CHRFAM7A diversifies human immune adaption through Ca signalling and actin cytoskeleton reorganization.

Background: Human restricted genes contribute to human specific traits in the immune system. CHRFAM7A, a uniquely human fusion gene, is a negative regulator of the α7 nicotinic acetylcholine receptor (α7 nAChR), the highest Ca conductor of the ACh receptors implicated in innate immunity. Understanding the mechanism of how CHRFAM7A affects the immune system remains unexplored.

Translational implications of CHRFAM7A, an elusive human-restricted fusion gene.

Genes restricted to humans may contribute to human-specific traits and provide a different context for diseases. CHRFAM7A is a uniquely human fusion gene and a negative regulator of the α7 nicotinic acetylcholine receptor (α7 nAChR). The α7 nAChR has been a promising target for diseases affecting cognition and higher cortical functions, however, the treatment effect observed in animal models failed to translate into human clinical trials.

Neuronal actin cytoskeleton gain of function in the human brain.

Background: While advancements in imaging techniques have led to major strides in deciphering the human brain, successful interventions are elusive and represent some of the most persistent translational gaps in medicine. Human restricted CHRFAM7A has been associated with neuropsychiatric disorders.

Methods: The physiological role of CHRFAM7A in human brain is explored using multiomics approach on 600 post mortem human brain tissue samples.

iPSC-Derived Microglia for Modeling Human-Specific DAMP and PAMP Responses in the Context of Alzheimer's Disease.

Neuroinflammation in Alzheimer's disease (AD) has been the focus for identifying targetable pathways for drug development. The role of amyloid beta (Aβ), a prototype of damage-associated molecular patterns (DAMPs), has been implicated in triggering an inflammatory response. As alpha7 nicotinic acetylcholine receptor (α7 nAChR) binds Aβ with high affinity, α7 nAChR may play a role in Aβ-induced neuroinflammation.

CHRFAM7A: A human specific fusion gene, accounts for the translational gap for cholinergic strategies in Alzheimer's disease.

Background: Cholinergic neuronal loss is one of the hallmarks of AD related neurodegeneration; however, preclinical promise of α7 nAChR drugs failed to translate into humans. CHRFAM7A, a uniquely human fusion gene, is a negative regulator of α7 nAChR and was unaccounted for in preclinical models.

Methods: Molecular methods: Function of CHRFAM7A alleles was studied in vitro in two disease relevant phenotypic readouts: electrophysiology and Aβ uptake.

iPSC model of CHRFAM7A effect on α7 nicotinic acetylcholine receptor function in the human context.

The α7 nicotinic acetylcholine receptor (α7nAChR) has been a promising target for diseases affecting cognition and higher cortical functions; however, the effect observed in animal models failed to translate into human clinical trials identifying a translational gap. CHRFAM7A is a human-specific fusion gene with properties that enable incorporation into the α7nAChR and, being human specific, CHRFAM7A effect was not accounted for in preclinical studies. We hypothesized that CHRFAM7A may account for this translational gap and understanding its function may offer novel insights when exploring α7nAChR as a drug target.

Timing of Wnt Inhibition Modulates Directed Differentiation of Medial Ganglionic Eminence Progenitors from Human Pluripotent Stem Cells.

differentiation of human pluripotent stem cell into relevant cell types is a desirable model system that has the human biological context, is a renewable source, and is scalable. GABA interneurons and basal forebrain cholinergic neurons, derivates of the medial ganglionic eminence (MGE), are implicated in diverse neuropsychiatric diseases. Various protocols have been proposed to generate MGE progenitors: the embryoid body- (EB-) based rosette-derived (RD), the adherent (AdD), and the nonadherent (NAdD) approaches.

Selective expression of myosin IC Isoform A in mouse and human cell lines and mouse prostate cancer tissues.

Myosin IC is a single headed member of the myosin superfamily. We recently identified a novel isoform and showed that the MYOIC gene in mammalian cells encodes three isoforms (isoforms A, B, and C). Furthermore, we demonstrated that myosin IC isoform A but not isoform B exhibits a tissue specific expression pattern.

Tissue specific expression of myosin IC isoforms.

Background: Myosin IC is a single headed member of the myosin superfamily that localizes to the cytoplasm and the nucleus and is implicated in a variety of processes in both compartments. We recently identified a novel isoform of myosin IC and showed that the MYOIC gene in mammalian cells encodes three isoforms (isoforms A, B, and C) that differ only in the addition of short isoform-specific N-terminal peptides. The expression pattern of the isoforms and the mechanisms of expression regulation remain unknown.

Identification of signals that facilitate isoform specific nucleolar localization of myosin IC.

Myosin IC is a single headed member of the myosin superfamily that localizes to the cytoplasm and the nucleus, where it is involved in transcription by RNA polymerases I and II, intranuclear transport, and nuclear export. In mammalian cells, three isoforms of myosin IC are expressed that differ only in the addition of short isoform-specific N-terminal peptides. Despite the high sequence homology, the isoforms show differences in cellular distribution, in localization to nuclear substructures, and in their interaction with nuclear proteins through yet unknown mechanisms.

Identification and characterization of a novel myosin Ic isoform that localizes to the nucleus.

In vertebrates, two myosin Ic isoforms that localize to the cytoplasm and to the nucleus have been characterized. The isoform that predominantly localizes to the nucleus is called nuclear myosin I (NMI). NMI has been identified as a key factor involved in nuclear processes such as transcription by RNA polymerases I and II and intranuclear transport processes.

Cofilin and slingshot localization in the epithelium of uterine endometrium changes during the menstrual cycle and in endometriosis.

Regulation of the actin cytoskeleton is essential for epithelial cell polarity and protein trafficking within human uterine epithelium. The actin-binding protein cofilin is involved in regulation of actin dynamics by promoting actin branching and cytoskeleton reorganization. Dual immunohistochemical staining of cofilin and G-actin (represented by DNAse I staining) revealed cofilin-G-actin colocalization in the apical side of luminal epithelial cells of human uterine endometrium during the proliferative phase of the menstrual cycle.

Human transcriptional coactivator with PDZ-binding motif (TAZ) is downregulated during decidualization.

Transcriptional coactivator with PDZ-binding motif (TAZ) is known to bind to a variety of transcription factors to control cell differentiation and organ development. However, its role in uterine physiology has not yet been described. To study its regulation during the unique process of differentiation of fibroblasts into decidual cells (decidualization), we utilized the human uterine fibroblast (HuF) in vitro cell model.

Manipulating actin dynamics affects human in vitro decidualization.

The differentiation of uterine stromal fibroblasts into decidual cells is critical for establishing pregnancy. This process, called decidualization, requires the reorganization of the actin cytoskeleton, which mainly depends on actin dynamics and the phosphorylation status of the myosin light chain. We manipulated actin dynamics with jasplakinolide (100 nM) and latrunculin B (1 microM), both of which significantly inhibited the synthesis of decidualization markers induced by 6 days of treatment with embryo-mimicking stimulus interleukin 1beta (IL1B) and steroid hormones (SHs; 17beta-estradiol and medroxyprogesterone acetate) in the human uterine fibroblast (HuF) in vitro model.

Multipotent properties of myofibroblast cells derived from human placenta.

Human uterine fibroblasts (HuF) isolated from the maternal part (decidua parietalis) of a term placenta provide a useful model of in vitro cell differentiation into decidual cells (decidualization, a critical process for successful pregnancy). After isolation, the cells adhere to plastic and have either a small round or spindle-shaped morphology that later changes into a flattened pattern in culture. HuF robustly proliferate in culture until passage 20 and form colonies when plated at low densities.

Increased phosphorylation of myosin light chain prevents in vitro decidualization.

Differentiation of stromal cells into decidual cells, which is critical to successful pregnancy, represents a complex transformation requiring changes in cytoskeletal architecture. We demonstrate that in vitro differentiation of human uterine fibroblasts into decidual cells includes down-regulation of alpha-smooth muscle actin and beta-tubulin, phosphorylation of focal adhesion kinase, and redistribution of vinculin. This is accompanied by varied adhesion to fibronectin and a modified ability to migrate.

Inhibiting myosin light chain kinase induces apoptosis in vitro and in vivo.

Previous short-term studies have correlated an increase in the phosphorylation of the 20-kDa light chain of myosin II (MLC20) with blebbing in apoptotic cells. We have found that this increase in MLC20 phosphorylation is rapidly followed by MLC20 dephosphorylation when cells are stimulated with various apoptotic agents. MLC20 dephosphorylation is not a consequence of apoptosis because MLC20 dephosphorylation precedes caspase activation when cells are stimulated with a proapoptotic agent or when myosin light chain kinase (MLCK) is inhibited pharmacologically or by microinjecting an inhibitory antibody to MLCK.

Increased baclofen-stimulated G protein coupling and deactivation in rat brain cortex during development.

The number and affinity of GABA(B) receptors (assayed by the specific antagonist [(3)H]CGP54626A) was unchanged when compared in carefully washed cerebrocortical membranes from young (12-day-old) and adult (90-day-old) rats. In contrast, high-affinity GTPase activity, both basal and baclofen-stimulated was significantly higher (by 45% and 56%, respectively) in adult than in young rats. Similar results were obtained by concomitant determination of agonist (baclofen)-stimulated GTP gamma S binding.

Modulation of adenylyl cyclase activity by baclofen in the developing rat brain: difference between cortex, thalamus and hippocampus.

Ontogenetic changes in the levels of GABA(B) receptors and their ability to modulate adenylyl cyclase (AC) activity were analyzed in rat cortex, thalamus and hippocampus. The relative numbers of GABA(B) receptors (measured as saturable, high-affinity [(3)H](-)baclofen binding sites) in cortex and thalamus were high already at postnatal day 1 (PD 1) and they reached a maximum at PD 25 and PD 12, respectively. There were no detectable high-affinity [(3)H](-)baclofen binding sites in hippocampus between birth and PD 12 and low-affinity [(3)H](-)baclofen binding attained at PD 12 did not change in adulthood (PD 90).

Ontogenetic development of the G protein-mediated adenylyl cyclase signalling in rat brain.

Maturation of the brain adenylyl cyclase (AC) signalling system was investigated in the developing rat cortex, thalamus and hippocampus. Expression of AC type II, IV and VI measured by Western blot dramatically increased in all tested brain regions during the first 3 weeks after birth and these levels were maintained in adulthood. AC type I did not change during ontogenesis.

Opposing changes of trimeric G protein levels during ontogenetic development of rat brain.

Developmental changes in the distribution of guanine nucleotide-binding regulatory proteins (G proteins) were investigated in the rat brain during postnatal development. Using a standard or high-resolution urea-SDS-PAGE and specific polyclonal antipeptide antibodies oriented against G(i)alpha1/G(i)alpha2, G(i)alpha3, G(s)alpha, G(o)alpha1/G(o)alpha2, G(q)alpha/G(11)alpha and Gbeta subunit, all these proteins were determined by quantitative immunoblotting in homogenates prepared from cortex, thalamus, hippocampus and pituitary of 1-, 7-, 12-, 18-, 25- and 90-day-old animals. The levels of the majority of G protein alpha subunits, namely G(i)alpha1, G(i)alpha2, G(i)alpha3, G(o)alpha1, G(o)alpha2, G(q)alpha, G(11)alpha and Gbeta, were high already at birth.