Polyacrylic-Coated Solid Nanoparticles Increase the Aquaporin Permeability to Hydrogen Peroxide.

Aquaporins (AQPs) allow the diffusion of hydrogen peroxide (H2O2) and act as ROS scavenging systems, which are important for controlling the redox state of cells. Recently, cerium oxide nanoparticles were found to increase the water and H2O2 permeability by modulating AQPs. To further analyze the action of nanoparticles (NPs) on AQP, we examined the effect of the NPs presenting different core compositions (CeO2, Gd2O3, Fe3O4, and TiO2), hydrodynamic sizes, and surface functionalization. The NPs produced an increase in H2O and H2O2 permeability as a general trend. The hydrodynamic sizes of the NPs in the range of 22-100 nm did not produce any significant effect. The chemical nature of the NPs' core did not modify the effect and its intensity. On the other hand, the NPs' functionalized surface plays a major role in influencing both water and H2O2 permeability. The results suggest that NPs can play a significant role in controlling oxidative stress in cells and might represent an innovative approach in the treatment of a number of pathologies associated with an increased oxidative status.

Oxytocin Prevents the Development of 3-NP-Induced Anxiety and Depression in Male and Female Rats: Possible Interaction of OXTR and mGluR2.

Huntington disease (HD) is a progressive neurological disorder with dominant motor symptoms. It also has psychiatric manifestations, like anxiety and depression, that can emerge themselves before motor symptoms and impose a major burden on patients. Oxytocin (OXT) is a newly emerged treatment for disorders like autism and schizophrenia and recently is using to alleviate depression and anxiety. In the current study, we investigated the behavioral and molecular effects of OXT on the development of anxiety and depression in 3-nitropropionic acid (3-NP)-induced model of HD. Anxiety- and depression-like behaviors as well as the levels of oxytocin receptor (OXTR), metabotropic glutamate receptor (mGluR) 2, mGluR5, and glutathione (GSH) were measured in striatum, hippocampus, prefrontal cortex, and amygdala. Also, we questioned if sex had any modulatory effect. We found that 3-NP increased anxiety and depression compared to controls. It also reduced the levels of OXTR and mGluR2, increased mGluR5, and reduced GSH in studied brain regions. Pretreatment with OXT before the injection of 3-NP ameliorated anxiety and depression. Additionally, it protected the brain from developing low levels of OXTR, mGluR2, and GSH and high levels of mGluR5 in studied regions. The protective effects of OXT were similar between male and female animals. These data suggest that OXTR, mGluR2, mGluR5, and GSH may contribute to psychiatric manifestations of HD. In addition, pretreatment with OXT could prevent the mood changes in male and female rats.

Melanocortin 4 receptor activation protects striatal neurons and glial cells from 3-nitropropionic acid toxicity.

α-Melanocyte stimulating hormone (α-MSH) is a melanocortin which exerts potent anti-inflammatory and anti-apoptotic effects. Melanocortin 4 receptors (MC4R) are abundantly expressed in the brain and we previously demonstrated that [Nle(4), D-Phe(7)]melanocyte-stimulating hormone (NDP-MSH), an α-MSH analogue, increased expression of brain derived-neurotrophic factor (BDNF), and peroxisome proliferator-activated receptor-γ (PPAR-γ). We hypothesized that melanocortins could affect striatal cell survival through BDNF and PPAR-γ. First, we determined the expression of these factors in the striatum. Acute intraperitoneal administration (0.5 mg/kg) of α-MSH increased the levels of BDNF mRNA in rat striatum but not in rat cerebral cortex. Also, protein expression of PPAR-γ and MC4R was increased by acute treatment with α-MSH in striatum but not in cortex. No changes were observed by 48 h treatment. Next, we evaluated melanocortins effect on neuron and glial survival. 3-nitropropionic acid (3-NP), which is known to induce striatal degeneration, was used to induce cell death in the rat striatal cell line ST14A expressing mutant human huntingtin (Q120) or in ST14A cells expressing normal human huntingtin (Q15), in primary cultured astrocytes, and in BV2 cells. NDP-MSH protected Q15 cells, astrocytes and BV2 cells from death by 3-NP whereas it did not fully protect Q120 cells. Protection of Q15 cells and astrocytes was blocked by a MC4R specific inhibitor (JKC-363) and a PPAR-γ antagonist (GW9662). The BDNF receptor antagonist (ANA-12) abolished NDP-MSH protective effect in astrocytes but not in Q15 cells. We demonstrate for the first time that melanocortins, acting through PPAR-γ and BDNF, protect neurons and glial cells from 3-NP toxicity.

Osteopontin and its spatiotemporal relationship with glial cells in the striatum of rats treated with mitochondrial toxin 3-nitropropionic acid: possible involvement in phagocytosis.

BACKGROUND: Osteopontin (OPN, SPP1) is upregulated in response to acute brain injury, and based on its immunoreactivity, two distinct forms have been identified: intracellular OPN within brain macrophages and small granular OPN, identified as OPN-coated degenerated neurites. This study investigates the spatiotemporal relationship between punctate OPN deposition and astroglial and microglial reactions elicited by 3-nitropropionic acid (3-NP). METHODS: Male Sprague-Dawley rats were intraperitoneally injected with mitochondrial toxin 3-NP and euthanized at 3, 7, 14, and 28 days. Quantitative and qualitative light and electron microscopic techniques were used to assess the relationship between OPN and glial cells. Statistical significance was determined by Student's t test or a one-way analysis of variance followed by Tukey's multiple comparisons test. RESULTS: Punctate OPN-immunoreactive profiles were synthesized and secreted by amoeboid-like brain macrophages in the lesion core, but not by reactive astrocytes and activated microglia with a stellate shape in the peri-lesional area. Punctate OPN accumulation was detected only in the lesion core away from reactive astrocytes in the peri-lesional area at day 3, but had direct contact with, and even overlapped with astroglial processes at day 7. The distance between the OPN-positive area and the astrocytic scar significantly decreased from days 3 to 7. By days 14 and 28 post-lesion, when the glial scar was fully formed, punctate OPN distribution mostly overlapped with the astrocytic scar. Three-dimensional reconstructions and quantitative image analysis revealed numerous granular OPN puncta inside the cytoplasm of reactive astrocytes and brain macrophages. Reactive astrocytes showed prominent expression of the lysosomal marker lysosomal-associated membrane protein 1, and ultrastructural analysis confirmed OPN-coated degenerating neurites inside astrocytes, suggesting the phagocytosis of OPN puncta by reactive astrocytes after injury. CONCLUSIONS: Punctate OPN-immunoreactive profiles corresponded to OPN-coated degenerated neurites, which were closely associated with, or completely engulfed by, the reactive astrocytes forming the astroglial scar in 3-NP lesioned striatum, suggesting that OPN may cause astrocytes to migrate towards these degenerated neurites in the lesion core to establish physical contact with, and possibly, to phagocytose them. Our results provide novel insights essential to understanding the recovery and repair of the central nervous system tissue.

Progressive accumulation of autofluorescent granules in macrophages in rat striatum after systemic 3-nitropropionic acid: a correlative light- and electron-microscopic study.

A variety of tissue biomolecules and intracellular structures are known to be autofluorescent. However, autofluorescent signals in brain tissues often confound analysis of the fluorescent markers used for immunohistochemistry. While investigating tissue and cellular pathologies induced by 3-nitropropionic acid, a mitochondrial toxin selective for striatal neurons, we encountered many autofluorescent signals confined to the lesion core. These structures were excited by blue (wavelength = 488 nm) and yellow-orange (555 nm), but not by red (639 nm) or violet (405 nm) lasers, indicating that this autofluorescence overlaps with the emission spectra of commonly used fluorophores. Almost all of the autofluorescence was localized in activated microglia/macrophages, while reactive astrocytes emitted no detectable autofluorescence. Amoeboid brain macrophages filled with autofluorescent granules revealed very weak expression of the microglial marker, ionized calcium-binding adaptor molecule 1 (Iba1), while activated microglia with evident processes and intense Iba1 immunoreactivity contained scant autofluorescent granules. In addition, immunolabeling with two lysosomal markers, ED1/CD68 and lysosomal-associated membrane protein 1, showed a pattern complementary with autofluorescent signals in activated microglia/macrophages, implying that the autofluorescent structures reside within cytoplasm free of intact lysosomes. A correlative light- and electron-microscopic approach finally revealed the ultrastructural identity of the fluorescent granules, most of which matched to clusters of lipofuscin-like inclusions with varying morphology. Thus, autofluorescence in the damaged brain may reflect the presence of lipofuscin-laden brain macrophages, which should be taken into account when verifying any fluorescent signals that are likely to be correlated with activated microglia/macrophages after brain insults.

Oncostatin M-dependent Mcl-1 induction mediated by JAK1/2-STAT1/3 and CREB contributes to bioenergetic improvements and protective effects against mitochondrial dysfunction in cortical neurons.

Oncostatin M (OSM), a cytokine in the interleukin-6 (IL-6) family, has been proposed to play a protective role in the central nervous system, such as attenuation of excitotoxicity induced by N-methyl-D-aspartate (NMDA) and glutamate. However, the potential neuroprotective effects of OSM against mitochondrial dysfunction have never been reported. In the present study, we tested the hypothesis that OSM may confer neuronal resistance against 3-nitropropionic acid (3-NP), a plant toxin that irreversibly inhibits the complex II of the mitochondrial electron transport chain, and characterized the underlying molecular mechanisms. We found that OSM preconditioning dose- and time-dependently protected cortical neurons against 3-NP toxicity. OSM stimulated expression of myeloid cell leukemia-1 (Mcl-1), an anti-apoptotic Bcl-2 family member expressed in differentiating myeloid cells, that required prior phosphorylation of Janus kinase-1 (JAK1), JAK2, extracellular signal-regulated kinase-1/2 (ERK1/2), signal transducer and activator of transcription-3 (STAT3), STAT1, and cAMP-response element-binding protein (CREB). Pharmacological inhibitors of JAK1, JAK2, ERK1/2, STAT3, STAT1, and CREB as well as the siRNA targeting at STAT3 and Mcl-1 all abolished OSM-dependent 3-NP resistance. Finally, OSM-dependent Mcl-1 induction contributed to the enhancements of mitochondrial bioenergetics including increases in spare respiratory capacity and ATP production. In conclusion, our findings indicated that OSM induces Mcl-1 expression via activation of ERK1/2, JAK1/2, STAT1/3, and CREB; furthermore, OSM-mediated Mcl-1 induction contributes to bioenergetic improvements and neuroprotective effects against 3-NP toxicity in cortical neurons. OSM may thus serve as a novel neuroprotective agent against mitochondrial dysfunction commonly associated with pathogenic mechanisms underlying neurodegeneration.

Characteristic Changes of Astrocyte and Microglia in Rat Striatum Induced by 3-NP and MCAO.

Our previous studies had confirmed that both 3-NP and MCAO induced the behavioral defect as well as striatal neuronal injury and loss in experimental rats. This study aimed to examine different response forms of striatal astrocyte and microglia in 3-NP and MCAO rat models. The present results showed that the immunoreaction for GFAP was extremely weak in the lesioned core of striatum, but in the transition zone of 3-NP model and the penumbra zone of MCAO model, GFAP+ cells showed strong hypertrophic and proliferative changes. Statistical analysis for the number, size and integral optical density (IOD) of GFAP+ cells showed significant differences when compared with their controls and compared between the core and the transition zone or the penumbra zone, respectively, but no differences between the 3-NP and MCAO groups. However, Iba-1+ cells showed obvious hypertrophy and proliferation in the injured striatum in the 3-NP and the MCAO models, especially in the transition zone of 3-NP model and the penumbra zone of MCAO model. These Iba-1+ cells displayed two characteristic forms as branching cells with thick processes and amoeboid cells with thin processes. Statistical analysis showed that the number, size and IOD of Iba-1+ cells were significantly increased in the cores and the transition zone of 3-NP group and the penumbra zone of MCAO group than that of the controls, and the immune response of Iba-1 was stronger in the MCAO group than in the 3-NP group. The present results suggested that characteristic responses of astrocyte and microglia in the 3-NP and the MCAO models display their different effects on the pathological process of brain injury.

Forkhead transcription factor FOXO3a levels are increased in Huntington disease because of overactivated positive autofeedback loop.

Huntington disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by an increased number of CAG repeats in the HTT gene coding for huntingtin. Decreased neurotrophic support and increased mitochondrial and excitotoxic stress have been reported in HD striatal and cortical neurons. The members of the class O forkhead (FOXO) transcription factor family, including FOXO3a, act as sensor proteins that are activated upon decreased survival signals and/or increased cellular stress. Using immunocytochemical screening in mouse striatal Hdh(7/7) (wild type), Hdh(7/109) (heterozygous for HD mutation), and Hdh(109/109) (homozygous for HD mutation) cells, we identified FOXO3a as a differentially regulated transcription factor in HD. We report increased nuclear FOXO3a levels in mutant Hdh cells. Additionally, we show that treatment with mitochondrial toxin 3-nitropropionic acid results in enhanced nuclear localization of FOXO3a in wild type Hdh(7/7) cells and in rat primary cortical neurons. Furthermore, mRNA levels of Foxo3a are increased in mutant Hdh cells compared with wild type cells and in 3-nitropropionic acid-treated primary neurons compared with untreated neurons. A similar increase was observed in the cortex of R6/2 mice and HD patient post-mortem caudate tissue compared with controls. Using chromatin immunoprecipitation and reporter assays, we demonstrate that FOXO3a regulates its own transcription by binding to the conserved response element in Foxo3a promoter. Altogether, the findings of this study suggest that FOXO3a levels are increased in HD cells as a result of overactive positive feedback loop.

Mitochondrial impairment and oxidative stress compromise autophagosomal degradation of α-synuclein in oligodendroglial cells.

α-Synuclein (α-syn)-containing glial cytoplasmic inclusions originating in oligodendrocytes are characteristically observed in multiple system atrophy. The mechanisms of glial cytoplasmic inclusion formation remain rather elusive. α-Syn over-expression, uptake from the environment, oxidative stress or impairment of the proteolytic degradation systems have been discussed. Here, we investigated whether in oligodendrocytes autophagy plays a major role in the degradation and aggregation of endogenously expressed α-syn and of α-syn taken up from the extracellular environment. Furthermore, we studied whether in cells with impaired mitochondria the accumulation and aggregation of exogenously added α-syn is promoted. Using primary cultures of rat brain oligodendrocytes and an oligodendroglial cell line, genetically engineered to express green fluorescent protein-microtubule-associated light chain 3 with or without α-syn to monitor the autophagic flux, we demonstrate that both exogenously applied α-syn and α-syn stably expressed endogenously are effectively degraded by autophagy and do not affect the autophagic flux per se. Mitochondrial impairment with the protonophore carbonyl cyanide 3-chlorophenylhydrazone or 3-nitropropionic acid disturbs the autophagic pathway and leads to the accumulation of exogenously applied α-syn and enhances its propensity to form aggregates intracellularly. Thus, mitochondrial dysfunction and oxidative stress, which occur over time and are significant pathological features in synucleinopathies, have an impact on the autophagic pathway and participate in pathogenesis. Glial cytoplasmic inclusions are characteristically observed in multiple system atrophy, their occurrence might be related to failure in protein degradation systems. Here, we show that in oligodendrocytes autophagy is the major route of α-synuclein degradation which is either endogenously expressed or added exogenously (1, 2). Mitochondrial impairment (3) disturbs the autophagic flux and leads to the accumulation of exogenously applied α-synuclein, and enhances its propensity to form aggregates intracellulary (4).

Copper mediated follicular atresia: Implications for granulosa cell death.

3-nitropropanoic acid is a potent oxidative stress inducer that is conventionally regarded as a regulator of follicular atresia by regulating granulosa cells (GCs) death through the apoptosis pathway. There has been no research investigating the impact of copper metal overload induced Cuproptosis in ovarian GCs as a factor contributing to hindered follicular development.To elucidate whether 3-NP-induced oxidative stress plays a contributory role in promoting Cuproptosis, and discuss the role of Cuproptosis in the development of ovarian follicles.We conducted an analysis of cuproptosis occurrence in murine GCs and C57BL/6 J mice under the influence of 3-NP and 3-NP with added exogenous copper.The results revealed that 3-NP serving as a robust facilitator of exogenous copper uptake by upregulating the expression of copper transporter 1 (CTR1). In turn, culminated in the accumulation of intracellular copper within mouse granulosa cells (mGCs). Furthermore, 3-NP promoted mitochondrial permeability transition pore opening and concurrently reduced the stability of lipoic acid proteins. These actions collectively induced the oligomerization of Dihydrolipoamide S-Acetyltransferase (DLAT), ultimately leading to cuproptosis in GCs and consequent follicular atresia. Heavy metal copper and fungal decomposition product 3-NP, induce ovarian atresia via cuproptosis, modulating the reproductive performance of female animals.

Neuroprotective effect of human cord blood-derived extracellular vesicles by improved neuromuscular function and reduced gliosis in a rat model of Huntington's disease.

Huntington's disease (HD) is a hereditary condition characterized by the gradual deterioration of nerve cells in the striatum. Recent scientific investigations have revealed the promising potential of Extracellular vesicles (EVs) as a therapy to mitigate inflammation and enhance motor function. This study aimed to examine the impact of administering EVs derived from human umbilical cord blood (HUCB) on the motor abilities and inflammation levels in a rat model of HD. After ultracentrifugation to prepare EVs from HUCB to determine the nature of the obtained contents, the expression of CD markers 81 and 9, the average size and also the morphology of its particles were investigated by DLS and Transmission electron microscopy (TEM). Then, in order to induce the HD model, 3-nitropropionic acid (3-NP) neurotoxin was injected intraperitoneal into the rats, after treatment by HUCB-EVs, rotarod, electromyogram (EMG) and the open field tests were performed on the rats. Finally, after rat sacrifice and the striatum was removed, Hematoxylin and eosin staining (H&E), stereology, immunohistochemistry, antioxidant tests, and western blot were performed. Our results showed that the contents of the HUCB-EVs express the CD9 and CD81 markers and have spherical shapes. In addition, the injection of HUCB-EVs improved motor and neuromuscular function, reduced gliosis, increased antioxidant activity and inflammatory factor, and partially prevented the decrease of neurons. The findings generally show that HUCB-EVs have neuroprotective effects and reduce neuroinflammation from the toxic effects of 3-NP, which can be beneficial for the recovery of HD.

Muscle quality, physical performance, and comorbidity are predicted by circulating procollagen type III N-terminal peptide (P3NP): the InCHIANTI follow-up study.

Sarcopenia is characterized by skeletal muscle quantitative and qualitative alterations. A marker of collagen turnover, procollagen type III N-terminal peptide (P3NP), seems to be related to those conditions. This study aims to assess the predictive role of P3NP in muscle density and physical performance changes. In the InCHIANTI study, a representative sample from the registry lists of two towns in Tuscany, Italy, was recruited. Baseline data was collected in 1998, and follow-up visits were conducted every 3 years. Out of the 1453 participants enrolled at baseline, this study includes 1052 participants. According to P3NP median levels, population was clustered in two groups; 544 (51.7%) of the 1052 subjects included were classified in the low median levels (LM-P3NP); at the baseline, they were younger, had higher muscle density, and performed better at the Short Physical Performance Battery (SPPB), compared to the high-median group (HM-P3NP).LM-P3NP cases showed a lower risk to develop liver chronic diseases, CHF, myocardial infarction, and osteoarthritis. HM-P3NP levels were associated with a longitudinal reduction of muscle density, and this effect was potentiated by the interaction between P3NP and leptin. Moreover, variation in physical performance was inversely associated with high level of P3NP, and directly associated with high fat mass, and with the interaction between P3NP and muscle density. Our data indicate that P3NP is associated with the aging process, affecting body composition, physical performance, and clinical manifestations of chronic degenerative age-related diseases.

Bone Turnover Markers during Growth Hormone Therapy for Short Stature Children Born Small for Gestational Age.

Growth hormone therapy (GHT) can improve growth velocity and final height, but can also accelerate the process of bone growth, which is related to structural bone modeling in both formation and resorption. This study evaluated the capacity of bone turnover markers to predict early growth response to one year of GHT in short stature children born small for gestational age (SGA). This study included 25 prepubertal children born SGA. We estimated P1NP (N-terminal procollagen type 1), CTX (C-terminal telopeptide of collagen type 1), P3NP (N-terminal procollagen type 3), NT-pro-CNP (amino-terminal C-type natriuretic peptide) and Ca-P metabolism using standard ECLIA (electrochemiluminescence), RIA (radioimmunoassay), and ELISA (enzyme-linked immunosorbent assay) methods. A statistically significant increase in bone resorption markers (CTX) was found at both 6 and 12 months. P1NP bone markers were increased at 6 months and after 12 months of therapy. The P3NP marker for collagen synthesis also increased after 12 months of therapy. We obtained significant increases in phosphorus levels at 6 and 12 months, and similar ALP (alkaline phosphatase) increases. We found a significant correlation between height (cm) and CTX after 6-12 months, as well as a P1NP/height (SD) correlation after 12 months. Calcium levels significantly correlated with height (SD) after 12 months. We found strong reactions of bone resorption and bone formation markers during growth hormone therapy, which may determine their selection as predictors of GHT outcome in children born SGA. However, the issue requires further research.

Analytical Method Development and Dermal Absorption of 4-Amino-3-Nitrophenol (4A3NP), a Hair Dye Ingredient under the Oxidative or Non-Oxidative Condition.

The chemical 4-amino-3-nitrophenol (4A3NP) is classified as an amino nitrophenol and is primarily utilized as an ingredient in hair dye colorants. In Korea and Europe, it is exclusively used in non-oxidative or oxidative hair dye formulations, with maximum allowable concentrations of 1% and 1.5%, respectively. Despite this widespread use, risk assessment of 4A3NP has not been completed due to the lack of proper dermal absorption data. Therefore, in this study, both the analytical method validation and in vitro dermal absorption study of 4A3NP were conducted following the guidelines provided by the Korea Ministry of Food and Drug Safety (MFDS). Before proceeding with the dermal absorption study, analytical methods were developed for the quantitation of 4A3NP through multiple reaction monitoring (MRM) via liquid chromatography-mass spectrometry (LC-MS) in various matrices, including swab wash (WASH), stratum corneum (SC), skin (SKIN, comprising the dermis and epidermis), and receptor fluid (RF). These developed methods demonstrated excellent linearity (R2 = 0.9962-0.9993), accuracy (93.5-111.73%), and precision (1.7-14.46%) in accordance with the validation guidelines.The dermal absorption of 4A3NP was determined using Franz diffusion cells with mini-pig skin as the barrier. Under both non-oxidative and oxidative (6% hydrogen peroxide (H2O2): water, 1:1) hair dye conditions, 1% and 1.5% concentrations of 4A3NP were applied to the skin at a rate of 10 µL/cm2, respectively. The total dermal absorption rates of 4A3NP under non-oxidative (1%) and oxidative (1.5%) conditions were determined to be 5.62 ± 2.19% (5.62 ± 2.19 µg/cm2) and 2.83 ± 1.48% (4.24 ± 2.21 µg/cm2), respectively.

Biosynthesis of ZnO, Bi2O3 and ZnO-Bi2O3 bimetallic nanoparticles and their cytotoxic and antibacterial effects.

This work introduces an easy method for producing Bi2O3, ZnO, ZnO-Bi2O3 nanoparticles (NPs) by Biebersteinia Multifida extract. Our products have been characterized through the outcomes which recorded with using powder X-ray diffractometry (PXRD), Raman, energy dispersive X-ray (EDX), field emission-scanning electron microscopy (FE-SEM), and Fourier-transform infrared (FT-IR) techniques. The finding of SEM presented porous structure and spherical morphology for Bi2O3 and ZnO NPs, respectively. While FE-SEM image of bimetallic nanoparticles showed both porous and spherical morphologies for them; so that spherical particles of ZnO have sat on the porous structure of Bi2O3 NPs. According to the PXRD results, the crystallite sizes of Bi2O3, ZnO and ZnO-Bi2O3 NPs have been obtained 57.69, 21.93, and 43.42 nm, respectively. Antibacterial performance of NPs has been studied on Staphylococcus epidermidis and Pseudomonas aeruginosa bacteria, to distinguish the minimum microbial inhibitory concentration (MIC). Antimicrobial outcomes have showed a better effect for ZnO-Bi2O3 NPs. Besides, wondering about the cytotoxic action against cancer cell lines, the MTT results have verified the intense cytotoxic function versus breast cancer cells (MCF-7). According to these observations, obtained products can prosper medical and biological applications.

Neurotrophin-3 Rescues Striatal Synaptic Plasticity in Model of Neurodegeneration by PLC Signaling Activation.

BACKGROUND: Neurotrophins are essential factors for neural growth and function; they play a crucial role in neurodegenerative diseases where their expression levels are altered. Our previous research has demonstrated changes in synaptic plasticity and neurotrophin expression levels in a pharmacological model of Huntington's disease induced by 3-nitropropionic acid (3-NP). In the 3- NP-induced HD model, corticostriatal Long Term Depression (LTD) was impaired, but neurotrophin-3 (NT-3) restored striatal LTD. This study delves into the NT-3-induced signaling pathways involved in modulating and restoring striatal synaptic plasticity in cerebral slices from 3-NPinduced striatal degeneration in mice in vivo. METHODS: Phospholipase C (PLC), phosphatidylinositol-3-kinase (PI3K), and mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathways activated by NT-3 were analyzed by means of field electrophysiological recordings in brain slices from control and 3-NP treated in the presence of specific inhibitors of the signaling pathways. RESULTS: Using specific inhibitors, PLC, PI3K, and MEK/ERK signaling pathways contribute to NT3-mediated plasticity modulation in striatal tissue slices recorded from control animals. However, in the neurodegeneration model induced by 3-NP, the recovery of striatal LTD induced by NT-3 was prevented only by the PLC inhibitor. Moreover, the PLC signaling pathway appeared to trigger downstream activation of the endocannabinoid system, evidenced by AM 251, an inhibitor of the CB1 receptor, also hindered NT-3 plasticity recovery. CONCLUSION: Our finding highlights the specific involvement of the PLC pathway in the neuroprotective effects of NT-3 in mitigating synaptic dysfunction under neurodegenerative conditions.

Connection between Cardiac Fibrosis Biomarkers and Echocardiography Parameters in Advanced Chronic Kidney Disease Patients.

BACKGROUND: Myocardial fibrosis represents a mainstay pathway in the pathophysiology of uremic cardiomyopathy. This process leads to structural and functional changes in the heart, which can be detected by echocardiography. The purpose of our study was to determine the association between four echocardiographic parameters (ejection fraction (EF), global longitudinal strain (GLS), mean E/e' ratio, and left atrial volume indexed) and biomarkers associated with cardiac fibrosis, such as procollagen type I carboxy-terminal propeptide (PICP), procollagen type III N-terminal peptide (P3NP), and galectin-3 (Gal-3) in patients with end-stage renal disease (ESRD). METHODS: 140 patients with ESRD were enrolled and investigated by echocardiography and the serum levels of the aforementioned biomarkers were determined at baseline. RESULTS: The mean EF was 53.63 ± 8%, the mean GLS was -10.2 ± 5.3%, the mean E/e' ratio was 9.8 ± 4.3, and the mean left atrial volume indexed (LAVI) was 45.8 ± 14.2 mL/m2. The average levels for PICP, P3NP, and Gal-3 were 457.2 ± 240 µg/L, 242 ± 199.9 µg/L, and 10.7 ± 3.7 ng/mL, respectively. In regression analysis, PICP was strongly associated with all four echocardiographic parameters (EF: p = 0.0002, R2 = 0.69; GLS: p = 0.00001, R2 = 0.81; mean E/e': p = 0.00002; R2 = 0.89; LAVI: p = 0.003; R2 = 0.73). P3NP and Gal-3 were only associated with the EF (p = 0.01, R2 = 0.31 and p = 0.02; R2 = 0.35, respectively). CONCLUSION: Our study evidenced that PICP, a collagen-derived biomarker, is associated with important echocardiography parameters, suggesting that it can serve as an indicator of the presence of subclinical systolic and diastolic dysfunction in patients with advanced CKD.

Effect of pegbelfermin on NASH and fibrosis-related biomarkers and correlation with histological response in the FALCON 1 trial.

Background & Aims: FALCON 1 was a phase IIb study of pegbelfermin in patients with non-alcoholic steatohepatitis (NASH) and stage 3 fibrosis. This FALCON 1 post hoc analysis aimed to further assess the effect of pegbelfermin on NASH-related biomarkers, correlations between histological assessments and non-invasive biomarkers, and concordance between the week 24 histologically assessed primary endpoint response and biomarkers. Methods: Blood-based composite fibrosis scores, blood-based biomarkers, and imaging biomarkers were evaluated for patients with available data from FALCON 1 at baseline through week 24. SomaSignal tests assessed protein signatures of NASH steatosis, inflammation, ballooning, and fibrosis in blood. Linear mixed-effect models were fit for each biomarker. Correlations and concordance were assessed between blood-based biomarkers, imaging, and histological metrics. Results: At week 24, pegbelfermin significantly improved blood-based composite fibrosis scores (ELF, FIB-4, APRI), fibrogenesis biomarkers (PRO-C3 and PC3X), adiponectin, CK-18, hepatic fat fraction measured by MRI-proton density fat fraction, and all four SomaSignal NASH component tests. Correlation analyses between histological and non-invasive measures identified four main categories: steatosis/metabolism, tissue injury, fibrosis, and biopsy-based metrics. Concordant and discordant effects of pegbelfermin on the primary endpoint vs. biomarker responses were observed; the most clear and concordant effects were on measures of liver steatosis and metabolism. A significant association between hepatic fat measured histologically and by imaging was observed in pegbelfermin arms. Conclusions: Pegbelfermin improved NASH-related biomarkers most consistently through improvement of liver steatosis, though biomarkers of tissue injury/inflammation and fibrosis were also improved. Concordance analysis shows that non-invasive assessments of NASH support and exceed the improvements detected by liver biopsy, suggesting that greater consideration should be given to the totality of available data when evaluating the efficacy of NASH therapeutics. Clinical trial number: Post hoc analysis of NCT03486899. Impact and implications: FALCON 1 was a study of pegbelfermin vs. placebo in patients with non-alcoholic steatohepatitis (NASH) without cirrhosis; in this study, patients who responded to pegbelfermin treatment were identified through examination of liver fibrosis in tissue samples collected through biopsy. In the current analysis, non-invasive blood- and imaging-based measures of fibrosis, liver fat, and liver injury were used to determine pegbelfermin treatment response to see how they compared with the biopsy-based results. We found that many of the non-invasive tests, particularly those that measured liver fat, identified patients who responded to pegbelfermin treatment, consistent with the liver biopsy findings. These results suggest that there may be additional value in using data from non-invasive tests, along with liver biopsy, to evaluate how well patients with NASH respond to treatment.

A randomized, double-blind, placebo-controlled phase IIa trial of efruxifermin for patients with compensated NASH cirrhosis.

Background & Aims: Efruxifermin has shown clinical efficacy in patients with non-alcoholic steatohepatitis (NASH) and F1-F3 fibrosis. The primary objective of the BALANCED Cohort C was to assess the safety and tolerability of efruxifermin in patients with compensated NASH cirrhosis. Methods: Patients with NASH and stage 4 fibrosis (n = 30) were randomized 2:1 to receive efruxifermin 50 mg (n = 20) or placebo (n = 10) once-weekly for 16 weeks. The primary endpoint was safety and tolerability of efruxifermin. Secondary and exploratory endpoints included evaluation of non-invasive markers of liver injury and fibrosis, glucose and lipid metabolism, and changes in histology in a subset of patients who consented to end-of-study liver biopsy. Results: Efruxifermin was safe and well-tolerated; most adverse events (AEs) were grade 1 (n = 7, 23.3%) or grade 2 (n = 19, 63.3%). The most frequent AEs were gastrointestinal, including transient, mild to moderate diarrhea, and/or nausea. Significant improvements were noted in key markers of liver injury (alanine aminotransferase) and glucose and lipid metabolism. Sixteen-week treatment with efruxifermin was associated with significant reductions in non-invasive markers of fibrosis including Pro-C3 (least squares mean change from baseline [LSMCFB] -9 µg/L efruxifermin vs. -3.4 µg/L placebo; p = 0.0130) and ELF score (-0.4 efruxifermin vs. +0.4 placebo; p = 0.0036), with a trend towards reduced liver stiffness (LSMCFB -5.7 kPa efruxifermin vs. -1.1 kPa placebo; n.s.). Of 12 efruxifermin-treated patients with liver biopsy after 16 weeks, 4 (33%) achieved fibrosis improvement of at least one stage without worsening of NASH, while an additional 3 (25%) achieved resolution of NASH, compared to 0 of 5 placebo-treated patients. Conclusions: Efruxifermin appeared safe and well-tolerated with encouraging improvements in markers of liver injury, fibrosis, and glucose and lipid metabolism following 16 weeks of treatment, warranting confirmation in larger and longer term studies. Lay summary: Cirrhosis resulting from non-alcoholic steatohepatitis (NASH), the progressive form of non-alcoholic fatty liver disease, represents a major unmet medical need. Currently there are no approved drugs for the treatment of NASH. This proof-of-concept randomized, double-blind clinical trial demonstrated the potential therapeutic benefit of efruxifermin treatment compared to placebo in patients with cirrhosis due to NASH. Clinical Trial Number: NCT03976401.

Erythropoietin and Bacillus Calmette-Guérin Vaccination Mitigate 3-Nitropropionic Acid-Induced Huntington-like Disease in Rats by Modulating the PI3K/Akt/mTOR/P70S6K Pathway and Enhancing the Autophagy.

Oxidative stress and mitochondrial dysfunction are among the mechanisms expected to explain the pathogenesis of Huntington's disease. Erythropoietin (EPO) and the Bacillus Calmette-Guérin (BCG) vaccine have neuroprotective effects against neurodegenerative diseases; however, the full mechanisms of their action are currently unclear. Here, for the first time, we investigated the neuroprotective effect of BCG vaccination in Huntington-like disease induced by 3-nitropropionic acid (3-NP) and its combination with EPO. Male Wistar rats were randomized into five groups: saline-treated control; 3-NP group (20 mg/kg/day, i.p.) for 7 days; EPO-treated group (5000 IU/kg/day, i.p.) for 14 days after 3-NP administration; live BCG vaccine prophylactic group (5000 cfu/g, i.p.) 10 days prior to 3-NP administration; and live BCG vaccine (5000 cfu/g, i.p.) 10 days before 3-NP administration, followed by EPO treatment (5000 IU/kg/day, i.p.) for 14 days. In a histopathological examination, striatum neurodegeneration was evidenced in the 3-NP injected rats. Administration of 3-NP elevated the levels of p-PI3K, p-Akt, p-mTOR, p-P70S6K, BAX, malondialdehyde, nitric oxide, and cytochrome oxidase while reduced the levels of BCL-2, superoxide dismutase, reduced glutathione, and the autophagy marker microtubule-associated protein light chain 3 in the striatum. EPO and BCG ameliorated the biochemical, histopathological, and behavioral derangements induced by 3-NP, with prominent neuroprotection observed in rats administered the BCG prophylactic combined with EPO treatment. These results highlight the role played by EPO and BCG in the management of 3-NP-induced Huntington-like disease by inhibiting the PI3K/Akt/mTOR/P70S6K pathway and enhancing the autophagy.