Autophagy initiation triggers p150Glued-AP-2ß interaction on the lysosomes and facilitates their transport.

The endocytic adaptor protein 2 (AP-2) complex binds dynactin as part of its noncanonical function, which is necessary for dynein-driven autophagosome transport along microtubules in neuronal axons. The absence of this AP-2-dependent transport causes neuronal morphology simplification and neurodegeneration. The mechanisms that lead to formation of the AP-2-dynactin complex have not been studied to date. However, the inhibition of mammalian/mechanistic target of rapamycin complex 1 (mTORC1) enhances the transport of newly formed autophagosomes by influencing the biogenesis and protein interactions of Rab-interacting lysosomal protein (RILP), another dynein cargo adaptor. We tested effects of mTORC1 inhibition on interactions between the AP-2 and dynactin complexes, with a focus on their two essential subunits, AP-2ß and p150Glued. We found that the mTORC1 inhibitor rapamycin enhanced p150Glued-AP-2ß complex formation in both neurons and non-neuronal cells. Additional analysis revealed that the p150Glued-AP-2ß interaction was indirect and required integrity of the dynactin complex. In non-neuronal cells rapamycin-driven enhancement of the p150Glued-AP-2ß interaction also required the presence of cytoplasmic linker protein 170 (CLIP-170), the activation of autophagy, and an undisturbed endolysosomal system. The rapamycin-dependent p150Glued-AP-2ß interaction occurred on lysosomal-associated membrane protein 1 (Lamp-1)-positive organelles but without the need for autolysosome formation. Rapamycin treatment also increased the acidification and number of acidic organelles and increased speed of the long-distance retrograde movement of Lamp-1-positive organelles. Altogether, our results indicate that autophagy regulates the p150Glued-AP-2ß interaction, possibly to coordinate sufficient motor-adaptor complex availability for effective lysosome transport.

Targeting the EGFR pathway: An alternative strategy for the treatment of tuberous sclerosis complex?

INTRODUCTION: Tuberous sclerosis complex (TSC) is caused by variants in TSC1/TSC2, leading to constitutive activation of the mammalian target of rapamycin (mTOR) complex 1. Therapy with everolimus has been approved for TSC, but variations in success are frequent. Recently, caudal late interneuron progenitor (CLIP) cells were identified as a common origin of the TSC brain pathologies such as subependymal giant cell astrocytomas (SEGA) and cortical tubers (CT). Further, targeting the epidermal growth factor receptor (EGFR) with afatinib, which is expressed in CLIP cells, reduces cell growth in cerebral TSC organoids. However, investigation of clinical patient-derived data is lacking. AIMS: Observation of EGFR expression in SEGA, CT and focal cortical dysplasia (FCD) 2B human brain specimen and investigation of whether its inhibition could be a potential therapeutic intervention for these patients. METHODS: Brain specimens of 23 SEGAs, 6 CTs, 20 FCD2Bs and 17 controls were analysed via immunohistochemistry to characterise EGFR expression, cell proliferation (via Mib1) and mTOR signalling. In a cell-based assay using primary patient-derived cells (CT n = 1, FCD2B n = 1 and SEGA n = 4), the effects of afatinib and everolimus on cell proliferation and cell viability were observed. RESULTS: EGFR overexpression was observed in histological sections of SEGA, CT and FCD2B patients. Both everolimus and afatinib decreased the proliferation and viability in primary SEGA, tuber and FCD2B cells. CONCLUSION: Our study demonstrates that EGFR suppression might be an effective alternative treatment option for SEGAs and tubers, as well as other mTOR-associated malformations of cortical development, including FCD2B.

Liquid semen storage-induced alteration in the protein composition of turkey (Meleagris gallopavo) spermatozoa.

Liquid storage of turkey semen without the loss of fertilizing ability is of practical interest to the poultry industry. However, fertility rates from liquid-stored turkey semen decline within a few hours. A clear cause of the decline in spermatozoa quality remains unidentified. Therefore, the purpose of the present study was to monitor the dynamics of proteomic changes in spermatozoa during 48 h of liquid storage by 2-dimensional difference in-gel electrophoresis coupled with matrix-assisted laser desorption/ionization mass spectrometry. A total of 57 protein spots were differentially expressed between fresh and stored spermatozoa; 42 spots were more and 15 were less abundant after 48 h of semen storage. Raw proteomic data are available via ProteomeXchange with identifier PXD043050. The selected differentially expressed proteins (DEPs) were validated by western blotting and localized in specific spermatozoa structures by immunofluorescence, such as the head (acrosin and tubulin α), midpiece (acrosin, aconitate hydratase 2, and glycerol-3-phosphate dehydrogenase) and tail (tubulin α). Most of the DEPs that changed in response to liquid storage were related to flagellum-dependent cell motility, energy derivation through oxidation of organic compounds and induction of fertilization, suggesting the complexity of the processes leading to the decrease in stored semen quality. The damaging effect of liquid storage on spermatozoa flagellum manifested as more microtubule proteins, such as tubulins and tektins, most likely formed by posttranslational modifications, tubulin α relocation from the tail to the sperm head, which appeared after 48 h of semen storage, and decreases in fibrous shelf proteins at the same time. Motility could be affected by dysregulation of Ca2+-binding proteins and disturbances in energy metabolism in spermatozoa flagellum. Regarding sperm mitochondria, DEPs involved in energy derivation through the oxidation of organic compounds indicated disturbances in fatty acid beta oxidation and the tricarboxylic acid cycle as possible reasons for energy deficiency during liquid storage. Disturbances in acrosin and 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase zeta may be involved in rapid declines in the fertility potential of stored turkey spermatozoa. These results showed the complexity of the processes leading to a decrease in stored semen quality and broadened knowledge of the detrimental effects of liquid storage on turkey spermatozoa physiology.

Astrocytic ß-catenin signaling via TCF7L2 regulates synapse development and social behavior.

The Wnt/ß-catenin pathway contains multiple high-confidence risk genes that are linked to neurodevelopmental disorders, including autism spectrum disorder. However, its ubiquitous roles across brain cell types and developmental stages have made it challenging to define its impact on neural circuit development and behavior. Here, we show that TCF7L2, which is a key transcriptional effector of the Wnt/ß-catenin pathway, plays a cell-autonomous role in postnatal astrocyte maturation and impacts adult social behavior. TCF7L2 was the dominant Wnt effector that was expressed in both mouse and human astrocytes, with a peak during astrocyte maturation. The conditional knockout of Tcf7l2 in postnatal astrocytes led to an enlargement of astrocytes with defective tiling and gap junction coupling. These mice also exhibited an increase in the number of cortical excitatory and inhibitory synapses and a marked increase in social interaction by adulthood. These data reveal an astrocytic role for developmental Wnt/ß-catenin signaling in restricting excitatory synapse numbers and regulating adult social behavior.

Generation of two human iPSC lines from dermal fibroblasts of adult- and juvenile-onset Huntington's disease patients and two healthy donors.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by a mutation in the HTT gene. To generate human-induced pluripotent stem cells (hiPSCs), we used dermal fibroblasts from 1 healthy adult control (K-Pic2), 1 HD manifest patient (M-T2), 1 healthy juvenile control (jK-N1), and 1 juvenile HD patient (jHD-V1). HD stage of patients was assessed by neurological tests and donors were without comorbidities and were non-smokers. Characterization showed that the obtained hiPSCs have the same number of CAG repeats as the parental fibroblast lines, express pluripotency markers and have the ability to differentiate into all 3 germ layers.

Specific expression of alternatively spliced genes in the turkey (Meleagris gallopavo) reproductive tract revealed their function in spermatogenesis and post-testicular sperm maturation.

The tissue-specific profile of alternatively spliced genes (ASGs) and their involvement in reproduction processes characteristic of turkey testis, epididymis, and ductus deferens were investigated for the first time in birds. Deep sequencing of male turkey reproductive tissue RNA samples (n = 6) was performed using Illumina RNA-Seq with 2 independent methods, rMATs and SUPPA2, for differential alternative splicing (DAS) event prediction. The expression of selected ASGs was validated using quantitative real-time reverse transcriptase-polymerase chain reaction. The testis was found to be the site of the highest number of posttranscriptional splicing events within the reproductive tract, and skipping exons were the most frequently occurring class of alternative splicing (AS) among the reproductive tract. Statistical analysis revealed 86, 229, and 6 DAS events in the testis/epididymis, testis/ductus deferens, and epididymis/ductus deferens comparison, respectively. Alternative splicing was found to be a mechanism of gene expression regulation within the turkey reproduction tract. In testis, modification was observed for spermatogenesis specific genes; the changes in 5' UTR could act as regulator of MEIG1 expression (a player during spermatocytes meiosis), and modification of 3' UTR led to diversification of CREM mRNA (modulator of gene expression related to the structuring of mature spermatozoa). Sperm tail formation can be regulated by changes in the 5' UTR of testicular SLC9A3R1 and gene silencing by producing dysfunctional variants of ODF2 in the testis and ATP1B3 in the epididymis. Predicted differentially ASGs in the turkey reproductive tract seem to be involved in the regulation of spermatogenesis, including acrosome formation and sperm tail formation and binding of sperm to the zona pellucida. Several ASGs were classified as cilia by actin and microtubule cytoskeleton organization. Such genes may play a role in the organization of sperm flagellum and post-testicular motility development. To our knowledge, this is the first functional investigation of alternatively spliced genes associated with tissue-specific processes in the turkey reproductive tract.

MicroRNA Expression Profile in TSC Cell Lines and the Impact of mTOR Inhibitor.

The aim of this study was to assess the potential implication of microRNA on tuberous sclerosis (TSC) pathogenesis by performing microRNA profiling on cell lines silencing TSC1 or TSC2 genes using qPCR panels, before and after incubation with rapamycin. Significant differences in expression were observed between samples before and after rapamycin treatment in nineteen miRNAs in TSC1, five miRNAs in TSC2 and seven miRNAs in controls. Of miRNAs dysregulated before rapamycin treatment, three normalized after treatment in the TSC1 group (miR-21-3p, miR-433-3p, let-7g-3p) and one normalized in the TSC2 group (miR-1224-3p). Of the miRNAs dysregulated before rapamycin treatment in the TSC1 and TSC2 groups, two did not normalize after treatment (miR-33a-3p, miR-29a-3p). The results of the possible targets indicated that there are four common genes with seed regions susceptible to regulation by those miRNAs: ZBTB20, PHACTR2, PLXNC1 and ATP1B4. Our data show no changes in mRNA expression of these targets after rapamycin treatment. In conclusion, results of our study indicate the involvement of miRNA dysregulation in the pathogenesis of TSC. Some of the miRNA might be used as markers of treatment efficacy and autonomic miRNA as a target for future therapy.

Generation of three human iPSC lines from patients with Huntington's disease with different CAG lengths and human control iPSC line from a healthy donor.

Huntington's disease (HD) is a progressive neurodegenerative disorder with autosomal-dominant heritability that affect the central nervous system and peripheral tissues. The human-induced pluripotent stem cells (hiPSC) lines were generated from dermal fibroblasts of patients without comorbidities, non-smokers, at the pre-manifest (IIMCBi004-A), early-manifest (IIMCBi005-A), and manifest (IIMCBi006-A) HD stage assessed by neurological tests, as well as from a healthy donor (IIMCBi003-A). Characterization showed that the obtained hiPSC lines contained different CAG repeats consistent with the number of CAG repeats in original fibroblasts. Moreover, hiPSCs expressed pluripotency markers and were able to differentiate into three-germ layers in vitro.

Proteomic analysis of carp seminal plasma provides insights into the immune response to bacterial infection of the male reproductive system.

Aeromonas salmonicida is recognized as a significant bacterial pathogen in ulcerative disease of cyprinid fish. However, the mechanism of immunity to these bacteria in common carp is still not well understood, especially the immune regulation in the gonad to bacterial infection. The aims of our study were to analyze changes in the seminal plasma proteome following A. salmonicida infection in carp males. The observed pathological changes in the tissue (liver, spleen, kidney and testis) morphology and upregulation of immune-related genes (tnfa2, il6a) confirmed the successful infection challenge. Using mass spectrometry-based label-free quantitative proteomics, we identified 1402 seminal plasma proteins, and 44 proteins (20 up- and 24 downregulated) were found to be differentially abundant between infected and control males. Most differentially abundant proteins were involved in the immune response mechanisms, such as acute phase response, complement activation and coagulation, inflammation, lipid metabolism, cell-cell and cell-matrix adhesion, creatine-phosphate biosynthesis and germ cell-Sertoli cell junction signaling. Bacterial infection also caused profound changes in expression of selected genes in the testis and hematopoietic organs, which contributed to changes in seminal proteins. The altered seminal proteins and bacterial proteins in seminal plasma may serve as valuable markers of infection in the testis.

Transcriptome and Proteome Analysis Revealed Key Pathways Regulating Final Stage of Oocyte Maturation of the Turkey (Meleagris gallopavo).

In birds, the zona pellucida (ZP) matrix that surrounds the ovulated oocyte-called the inner perivitelline layer-is involved in sperm-zona interaction and successful fertilization. To identify the important genes and proteins connected with the final step of egg development, next-generation sequencing and two-dimensional electrophoresis, combined with mass spectrometry, were used for the analysis of mature oocytes at the F1 developmental stage. A total of 8161 genes and 228 proteins were annotated. Six subfamilies of genes, with codes ZP, ZP1-4, ZPD, and ZPAX, were identified, with the dominant expression of ZPD. The main expression site for ZP1 was the liver; however, granulosa cells may also participate in local ZP1 secretion. A ubiquitination system was identified in mature oocytes, where ZP1 was found to be the main ubiquitinated protein. Analysis of transcripts classified in estrogen receptor (ESR) signaling indicated the presence of ESR1 and ESR2, as well as a set of estrogen-dependent genes involved in both genomic and nongenomic mechanisms for the regulation of gene expression by estrogen. Oxidative phosphorylation was found to be a possible source of adenosine triphosphate, and the nuclear factor erythroid 2-related factor 2 signaling pathway could be involved in the response against oxidative stress. Oocyte-granulosa cell communication by tight, adherens, and gap junctions seems to be essential for the final step of oocyte maturation.

mTOR controls endoplasmic reticulum-Golgi apparatus trafficking of VSVg in specific cell types.

BACKGROUND: Mammalian/mechanistic target of rapamycin (mTOR) complexes are essential for cell proliferation, growth, differentiation, and survival. mTORC1 hyperactivation occurs in the tuberous sclerosis complex (TSC). mTORC1 localizes to the surface of lysosomes, where Rheb activates it. However, mTOR was also found on the endoplasmic reticulum (ER) and Golgi apparatus (GA). Recent studies showed that the same inputs regulate ER-to-GA cargo transport and mTORC1 (e.g., the level of amino acids or energy status of the cell). Nonetheless, it remains unknown whether mTOR contributes to the regulation of cargo passage through the secretory pathway. METHODS: The retention using selective hooks (RUSH) approach was used to image movement of model cargo (VSVg) between the ER and GA in various cell lines in which mTOR complexes were inhibited. We also investigated VSVg trafficking in TSC patient fibroblasts. RESULTS: We found that mTOR inhibition led to the overall enhancement of VSVg transport through the secretory pathway in PC12 cells and primary human fibroblasts. Also, in TSC1-deficient cells, VSVg transport was enhanced. CONCLUSIONS: Altogether, these data indicate the involvement of mTOR in the regulation of ER-to-GA cargo transport and suggest that impairments in exocytosis may be an additional cellular process that is disturbed in TSC.

Characterization and biological role of cysteine-rich venom protein belonging to CRISPs from turkey seminal plasma†.

Turkey semen contains cysteine-rich secretory proteins (CRISPs) that belong to the dominant seminal plasma proteins. We aimed to isolate and characterize CRISP from turkey seminal plasma and evaluate its possible involvement in yellow semen syndrome (YSS). YSS, which is well characterized, causes reduced fertility and hatchability. The protein was purified using hydrophobic interaction, gel filtration, and reverse phase chromatography. It then was subjected to identification by mass spectrometry, analysis of physicochemical properties, and specific antibody production. The biological function of the isolated protein was tested and included its effects on sperm motility and migration and sperm-egg interactions. Sperm motility was measured with the CASA system using Hobson Sperm Tracker. The reproductive tract of turkey toms was analyzed for gene expression; immunohistochemistry was used for protein localization in the male reproductive tract, spermatozoa, and inner perivitelline layer. The isolated protein was identified as cysteine-rich venom protein-like isoform X2 (CRVP X2; XP_010706464.1) and contained feature motifs of CRISP family proteins. Turkey CRVP X2 was present in both spermatozoa and seminal plasma. The extensive secretion of CRVP X2 by the epithelial cells of the epididymis and ductus deferens suggests its involvement in post-testicular sperm maturation. The internally localized CRVP X2 in the proximal part of the sperm tail might be responsible for stimulation of sperm motility. CRVP X2 on the sperm head might be involved in several events prior to fusion and may also participate in gamete fusion itself. Although the mechanisms by which CRVP X2 mediates fertilization are still unknown, the involvement of complementary sites cannot be excluded. The disturbance of CRVP X2 expression can serve as an etiologic factor of YSS in the turkey. This study expands the understanding of the detailed mechanism of fertilization in birds by clarifying the specific role of CRVP X2.

Establishment of two hiPSC lines (IIMCBi001-A and IIMCBi002-A) from dermal fibroblasts of healthy donors and characterization of their cell cycle.

Two human induced pluripotent stem cell (hiPSC) lines (IIMCBi001-A and IIMCBi002-A) were generated from dermal fibroblasts of healthy females 10 and 30 years old, respectively. For the reprogramming lentiviral vector expressing OCT4, SOX2, KLF4 and C-MYC was used. The generated hiPSCs showed typical embryonic stem cell-like morphology and correct diploid karyotype. Characterization of the hiPSC lines confirmed expression of pluripotency markers and demonstrated their ability to differentiate into the three-germ layers. Cell cycle analysis of the hiPSCs allowed to estimate population doubling time (DT), duration time of particular phases of the cell cycle and proportion of cells found at each phase.

Cytosolic translational responses differ under conditions of severe short-term and long-term mitochondrial stress.

Previous studies demonstrated that cells inhibit protein synthesis as a compensatory mechanism for mitochondrial dysfunction. Protein synthesis can be attenuated by 1) the inhibition of mTOR kinase, which results in a decrease in the phosphorylation of S6K1 and 4E-BP1 proteins, and 2) an increase in the phosphorylation of eIF2α protein. The present study investigated both of these pathways under conditions of short-term acute and long-term mitochondrial stress. Short-term responses were triggered in mammalian cells by treatment with menadione, antimycin A, or CCCP. Long-term mitochondrial stress was induced by prolonged treatment with menadione or rotenone and expression of genetic alterations, such as knocking down the MIA40 oxidoreductase or knocking out NDUFA11 protein. Short-term menadione, antimycin A, or CCCP cell treatment led to the inhibition of protein synthesis, accompanied by a decrease in mTOR kinase activity, an increase in the phosphorylation of eIF2α (Ser51), and an increase in the level of ATF4 transcription factor. Conversely, long-term stress led to a decrease in eIF2α (Ser51) phosphorylation and ATF4 expression and to an increase in S6K1 (Thr389) phosphorylation. Thus, under long-term mitochondrial stress, cells trigger long-lasting adaptive responses for protection against excessive inhibition of protein synthesis.

The level of microRNA 21 is upregulated by rapamycin in serum of tuberous sclerosis complex patients and subependymal giant cell astrocytoma (SEGA)-derived cell cultures.

Tuberous sclerosis complex (TSC) represents a genetic condition, in which the clinical manifestations are caused by the disinhibition of the mammalian target of rapamycin (mTOR) pathway due to mutations in the TSC1 (hamartin) or TSC2 (tuberin) genes. The deregulated mTOR activity leads to multi-site tumors, including subependymal giant cell astrocytoma (SEGA). SEGA is a brain tumor that affects around 15% of TSC patients. The aim of the study was to evaluate miR-21 expression in the serum of two groups of TSC patients: with or without SEGA tumors. We found no differences in the level of miR-21 depending on the presence of SEGA. Next, we studied the influence of prolonged rapamycin administration on miR-21 level in the blood serum of TSC patients (6-12 months of rapamycin) and in primary cultures of SEGA-derived cells treated with rapamycin in vitro. Here we show that rapamycin treatment leads to the upregulation of miR-21 in both patients' serum and in primary SEGA tumor cells in the culture indicating the regulatory relationship between rapamycin treatment and miR-21 expression.

Neural Stem Cell Dysfunction in Human Brain Disorders.

Neural stem cells (NSCs) give rise to the entire nervous system. Animal models suggest that defects in NSC proliferation and differentiation contribute to several brain disorders (e.g., microcephaly, macrocephaly, autism, schizophrenia, and Huntington's disease). However, animal models of such diseases do not fully recapitulate all disease-related phenotypes because of substantial differences in brain development between rodents and humans. Therefore, additional human-based evidence is required to understand the mechanisms that are involved in the development of neurological diseases that result from human NSC (hNSC) dysfunction. Human-induced pluripotent stem cells provide a new model to investigate the contribution of hNSCs to various neurological pathologies. In this chapter, we review the role of hNSCs in both neurodevelopment- and neurodegeneration-related human brain pathologies, with an emphasis on recent evidence that has been obtained using embryonic stem cell- or induced pluripotent stem cell-derived hNSCs and progenitors.

Adaptor Complex 2 Controls Dendrite Morphology via mTOR-Dependent Expression of GluA2.

The formation of dendritic arbors in neurons is a highly regulated process. Among the regulators of dendritogenesis are numerous membrane proteins that are eventually internalized via clathrin-mediated endocytosis. AP2 is an adaptor complex that is responsible for recruiting endocytic machinery to internalized cargo. Its direct involvement in dendritogenesis in mammalian neurons has not yet been tested. We found that the knockdown of AP2b1 (ß2-adaptin), an AP2 subunit, reduced the number of dendrites in developing rat hippocampal neurons and decreased α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluA2 levels by inhibiting mechanistic/mammalian target of rapamycin (mTOR). The dendritic tree abruption that was caused by AP2b1 knockdown was rescued by the overexpression of GluA2 or restoration of the activity of the mTOR effector p70S6 kinase (S6K1). Altogether, this work provides evidence that the AP2 adaptor complex is needed for the dendritogenesis of mammalian neurons and reveals that mTOR-dependent GluA2 biosynthesis contributes to this process.

GSK3ß Controls mTOR and Prosurvival Signaling in Neurons.

Glycogen synthase kinases-3ß (GSK3ß) is a key regulator of cell homeostasis. In neurons, GSK3ß contributes to control of neuronal transmission and plasticity. Despite extensive studies in non-neuronal cells, crosstalk between GSK3ß and other signaling pathways remains not well defined in neurons. In the present study, we report that GSK3ß positively affected the activity of effectors of mammalian target of rapamycin complex 1 (mTORC1) and complex 2 (mTORC2), in mature neurons in vitro and in vivo. GSK3ß also promoted prosurvival signaling and attenuated kainic acid-induced apoptosis. Our study identified GSK3ß as a positive regulator of prosurvival signaling, including the mTOR pathway, and indicates the possible neuroprotective role of GSK3ß in models of pharmacologically induced excitotoxicity.

Potassium ions in extender differentially influence the post-thaw sperm motility of salmonid fish.

Potassium ions are known to have an inhibitory effect on the sperm motility of salmonids. For this reason, the addition of K(+) to the extender is frequently applied. However, the effect of the addition of K(+) to the extender has not yet been tested. The aim of this study was to test the influence of potassium ion supplementation of the extender on the sperm motility parameters from five Salmonidae species (rainbow trout (Oncorhynchus mykiss), sex-reversed female rainbow trout, whitefish (Coregonus lavaretus), brown trout (Salmo trutta) and brook trout (Salvelinus fontinalis)). Semen samples were diluted in extender containing 0.18 M glucose in 9% methanol (GM) supplemented with 0, 20 or 40 mM potassium chloride. After thawing sperm were stored for 30, 60, 120 and 240 min at 4 °C. Our results demonstrated that the presence of potassium ions in the extender had a negative effect on percentage of motile sperm in four of the salmonid species. In contrast, potassium ions appeared to have a positive effect on percentage of post-thaw motile sperm in whitefish semen. However, this effect could be mimicked by changing the osmolality of the extender (which was achieved by increasing the glucose concentration to 0.22 M). The addition of potassium ions turned out to have no positive effect on post-thaw storage time. Our results suggest that osmolality, rather than potassium ions, seems to be essential for cryopreservation success of salmonids sperm. Further studies should focus on the effects of small changes in osmolality on the post-thaw quality of semen.