution of metabolites was observed with a significant accumulation of reduced, demethylated and glucuronide berberine in the brain after the oral administration of the Sucrosomial ® form. Glucuronide berberine plasma concentration was higher with Sucrosomial ® berberine compared to normal berberine. Finally, we observed similar increases of AMPK phosphorylation in the liver in response to the treatment with Sucrosomial ® berberine and berberine. Conclusions : The Sucrosomial ® formulation is an innovative and effective technology to improve berberine gastrointestinal (GI) absorption with proven in vitro and in vivo activity on insulin resistance.

Iron deficiency is one of the most common nutritional deficiencies worldwide and is often treated with oral iron supplements. However, commonly used supplements, including those based on ferrous iron salts, are associated with gastrointestinal side effects and unfavorable changes in the intestinal microbiome. Sucrosomial® iron is a novel iron formulation that is effective at treating iron deficiency, and with fewer gastrointestinal side effects, yet its effect on the gut microbiome has not been examined previously. Thus, we treated mice for two weeks with diets containing either Sucrosomial® iron or ferrous sulfate as the sole iron source and examined bacterial communities in the intestine using 16S Microbial Profiling of DNA extracted from feces collected both prior to and following dietary treatment. Mice treated with Sucrosomial® iron showed an increase in Shannon diversity over the course of the study. This was associated with a decrease in the abundance of the phylum Proteobacteria, which contains many pathogenic species, and an increase in short chain fatty acid producing bacteria such as Lachnospiraceae, Oscillibacter and Faecalibaculum. None of these changes were observed in mice treated with ferrous sulfate. These results suggest that Sucrosomial® iron may have a beneficial effect on the intestinal microbiome when compared to ferrous sulfate and that this form of iron is a promising alternative to ferrous iron salts for the treatment of iron deficiency., (© 2021. The Author(s).)

Background & Aims: Recent studies have verified that the SARS-CoV-2 infection (from December 2019 has affected 123 million people throughout the world and more than 3 million people in Italy), can have medium-term and long-term effects, collectively referred to as "post-Covid syndrome" or "long-Covid" characterized by chronic fatigue, followed by muscle weakness, dyspnea and headache. Chronic fatigue or chronic tiredness is a persistent symptom both in patients who have experienced a severe infection and in those who have experienced a mild form of infection. Studies conducted on both patients discharged from hospital and patients managed at home showed that there was no association between the severity of the Coronavirus disease (Covid-19) and the subsequent chronic fatigue symptom. The aim of this study was to evaluate the ability of a nutritional supplement based on vitamins, minerals, amino acids and plant extracts (Apportal®) intake, to ameliorate the general health status in particular the chronic fatigue symptom in subjects after SARS-CoV-2 negativity., Methods: Participants were advised to take one sachet daily of Apportal® for 28 consecutive days. At the beginning (T0), after 14 days (T1) and after 28 days (T2) of supplementation, general fatigue, mental fatigue and Quality of Life indexes were evaluated through specific questionnaires. The assessment of quality of life and health status were measured through the EuroQoL-5D questionnaire, chronic fatigue using the FACIT-Fatigue questionnaire and mental fatigue using the modified Chalder questionnaire., Results: 201 subjects were enrolled for the study; results showed a significant improvement in all indexes analyzed after 14 and 28 days of supplementation. The main significant improvement was observed after the first 14 days and it was further confirmed at 28 days as well. The RTE (Relative Treatment Effect) trend about quality of life, health status, FACIT-Fatigue and mental fatigue in the three questionnaires was statistically significant (Wald Statistic, p < 0.0001). The data of FACIT-questionnaire showed an improvement of at least 1 unit in 76.62% of subjects after 14 days and in 90.05% of subjects after 28 days. An improvement of 10-unit was found in about one third of subjects after 14 days and in half of the subjects after 28 days., Conclusions: This study shows that Apportal® can reduce chronic fatigue and improve quality of life and health status in subjects after SARS-CoV-2 negativity due to the synergistic effect of its components., Competing Interests: Declaration of competing interest Maria Sole Rossato and Germano Tarantino are Pharmanutra S.p.a. employees. Elisa Brilli is Alesco S.r.l. employee, a company included in Pharmanutra group. The other Authors declare that they have no conflict of interest., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Iron-refractory iron deficiency anemia (IRIDA) is an autosomal recessive disorder caused by genetic mutations on TMPRSS6 gene which encodes Matriptase2 (MT2). An altered MT2 cannot appropriately suppress hepatic BMP6/SMAD signaling in case of low iron, hence hepcidin excess blocks dietary iron absorption, leading to a form of anemia resistant to oral iron supplementation. In this study, using the IRIDA mouse model Mask, we characterized homozygous (msk/msk) compared to asymptomatic heterozygous (msk/wt) mice, assessing the major parameters of iron status in different organs, at different ages in both sexes. The effect of carbonyl iron diet was analyzed as control iron supplementation being used for many studies in mice. It resulted effective in both anemic control and msk/msk mice, as expected, even if there is no information about its mechanism of absorption. Then, we mainly compared two forms of oral iron supplement, largely used for humans: ferrous sulfate and Sucrosomial iron. In anemic control mice, the two oral formulations corrected hemoglobin levels from 11.40 ± 0.60 to 15.38 ± 1.71 g/dl in 2-4 weeks. Interestingly, in msk/msk mice, ferrous sulfate did not increase hemoglobin likely due to ferroportin/hepcidin-dependent absorption, whereas Sucrosomial iron increased it from 11.50 ± 0.60 to 13.53 ± 0.64 g/dl mainly in the first week followed by a minor increase at 4 weeks with a stable level of 13.30 ± 0.80 g/dl, probably because of alternative absorption. Thus, Sucrosomial iron, already used in other conditions of iron deficiency, may represent a promising option for oral iron supplementation in IRIDA patients., (© 2021 The Authors. American Journal of Hematology published by Wiley Periodicals LLC.)

Cetylated fatty acids (CFAs) are a group of fats that contain a single ester group within a hydrocarbon chain, which are reported to have beneficial biological effects. A novel mixture of CFAs produced by combining fatty acids derived from refined olive oil with cetyl esters (Lipocet®) is proposed for use as a food ingredient and was therefore subjected to a nonclinical safety assessment. The safety of Lipocet® was evaluated in a bacterial reverse mutation test and an in vitro mammalian cell micronucleus test, followed by a 90-day oral (gavage) toxicity study. In the 90-day study, Sprague-Dawley rats were administered with the vehicle (corn oil) or Lipocet® at 1,500, 3,000, or 4,500 mg/kg body weight/day for 90 days. A comparator reference control group received noncetylated fatty acids derived from olive oil at 4,500 mg/kg body weight/day to identify any effects that may be expected following consumption of high doses of fat. Lipocet® was nongenotoxic in vitro. In the 90-day study, changes observed in hematological and clinical biochemistry parameters were minor in nature and/or showed poor dose dependency. Histopathology findings in the gastrointestinal tract and lungs were noted but were not associated with a clear dose response and were likely incidental. Moreover, Lipocet® was just as well tolerated as the reference control. Therefore, 4,500 mg/kg body weight/day (the highest dose tested) was considered the no-observed-adverse-effect-level. These results support the safety of Lipocet® for use as a food ingredient., (© 2020 John Wiley & Sons, Ltd.)

Appropriate nutraceutical combinations may represent a valid approach to prevent vascular calcification associated with chronic kidney disease (CKD). In the present study, we tested the effect of a new nutraceutical combination named RenaTris ® , containing MK-7, magnesium carbonate, and Sucrosomial ® Iron, on vascular calcification in uremic rats. Rats were randomly divided into three groups, i.e. control (high-phosphate diet), uremic (high-phosphate diet containing 0.5% adenine), and supplemented uremic diet (0.5% adenine, MK-7, magnesium carbonate, and Sucrosomial ® Iron). After six weeks, sera and vascular calcification were examined. The uremic diet increased creatinine and phosphate levels and induced extensive vascular calcification. The uremic condition also induced a mild hypercholesterolemic condition (+52% of total cholesterol; p < 0.05). The supplemented uremic diet did not reduce creatinine, phosphate levels, or vascular calcification, however, we observed a significant hypocholesterolemic effect (-18.9% in supplemental uremic vs . uremic diet; p < 0.05). Similar to simvastatin, incubation of cultured human hepatoma cells (Huh7) with MK-7 significantly reduced cholesterol biosynthesis (-38%) and induced 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase and low-density lipoprotein receptor (LDLR) at both mRNA and protein levels. The effect of MK-7 on LDLR was counteracted by the co-incubation with squalene. Unlike simvastatin, MK-7 reduced PCSK9 in Huh7. These results indicated that the new nutraceutical combination significantly impacts cholesterol metabolism and its supplementation may help to control mild hypercholesterolemic conditions in CKD patients., Competing Interests: E.B. and G.T. are employees at PharmaNutra S.p.A.; N.F., M.P.A., M.G.L., N.B., G.V., M.S., S.S., C.N., H.C., A.B., E.F., M.R., and S.D.A. do not have any conflicts of interest to declare.

Iron deficiency (ID) is usually treated with oral iron salts, but up to 50% of patients complain of gastrointestinal side effects, leading to reduced treatment compliance. Intravenous (IV) iron formulations are increasingly safer, but there is still a risk of infusion and hypersensitivity reactions and the need for a venous access and infusion monitoring. Sucrosomial ® iron (SI) is an innovative oral iron formulation in which ferric pyrophosphate is protected by a phospholipid bilayer plus a sucrester matrix (sucrosome), which is absorbed through para-cellular and trans-cellular routes (M cells). This confers SI unique structural, physicochemical and pharmacokinetic characteristics, together with high iron bioavailability and excellent gastrointestinal tolerance. The analysis of available evidence supports oral SI iron as a valid option for ID treatment, which is more efficacious and better tolerated than oral iron salts. SI has also demonstrated similar effectiveness, with lower risks, in patients usually receiving IV iron (e.g., chronic kidney disease, cancer, bariatric surgery). Thus, oral SI emerges as a most valuable first option for treating ID, even more for subjects with intolerance to or inefficacy of iron salts. Moreover, SI should be also considered as an alternative to IV iron for initial and/or maintenance treatment in different patient populations., Competing Interests: S.G.-R. has nothing to declare; E.B. is an Alesco S.r.l. employee; G.T. is a Pharmanutra S.p.A. employee; M.M. has received industry-supplied honoraria for consultancies, lectures and/or travel support from Pharmacosmos, Vifor Pharma, Zambon, Pharmanutra, Sandoz and Celgene, and is member of the editorial board of the journals “Revista Española de Anestesiología y Reanimación”, “Medicina Intensiva” and “Blood Transfusion”.

Sucrosomial ® Iron is a recently developed formulation to treat iron deficiency based on ferric pyrophosphate covered by a matrix of phospholipids plus sucrose esters of fatty acids. Previous data indicated that Sucrosomial ® Iron is efficiently absorbed by iron-deficient subjects, even at low dosage, and without side effects. Its structural properties may suggest that it is absorbed by an intestinal pathway which is different to the one used by ionic iron. Although, studies in vitro showed that Sucrosomial ® Iron is readily absorbed, no animal models have been established to study this important aspect. To this aim, we induced iron deficient anemia in mice by feeding them with a low-iron diet, and then we treated them with either Sucrosomial ® Iron or sulfate iron by gavage for up to two weeks. Both iron formulations corrected anemia and restored iron stores in a two-week period, but with different kinetics. Ferrous Sulfate was more efficient during the first week and Sucrosomial ® Iron in the second week. Of note, when given at the same concentrations, Ferrous Sulfate induced the expression of hepcidin and four different inflammatory markers (Socs3, Saa1, IL6 and CRP), while Sucrosomial ® Iron did not. We conclude that anemic mice are interesting models to study the absorption of oral iron, and that Sucrosomial ® Iron is to be preferred over Ferrous Sulfate because of similar absorption but without inducing an inflammatory response.

The present study aimed to demonstrate that Sideral ® RM (SRM, Sucrosomial ® Raw Material Iron) is transported across the excised intestine via a biological mechanism, and to investigate the effect that this transport route may produce on oral iron absorption, which is expected to reduce the gastrointestinal (GI) side effects caused by the bioavailability of non-absorbed iron. Excised rat intestine was exposed to fluorescein isothiocyanate (FITC)-labeled SRM in Ussing chambers followed by confocal laser scanning microscopy to look for the presence of fluorescein-tagged vesicles of the FITC-labeled SRM. To identify FITC-labeled SRM internalizing cells, an immunofluorescence analysis for macrophages and M cells was performed using specific antibodies. Microscopy analysis revealed the presence of fluorescein positive particulate structures in tissues treated with FITC-labeled SRM. These structures do not disintegrate during transit, and concentrate in macrophage cells. Iron bioavailability was assessed by determining the time-course of Fe 3+ plasma levels. As references, iron contents in liver, spleen, and bone marrow were determined in healthy rats treated by gavage with SRM or ferric pyrophosphate salt (FP). SRM significantly increased both area under the curve (AUC) and clearance maxima (C max ) compared to FP, thus increasing iron bioavailability (AUC rel = 1.8). This led to increased iron availability in the bone marrow at 5 h after single dose gavage.

This paper presents a comparative evaluation of different oral ferric iron formulations for ability to retain Fe 3+ in simulated gastric fluid (SGF), be internalized by cells lining intestinal epithelium, and cross it to reach the bloodstream. In all formulations iron was ferric pyrophosphate, the excipients were different types and fractions of lecithin plus sucrose esters of fatty acids matrix (Sideral® RM; PRT1; PRT2) or lecithin without sucrester (SUN). Dissolution kinetics of formulations in SGF was studied by USP method. The ability of the formulations to promote iron intestinal absorption was evaluated by the Caco-2 cell model, measuring cellular ferritin content, and by the excised rat intestine model, yielding apparent permeability parameters (P app ). All formulations limited iron release in SGF to ≤10%. Sideral® RM was by far the most absorbed by Caco-2, as ferritin content was in the order: Sideral® RM≫PRT2>PRT1>SUN>control. The Fe 3+ crossing the intestinal barrier was in part reduced to Fe 2+ by epithelial enzymes, in part it was carried by formulation rearrangement into nano-structures able to protect it from reduction and apt for internalization by epithelium cells. P app parameters were in the order: Sideral® RM≫PRT1>PRT2>SUN=control. Relevance of transepithelial Fe 2+ carrier, DMT-1, to Fe 3+ transport was ruled out using a DMT-1 inhibitor. In conclusion, Sideral® RM retains iron in SGF, and is the most suitable for Fe 3+ internalization by Caco-2 cells, Fe 3+ protection from enzymatic reduction and promotion of Fe 3+ absorption across intestinal epithelium, non-mediated by DMT-1., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Modeling biological systems in vitro has contributed to clarification of complex mechanisms in simplified and controlled experimental conditions. Mouse embryonic stem (mES) cells can be successfully differentiated toward specific neuronal cell fates, thus representing an attractive tool to dissect, in vitro, mechanisms that underlie complex neuronal features. In this study, we generated and characterized a reporter mES cell line, called Tph2 GFP , in which the vital reporter GFP replaces the tryptophan hydroxylase 2 (Tph2) gene. Tph2 GFP mES cells selectively express GFP upon in vitro differentiation toward the serotonergic fate, they synthesize serotonin, possess excitable membranes, and show the typical morphological, morphometrical, and molecular features of in vivo serotonergic neurons. Thanks to the vital reporter GFP, we highlighted by time-lapse video microscopy several dynamic processes such as cell migration and axonal outgrowth in living cultures. Finally, we demonstrated that predifferentiated Tph2 GFP cells are able to terminally differentiate, integrate, and innervate the host brain when grafted in vivo. On the whole, the present study introduces the Tph2 GFP mES cell line as a useful tool allowing accurate developmental and dynamic studies and representing a reliable platform for the study of serotonergic neurons in health and disease.

Brain cholesterol biosynthesis and cholesterol levels are reduced in mouse models of Huntington's disease (HD), suggesting that locally synthesized, newly formed cholesterol is less available to neurons. This may be detrimental for neuronal function, especially given that locally synthesized cholesterol is implicated in synapse integrity and remodeling. Here, we used biodegradable and biocompatible polymeric nanoparticles (NPs) modified with glycopeptides (g7) and loaded with cholesterol (g7-NPs-Chol), which per se is not blood-brain barrier (BBB) permeable, to obtain high-rate cholesterol delivery into the brain after intraperitoneal injection in HD mice. We report that g7-NPs, in contrast to unmodified NPs, efficiently crossed the BBB and localized in glial and neuronal cells in different brain regions. We also found that repeated systemic delivery of g7-NPs-Chol rescued synaptic and cognitive dysfunction and partially improved global activity in HD mice. These results demonstrate that cholesterol supplementation to the HD brain reverses functional alterations associated with HD and highlight the potential of this new drug-administration route to the diseased brain., (© 2015 The Authors. Published under the terms of the CC BY 4.0 license.)

The Repressor Element 1 Silencing Transcription factor (REST/NRSF) is a master repressor of neuronal programs in non-neuronal lineages shown to function as a central regulator of developmental programs and stem cell physiology. Aberrant REST function has been associated with a number of pathological conditions. In cancer biology, REST has been shown to play a tumor suppressor activity in epithelial cancers but an oncogenic role in brain childhood malignancies such as neuroblastoma and medulloblastoma. Here we examined REST expression in human glioblastoma multiforme (GBM) specimens and its role in GBM cells carrying self-renewal and tumorigenic competence. We found REST to be expressed in GBM specimens, its presence being particularly enriched in tumor cells in the perivascular compartment. Significantly, REST is highly expressed in self-renewing tumorigenic-competent GBM cells and its knock down strongly reduces their self-renewal in vitro and tumor-initiating capacity in vivo and affects levels of miR-124 and its downstream targets. These results indicate that REST contributes to GBM maintenance by affecting its self-renewing and tumorigenic cellular component and that, hence, a better understanding of these circuitries in these cells might lead to new exploitable therapeutic targets.

The peroxisome-proliferator-activated receptor gamma coactivator 1 α (PGC1α) has been implicated in the pathogenesis of several neurodegenerative disorders, including Huntington's disease (HD). Recent data demonstrating white matter abnormalities in PGC1α knock-out (KO) mice prompted us to examine the role of PGC1α in CNS myelination and its relevance to HD pathogenesis. We found deficient postnatal myelination in the striatum of PGC1α KO mice, accompanied by a decrease in myelin basic protein (MBP). In addition, brain cholesterol, its precursors, and the rate-limiting enzymes for cholesterol synthesis, HMG CoA synthase (HMGCS1) and HMG CoA reductase (HMGCR), were also reduced in PGC1α KO mice. Moreover, knockdown of PGC1α in oligodendrocytes by lentiviral shRNA led to a decrease in MBP, HMGCS1, and Hmgcr mRNAs. Chromatin immunoprecipitations revealed the recruitment of PGC1α to MBP promoter in mouse brain, and PGC1α over-expression increased MBP and SREBP-2 promoter activity, suggesting that PGC1α regulates MBP and cholesterol synthesis at the transcriptional level. Importantly, expression of mutant huntingtin (Htt) in primary oligodendrocytes resulted in decreased expression of PGC1α and its targets HmgcS1, Hmgcr, and MBP. Decreased expression of MBP and deficient myelination were found postnatally and in adult R6/2 mouse model of HD. Diffusion tensor imaging detected white matter abnormalities in the corpus callosum of R6/2 mice, and electron microscopy revealed thinner myelin sheaths and increased myelin periodicity in BACHD [bacterial artificial chromosome (BAC)-mediated transgenic model for Huntington's disease] mice expressing full-length mutant Htt. Together, these data suggest that PGC1α plays a role in postnatal myelination and that deficient PGC1α activity in oligodendrocytes may contribute to abnormal myelination in HD.

Dopamine D2 receptor (D2R) signalling is implicated in limbic seizure propagation and seizure-induced neurodegeneration. D2R-/- mice display increased susceptibility to kainic acid (KA) seizures and seizure-induced apoptosis of hippocampal neurons. Here we further investigated the molecular pathways of KA-induced apoptosis in the D2R-/- hippocampus. Immunoblotting experiments showed a marked induction of caspase 3 in the D2R-/- but not in the wild-type hippocampus, 24 h after the administration of KA. The activation of the Akt/glycogen synthase kinase-3beta (GSK-3beta) pathway that has been implicated in neuronal apoptosis was also studied at the same time. No difference in Akt phosphorylation in the hippocampus was detected between the two genotypes, whereas GSK-3beta phosphorylation was markedly downregulated in D2R-/- mice. Our results suggest that GSK-3beta activation participates, independently of Akt, in the KA-induced apoptosis in the D2R-/- hippocampus.

Importins, also called karyopherins, belong to a large family of proteins involved in cytoplasm-to-nucleus transport. Transport machinery generally involves a complex formed by two different importin subtypes (alpha and beta). Both alpha and beta importins are expressed in the brain, and their expression and localization is regulated by physiological neuronal activity. Little is known about regulation of importin expression in brain pathological conditions. Here we studied the expression of importin beta1 (imp beta 1) in the rat hippocampus after acute and chronic seizures induced by the glutamate agonist kainic acid (KA). The overall content of imp beta 1 mRNA and protein did not change after acute KA seizures. However, acute KA seizures rapidly induced the translocation of imp beta 1 protein from the cytoplasm to the nucleus in pyramidal CA1 neurons. KA-induced imp beta 1 translocation was prevented by the NMDA (N-methyl-D-aspartic acid) receptor blocker MK-801. After chronic seizures, the overall levels of imp beta 1 mRNA and protein did not change in the whole hippocampus. Immunohistochemistry revealed a massive loss of imp beta 1-positive neurons in pyramidal layers (that degenerated after KA), whereas an increased number of imp beta 1-positive cells was detected in the stratum radiatum of rats with chronic seizures compared with control animals. Double-labeling experiments identified these cells as glial cells expressing the chondroitin sulfate proteoglycan NG2 (neuron/glial antigen 2), a glial subtype recently shown to regulate hippocampal neuron excitability. These data show a differential regulation of imp beta 1 expression after acute and chronic seizure activity in the rat hippocampus.

In the present study, we demonstrate a direct role for d-aspartate in regulating hippocampal synaptic plasticity. These evidences were obtained using two different experimental strategies which enabled a non-physiological increase of endogenous d-aspartate levels in the mouse hippocampus: a genetic approach based on the targeted deletion of d-aspartate oxidase gene and another based on the oral administration of d-aspartate. Overall, our results indicate that increased d-aspartate content does not affect basal properties of synaptic transmission but enhances long-term potentiation in hippocampal slices from both genetic and pharmacological animal models. Besides electrophysiological data, behavioral analysis suggests that altered levels of d-aspartate in the hippocampus do not perturb basal spatial learning and memory abilities, but may selectively interfere with the dynamic NMDAR-dependent processes underlying cognitive flexibility.

Sleep has traditionally been recognized as a precipitating factor for some forms of epilepsy, although differential diagnosis between some seizure types and parasomnias may be difficult. Autosomal dominant frontal lobe epilepsy is characterized by nocturnal seizures with hyperkinetic automatisms and poorly organized stereotyped movements and has been associated with mutations of the alpha 4 and beta 2 subunits of the neuronal nicotinic acetylcholine receptor. We performed a clinical and molecular genetic study of a large pedigree segregating sleep-related epilepsy in which seizures are associated with fear sensation, tongue movements, and nocturnal wandering, closely resembling nightmares and sleep walking. We identified a new genetic locus for familial sleep-related focal epilepsy on chromosome 8p12.3-8q12.3. By sequencing the positional candidate neuronal cholinergic receptor alpha 2 subunit gene (CHRNA2), we detected a heterozygous missense mutation, I279N, in the first transmembrane domain that is crucial for receptor function. Whole-cell recordings of transiently transfected HEK293 cells expressing either the mutant or the wild-type receptor showed that the new CHRNA2 mutation markedly increases the receptor sensitivity to acetylcholine, therefore indicating that the nicotinic alpha 2 subunit alteration is the underlying cause. CHRNA2 is the third neuronal cholinergic receptor gene to be associated with familial sleep-related epilepsies. Compared with the CHRNA4 and CHRNB2 mutations reported elsewhere, CHRNA2 mutations cause a more complex and finalized ictal behavior.