ERß mediates sex-specific protection in the App-NL-G-F mouse model of Alzheimer's disease.

Menopausal loss of neuroprotective estrogen is thought to contribute to the sex differences in Alzheimer's disease (AD). Activation of estrogen receptor beta (ERß) can be clinically relevant since it avoids the negative systemic effects of ERα activation. However, very few studies have explored ERß-mediated neuroprotection in AD, and no information on its contribution to the sex differences in AD exists. In the present study we specifically explored the role of ERß in mediating sex-specific protection against AD pathology in the clinically relevant App NL-G-F knock-in mouse model of amyloidosis, and if surgical menopause (ovariectomy) modulates pathology in this model. We treated male and female App NL-G-F mice with the selective ERß agonist LY500307 and subset of the females was ovariectomized prior to treatment. Memory performance was assessed and a battery of biochemical assays were used to evaluate amyloid pathology and neuroinflammation. Primary microglial cultures from male and female wild-type and ERß-knockout mice were used to assess ERß's effect on microglial activation and phagocytosis. We find that ERß activation protects against amyloid pathology and cognitive decline in male and female App NL-G-F mice. Ovariectomy increased soluble amyloid beta (Aß) in cortex and insoluble Aß in hippocampus, but had otherwise limited effects on pathology. We further identify that ERß does not alter APP processing, but rather exerts its protection through amyloid scavenging that at least in part is mediated via microglia in a sex-specific manner. Combined, we provide new understanding to the sex differences in AD by demonstrating that ERß protects against AD pathology differently in males and females, warranting reassessment of ERß in combating AD.

Magnesium-ibogaine therapy in veterans with traumatic brain injuries.

Traumatic brain injury (TBI) is a leading cause of disability. Sequelae can include functional impairments and psychiatric syndromes such as post-traumatic stress disorder (PTSD), depression and anxiety. Special Operations Forces (SOF) veterans (SOVs) may be at an elevated risk for these complications, leading some to seek underexplored treatment alternatives such as the oneirogen ibogaine, a plant-derived compound known to interact with multiple neurotransmitter systems that has been studied primarily as a treatment for substance use disorders. Ibogaine has been associated with instances of fatal cardiac arrhythmia, but coadministration of magnesium may mitigate this concern. In the present study, we report a prospective observational study of the Magnesium-Ibogaine: the Stanford Traumatic Injury to the CNS protocol (MISTIC), provided together with complementary treatment modalities, in 30 male SOVs with predominantly mild TBI. We assessed changes in the World Health Organization Disability Assessment Schedule from baseline to immediately (primary outcome) and 1 month (secondary outcome) after treatment. Additional secondary outcomes included changes in PTSD (Clinician-Administered PTSD Scale for DSM-5), depression (Montgomery-Åsberg Depression Rating Scale) and anxiety (Hamilton Anxiety Rating Scale). MISTIC resulted in significant improvements in functioning both immediately (Pcorrected < 0.001, Cohen's d = 0.74) and 1 month (Pcorrected < 0.001, d = 2.20) after treatment and in PTSD (Pcorrected < 0.001, d = 2.54), depression (Pcorrected < 0.001, d = 2.80) and anxiety (Pcorrected < 0.001, d = 2.13) at 1 month after treatment. There were no unexpected or serious adverse events. Controlled clinical trials to assess safety and efficacy are needed to validate these initial open-label findings. ClinicalTrials.gov registration: NCT04313712 .

Case report: Ibogaine reduced severe neuropathic pain associated with a case of brachial plexus nerve root avulsion.

Brachial plexus nerve root avulsion results from complete separation of the nerve root from the spinal cord and is one of the most challenging types of neuropathic pain, coinciding with motor, sensory and autonomic deficits. The severe pain and typical impossibility of root reattachment often leads to requests for amputation. Ibogaine is an indole alkaloid producing psychoactive effects through reported actions upon multiple neurotransmitter systems, including NMDA, κ- and µ-opioid receptors and σ2 receptor sites, along with stimulation of neurotrophic factors GDNF and BDNF. In this case report we describe a 53-year-old male with two decades of severe intractable pain due to brachial plexus nerve root avulsion from vehicular trauma who was successfully treated with both high dose inpatient and low dose outpatient administrations of ibogaine. Though promising for future study, the adverse effects of high dose ibogaine administrations may limit tolerability of this saturation protocol to the most refractory cases.

Optimized single-cell RNA sequencing protocol to study early genome activation in mammalian preimplantation development.

Here, we present a modification of single-cell tagged reverse transcription protocol to study gene expression on a single-cell level or with limited RNA input. We describe different enzymes for reverse transcription and cDNA amplification, modified lysis buffer, and additional clean-up steps before cDNA amplification. We also detail an optimized single-cell RNA sequencing method for handpicked single cells, or tens to hundreds of cells, as input material to study mammalian preimplantation development. For complete details on the use and execution of this protocol, please refer to Ezer et al.1.

Neuroprotection against Aminochrome Neurotoxicity: Glutathione Transferase M2-2 and DT-Diaphorase.

Glutathione is an important antioxidant that plays a crucial role in the cellular protection against oxidative stress and detoxification of electrophilic mutagens, and carcinogens. Glutathione transferases are enzymes catalyzing glutathione-dependent reactions that lead to inactivation and conjugation of toxic compounds, processes followed by subsequent excretion of the detoxified products. Degeneration and loss of neuromelanin-containing dopaminergic neurons in the nigrostriatal neurons generally involves oxidative stress, neuroinflammation, alpha-synuclein aggregation to neurotoxic oligomers, mitochondrial dysfunction, protein degradation dysfunction, and endoplasmic reticulum stress. However, it is still unclear what triggers these neurodegenerative processes. It has been reported that aminochrome may elicit all of these mechanisms and, interestingly, aminochrome is formed inside neuromelanin-containing dopaminergic neurons during neuromelanin synthesis. Aminochrome is a neurotoxic ortho-quinone formed in neuromelanin synthesis. However, it seems paradoxical that the neurotoxin aminochrome is generated during neuromelanin synthesis, even though healthy seniors have these neurons intact when they die. The explanation of this paradox is the existence of protective tools against aminochrome neurotoxicity composed of the enzymes DT-diaphorase, expressed in these neurons, and glutathione transferase M2-2, expressed in astrocytes. Recently, it has been reported that dopaminergic neurons can be protected by glutathione transferase M2-2 from astrocytes, which secrete exosomes containing the protective enzyme.

Effects of Oral Pirfenidone on Colon Anastomosis Healing and Adhesion Formation in Rats.

INTRODUCTION: Many experimental studies have examined multiple drugs or treatments to improve the healing of intestinal anastomoses. Synthetic prostacyclin analogs, immunosuppressants, erythropoietin, growth hormone, insulin-like growth factor type 1, synthetic metalloproteinases inhibitors, and hyperbaric oxygen therapy have produced promising results in low-risk models of anastomosis dehiscence. However, in high-risk models, only hyperbaric oxygen therapy has been shown to be useful. Pirfenidone (PFD), a commonly used antifibrosing drug, has not been shown to be effective for this purpose. Our objective was to evaluate the effects of PFD on anastomosis healing and adhesion genesis in a low-risk rat model of dehiscence of colonic anastomosis. METHODS: An experimental study was conducted on 40 healthy Wistar rats randomly assigned to the control group or PFD experimental group (20 rats in each group). Colon anastomosis was performed 3 cm above the peritoneal reflection using the same technique in all animals. Mechanical resistance was studied by measuring bursting pressure. Adhesions were evaluated macroscopic and histologically using common staining techniques. Animals received the first PFD dose 12 h after surgery at a dose of 500 mg/kg one a day (SID) for 5 consecutive days. On day 6, the animals were reoperated on to measure the bursting pressure in situ and to classify adhesions macroscopically, and the anastomosed colon was resected for histological analysis. RESULTS: There were no deaths, complications, or anastomosis dehiscence in either group. The mean bursting pressure was 120.8 ± 11 mm Hg and 135.5 ± 12.4 in the control and PFD groups, respectively (p < 0.001). The adhesions were less dense and had less inflammatory cell infiltration in the PFD group (p < 0.02 and 0.002, respectively). Collagen content was slightly higher in the PFD group (p = 0.04). CONCLUSIONS: Our results revealed favorable effects of PFD in this low-risk colon anastomosis model; for example, the bursting pressure was higher, and the macroscopic adhesions were soft and exhibited less inflammatory infiltration and higher collagen content in the PFD group than in the control group. The results showing that PFD treatment was associated with better healing of minor adhesions seem to be paradoxical because the therapeutic indications for this drug are directed at treating fibrosing diseases.

Astrocytes protect dopaminergic neurons against aminochrome neurotoxicity.

Astrocytes protect neurons by modulating neuronal function and survival. Astrocytes support neurons in several ways. They provide energy through the astrocyte-neuron lactate shuttle, protect neurons from excitotoxicity, and internalize neuronal lipid droplets to degrade fatty acids for neuronal metabolic and synaptic support, as well as by their high capacity for glutamate uptake and the conversion of glutamate to glutamine. A recent reported astrocyte system for protection of dopamine neurons against the neurotoxic products of dopamine, such as aminochrome and other o-quinones, were generated under neuromelanin synthesis by oxidizing dopamine catechol structure. Astrocytes secrete glutathione transferase M2-2 through exosomes that transport this enzyme into dopaminergic neurons to protect these neurons against aminochrome neurotoxicity. The role of this new astrocyte protective mechanism in Parkinson´s disease is discussed.

Estrogen receptor beta and neural development.

The female sex hormone estradiol (E2, 17ß-estradiol) has important functions in the developing brain. In addition to regulating sexual differentiation of the brain, E2 participates in the development of brain areas involved in functions unrelated to reproduction, such as cognition. E2 signals mainly thorough two estrogen receptors; estrogen receptor alpha (ERα) and beta (ERß). While ERα has distinct functions for sexual imprinting of the developing brain, ERß is considered to participate in the development of brain areas related to cognitive function. In this chapter we will focus on ERß's role during neural development. We will discuss the contributions of sex chromosomal and sex hormonal effects in this process and place it in relation to recent data on ERß obtained from stem cell models. Finally, we will discuss the lessons learned from mouse and stem cell models in understanding ERß's role in neural development and how new stem cell models, by addressing the human relevance, may help to advance our progress in this field.

Cellular Trafficking of Glutathione Transferase M2-2 Between U373MG and SHSY-S7 Cells is Mediated by Exosomes.

The enzyme glutathione transferase M2-2, expressed in human astrocytes, increases its expression in the presence of aminochrome and catalyzes the conjugation of aminochrome, preventing its toxic effects. Secretion of the enzyme glutathione transferase M2-2 from U373MG cells, used as a cellular model for astrocytes, has been reported, and the enzyme is taken up by neuroblastoma SYSH-S7 cells and provide protection against aminochrome. The present study provides evidence that glutathione transferase M2-2 is released in exosomes from U373MG cells, thereby providing a means for intercellular transport of the enzyme. With particular relevance to Parkinson disease and other degenerative conditions, we propose a new mechanism by which astrocytes may protect dopaminergic neurons against the endogenous neurotoxin aminochrome.

Hematopoietic stem cell- and induced pluripotent stem cell-derived CAR-NK cells as reliable cell-based therapy solutions.

Hematopoietic stem cell- (HSC) and induced pluripotent stem (iPS) cell-derived natural killer (NK) cells containing engineered functions, such as chimeric antigen receptors (CAR), offer great promise for the treatment of seemingly incurable oncological malignancies. Today, some of the main challenges of CAR cell-based therapeutics are the long manufacturing time and safety of the cell sources used. Additional challenges include avoiding graft vs host disease (GVHD) and cytokine release syndrome (CRS). Here, we show compelling evidence for the use of NK cell therapeutics as a reliable off-the-shelf option, as they address key issues. Furthermore, we highlight how iPS cells and directed differentiation toward HSC and NK cells address industrial scalability and safety.

Generation of nonviral integration-free human iPS cell line KISCOi001-A from normal human fibroblasts, under defined xeno-free and feeder-free conditions.

KISCOi001-A is a healthy feeder-free and fully characterized human induced pluripotent stem (iPS) cell line cultured under xeno-free and defined conditions. The cell line is generated from normal human foreskin fibroblasts with non-integrating episomal plasmid vectors encoding OCT4, SOX2, KLF4, NANOG, LIN28, nontransforming L-MYC and dominant negative p53. The generated iPS cells are transgene-free and their pluripotency is confirmed by the expression of stem cell markers and capacity to differentiate into the cells of ectoderm, endoderm and mesoderm while their identity and karyotype stability is confirmed with Genomic assays.

Motor Function Deficits in the Estrogen Receptor Beta Knockout Mouse: Role on Excitatory Neurotransmission and Myelination in the Motor Cortex.

BACKGROUND: Male estrogen receptor beta (ERß) knockout (BERKO) mice display anxiety and aggression linked to, among others, altered serotonergic signaling in the basolateral amygdala and dorsal raphe, impaired cortical radial glia migration, and reduced GABAergic signaling. The effects on primary motor cortex (M1 cortex) and locomotor activity as a consequence of ERß loss have not been investigated. OBJECTIVE: The aim of this study was to determine whether locomotor activity is altered as a consequence of the changes in the M1 cortex. METHODS: The locomotor activity of male wild-type (WT) and BERKO mice was evaluated using the open-field and rotarod tests. Molecular changes in the M1 cortex were analyzed by RNA sequencing, electron microscopy, electrophysiology, and immunohistological techniques. In addition, we established oligodendrocyte (OL) cultures from WT and BERKO mouse embryonic stem cells to evaluate OL function. RESULTS: Locomotor profiling revealed that BERKO mice were more active than WT mice but had impaired motor coordination. Analysis of the M1 cortex pointed out differences in synapse function and myelination. There was a reduction in GABAergic signaling resulting in imbalanced excitatory and inhibitory neurotransmission as well as a defective OL differentiation accompanied by myelin defects. The effects of ERß loss on OL differentiation were confirmed in vitro. CONCLUSION: ERß is an important regulator of GABAergic interneurons and OL differentiation, which impacts on adult M1 cortex function and may be linked to increased locomotor activity and decreased motor coordination in BERKO mice.

Suppression of lymphoma growth by the xenoestrogens bisphenol A and genistein.

Well-defined physiological functions of estrogens are mediated via nuclear estrogen receptors α (ESR1) and ß (ESR2). With regard to hematological malignancies, expression of ESR2 has been found in both B and T cell lymphomas. In addition to endogenous estrogens or selective ESR2 agonists, ESR2 signaling may be affected by both environmental synthetic estrogen-mimicking compounds and dietary phytoestrogens. In the present study, we demonstrate that oral exposure with either the synthetic compound bisphenol A (BPA) or the dietary phytoestrogen genistein reduced the growth of grafted murine T cell (EG7) and human B cell (Granta-519 mantle cell) lymphomas which both express ESR2. Suppression of lymphoma growth was due to reduced proliferation (BPA and genistein) and induction of apoptosis (genistein). Inhibition of lymphoma growth was seen at a BPA dose of 50 µg/kg body weight (BW)/day considered to be safe human exposure dose or a genistein dose of 1 mg/kg BW/day orally, which is reached in soy-rich diets. Thus, our study indicates that the environmental xenoestrogens BPA and genistein have anti-proliferative effects on ESR2-expressing lymphomas. Our data suggest that phytoestrogens may be considered as a dietary supplement for lymphoma patients and possibly for prevention of lymphoid malignancies.

Fluoxetine Affects Differentiation of Midbrain Dopaminergic Neurons In Vitro.

Recent meta-analyses found an association between prenatal exposure to the antidepressant fluoxetine (FLX) and an increased risk of autism in children. This developmental disorder has been related to dysfunctions in the brains' rewards circuitry, which, in turn, has been linked to dysfunctions in dopaminergic (DA) signaling. The present study investigated if FLX affects processes involved in dopaminergic neuronal differentiation. Mouse neuronal precursors were differentiated into midbrain dopaminergic precursor cells (mDPCs) and concomitantly exposed to clinically relevant doses of FLX. Subsequently, dopaminergic precursors were evaluated for expression of differentiation and stemness markers using quantitative polymerase chain reaction. FLX treatment led to increases in early regional specification markers orthodenticle homeobox 2 (Otx2) and homeobox engrailed-1 and -2 (En1 and En2). On the other hand, two transcription factors essential for midbrain dopaminergic (mDA) neurogenesis, LIM homeobox transcription factor 1 α (Lmx1a) and paired-like homeodomain transcription factor 3 (Pitx3) were downregulated by FLX treatment. The stemness marker nestin (Nes) was increased, whereas the neuronal differentiation marker ß3-tubulin (Tubb3) decreased. Additionally, we observed that FLX modulates the expression of several genes associated with autism spectrum disorder and downregulates the estrogen receptors (ERs) α and ß Further investigations using ERß knockout (BERKO) mDPCs showed that FLX had no or even opposite effects on several of the genes analyzed. These findings suggest that FLX affects differentiation of the dopaminergic system by increasing production of dopaminergic precursors, yet decreasing their maturation, partly via interference with the estrogen system.

Role of estrogen receptor beta in neural differentiation of mouse embryonic stem cells.

The ability to propagate mature cells and tissue from pluripotent stem cells offers enormous promise for treating many diseases, including neurodegenerative diseases. Before such cells can be used successfully in neurodegenerative diseases without causing unwanted cell growth and migration, genes regulating growth and migration of neural stem cells need to be well characterized. Estrogen receptor beta (ERß) is essential for migration of neurons and glial cells in the developing mouse brain. To examine whether ERß influences differentiation of mouse embryonic stem cells (mESC) into neural lineages, we compared control and ERß knockout (BERKO) mESCs at defined stages of neural development and examined the effects of an ERß-selective ligand (LY3201) with a combination of global and targeted gene-expression profiling and the expression of key pluripotency markers. We found that ERß was induced in embryoid bodies (EBs) and neural precursor cells (NPCs) during development. Proliferation was higher in BERKO NPCs and was inhibited by LY3201. Neurogenesis was reduced in BERKO ES cells, and oligodendrogliogenesis was enhanced. BERKO EBs expressed higher levels of key ectodermal and neural progenitor markers and lower levels of markers for mesoderm and endoderm lineages. ERß-regulated factors are involved in cell adhesion, axon guidance, and signaling of Notch and GABA receptor pathways, as well as factors important for the differentiation of neuronal precursors into dopaminergic neurons (Engrailed 1) and for the oligodendrocyte fate acquisition (Olig2). Our data suggest that ERß is an important component for differentiation into midbrain neurons as well as for preventing precocious oligodendrogliogenesis.

Aminochrome induces dopaminergic neuronal dysfunction: a new animal model for Parkinson's disease.

L-Dopa continues to be the gold drug in Parkinson's disease (PD) treatment from 1967. The failure to translate successful results from preclinical to clinical studies can be explained by the use of preclinical models which do not reflect what happens in the disease since these induce a rapid and extensive degeneration; for example, MPTP induces a severe Parkinsonism in only 3 days in humans contrasting with the slow degeneration and progression of PD. This study presents a new anatomy and develops preclinical model based on aminochrome which induces a slow and progressive dysfunction of dopaminergic neurons. The unilateral injection of aminochrome into rat striatum resulted in (1) contralateral rotation when the animals are stimulated with apomorphine; (2) absence of significant loss of tyrosine hydroxylase-positive neuronal elements both in substantia nigra and striatum; (3) cell shrinkage; (4) significant reduction of dopamine release; (5) significant increase in GABA release; (6) significant decrease in the number of monoaminergic presynaptic vesicles; (7) significant increase of dopamine concentration inside of monoaminergic vesicles; (8) significant increase of damaged mitochondria; (9) significant decrease of ATP level in the striatum (10) significant decrease in basal and maximal mitochondrial respiration. These results suggest that aminochrome induces dysfunction of dopaminergic neurons where the contralateral behavior can be explained by aminochrome-induced ATP decrease required both for anterograde transport of synaptic vesicles and dopamine release. Aminochrome could be implemented as a new model neurotoxin to study Parkinson's disease.

Inhibition of estrogen biosynthesis enhances lymphoma growth in mice.

Most lymphomas show higher incidence and poorer prognosis in males compared to females. However, the endocrine contribution to this gender difference is not entirely known. Here we show that castration accelerates lymphoma growth in C57BL6 male mice grafted with murine EG7 T cell lymphoma cells. However, the androgen receptor antagonist Bicalutamide did not affect lymphoma growth, suggesting no impact of androgen receptor signaling on lymphoma progression. In contrast, inhibition of androgen-to-estrogen conversion by the aromatase inhibitor (AI) Letrozole induced faster lymphoma growth in mice, suggesting that androgens impact lymphoma growth through its conversion to estrogens. This was supported by the inability of dihydrotestosterone, which is not converted to estrogens by aromatase, to influence lymphoma growth in castrated male mice. Lymphoma growth was also stimulated in immunocompromised mice grafted with human B cell lymphoma (Granta-519) and treated with either reversible or irreversible AIs, showing that the blockage of estrogen synthesis caused enhanced growth of both murine T and human B cell lymphomas and with different AIs. Additionally, AI-treated EG7 lymphomas showed accelerated growth not only in male but also in intact female mice. Altogether, our results demonstrate that aromatase inhibition accelerates lymphoma growth but not androgens per se, highlighting a protective role of estrogens in lymphoma pathogenesis. These results also raise concern that the use of AIs in women with breast cancer might enhance lymphoma progression.

Estrogen receptor ß controls MMP-19 expression in mouse ovaries during ovulation.

Estrogen receptor beta (ERß/ESR2) has a central role in mouse ovaries, as ERß knockout (BERKO) mice are subfertile due to an increase in fibrosis around the maturing follicle and a decrease in blood supply. This has a consequence that these follicles rarely rupture to release the mature oocyte. Matrix metalloproteinases (MMPs) are modulators of the extracellular matrix, and the expression of one specific MMP, MMP-19, is normally increased in granulosa cells during their maturation until ovulation. In this study, we demonstrate that MMP-19 levels are downregulated in BERKO mouse ovaries. Using human MCF-7 cells that overexpress ERß, we could identify MMP-19 to be a transcriptional target of ligand-bound activated ERß acting on a specificity protein-1 binding site. These data provide a molecular explanation for the observed follicle rupture defect that contributes to the subfertility of female BERKO mice.

Characterization of stem-like cells in mucoepidermoid tracheal paediatric tumor.

Stem cells contribute to regeneration of tissues and organs. Cells with stem cell-like properties have been identified in tumors from a variety of origins, but to our knowledge there are yet no reports on tumor-related stem cells in the human upper respiratory tract. In the present study, we show that a tracheal mucoepidermoid tumor biopsy obtained from a 6 year-old patient contained a subpopulation of cells with morphology, clonogenicity and surface markers that overlapped with bone marrow mesenchymal stromal cells (BM-MSCs). These cells, designated as MEi (mesenchymal stem cell-like mucoepidermoid tumor) cells, could be differentiated towards mesenchymal lineages both with and without induction, and formed spheroids in vitro. The MEi cells shared several multipotent characteristics with BM-MSCs. However, they displayed differences to BM-MSCs in growth kinectics and gene expression profiles relating to cancer pathways and tube development. Despite this, the MEi cells did not possess in vivo tumor-initiating capacity, as proven by the absence of growth in situ after localized injection in immunocompromised mice. Our results provide an initial characterization of benign tracheal cancer-derived niche cells. We believe that this report could be of importance to further understand tracheal cancer initiation and progression as well as therapeutic development.

Differences in gene expression between mouse and human for dynamically regulated genes in early embryo.

Infertility is a worldwide concern that can be treated with in vitro fertilization (IVF). Improvements in IVF and infertility treatment depend largely on better understanding of the molecular mechanisms for human preimplantation development. Several large-scale studies have been conducted to identify gene expression patterns for the first five days of human development, and many functional studies utilize mouse as a model system. We have identified genes of possible importance for this time period by analyzing human microarray data and available data from online databases. We selected 70 candidate genes for human preimplantation development and investigated their expression in the early mouse development from oocyte to the 8-cell stage. Maternally loaded genes expectedly decreased in expression during development both in human and mouse. We discovered that 25 significantly upregulated genes after fertilization in human included 13 genes whose orthologs in mouse behaved differently and mimicked the expression profile of maternally expressed genes. Our findings highlight many significant differences in gene expression patterns during mouse and human preimplantation development. We also describe four cancer-testis antigen families that are also highly expressed in human embryos: PRAME, SSX, GAGE and MAGEA.