ABSTRACT
Lymphoid tumor patients often exhibit resistance to standard therapies or experience relapse post-remission. Relapse is driven by Tumor Initiating Cells (TICs), a subset of tumor cells capable of regrowing the tumor and highly resistant to therapy. Growing cells in 3D gels is a method to discern tumorigenic cells because it strongly correlates with tumorigenicity. The finding that TICs, rather than differentiated tumor cells, grow in 3D gels offers a unique opportunity to unveil TIC-specific signaling pathways and therapeutic targets common to various cancer types. Here, we show that culturing lymphoid cells in 3D gels triggers reactive oxygen species (ROS) production, leading to non-tumor lymphoid cell death while enabling the survival and proliferation of a subset of lymphoma/leukemia cells, TICs or TIC-like cells. Treatment with the antioxidant N-acetylcysteine inhibits this lethality and promotes the growth of primary non-tumor lymphoid cells in 3D gels. A subset of lymphoma cells, characterized by an increased abundance of the antioxidant glutathione, escape ROS-induced lethality, a response not seen in non-tumor cells. Reducing glutathione production in lymphoma cells, either through pharmacological inhibition of glutamate cysteine ligase (GCL), the enzyme catalyzing the rate-limiting step in glutathione biosynthesis, or via knockdown of GCLC, the GCL catalytic subunit, sharply decreased cell growth in 3D gels and xenografts. Tumor cells from B-cell lymphoma/leukemia patients and λ-MYC mice, a B-cell lymphoma mouse model, overproduce glutathione. Importantly, pharmacological GCL inhibition hindered lymphoma growth in female λ-MYC mice, suggesting that this treatment holds promise as a therapeutic strategy for female lymphoma/leukemia patients.
Subject(s)
Glutathione , Lymphoma , Neoplastic Stem Cells , Reactive Oxygen Species , Animals , Humans , Female , Lymphoma/pathology , Lymphoma/metabolism , Lymphoma/drug therapy , Glutathione/metabolism , Mice , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Neoplastic Stem Cells/drug effects , Reactive Oxygen Species/metabolism , Male , Cell Line, Tumor , Cell Survival/drug effects , Cell Proliferation/drug effects , Carcinogenesis/pathology , Carcinogenesis/drug effects , Carcinogenesis/metabolismABSTRACT
BACKGROUND: SINO syndrome (Spastic paraplegia, Intellectual disability, Nystagmus and Obesity) is a rare autosomal dominant condition caused by heterozygous variants in KIDINS220. A total of 12 individuals are reported, comprising eight with SINO and four with an autosomal recessive condition attributed to bi-allelic KIDINS220 variants. METHODS: In our international cohort, we have comprised 14 individuals, carrying 13 novel pathogenic KIDINS220 variants in heterozygous form. We assessed clinical and molecular data of our cohort and previously reported individuals and based on functional experiments reached a better understanding of the pathogenesis behind KIDINS220-related disease. RESULTS: Using fetal tissue and in vitro assays, we demonstrate that the variants generate KIDINS220 truncated forms that mislocalize in punctate intracellular structures, with decreased levels of the full-length protein, suggesting a trans-dominant negative effect. 92% had their diagnosis within three years, with symptoms of developmental delay, spasticity, hypotonia, lack of eye contact and nystagmus. We identified a KIDINS220 variant associated with fetal hydrocephalus and show that 58% of examined individuals present brain ventricular dilatation. We extend the phenotypic spectrum of SINO syndrome to behavioral manifestations not previously highlighted. CONCLUSION: Our study provides further insights into the clinical spectrum, etiology and predicted functional impact of KIDINS220 variants.
ABSTRACT
In the adult mammalian brain, neural stem cells (NSCs) located in highly restricted niches sustain the generation of new neurons that integrate into existing circuits. A reduction in adult neurogenesis is linked to ageing and neurodegeneration, whereas dysregulation of proliferation and survival of NSCs have been hypothesized to be at the origin of glioma. Thus, unravelling the molecular underpinnings of the regulated activation that NSCs must undergo to proliferate and generate new progeny is of considerable relevance. Current research has identified cues promoting or restraining NSCs activation. Yet, whether NSCs depend on external signals to survive or if intrinsic factors establish a threshold for sustaining their viability remains elusive, even if this knowledge could involve potential for devising novel therapeutic strategies. Kidins220 (Kinase D-interacting substrate of 220 kDa) is an essential effector of crucial pathways for neuronal survival and differentiation. It is dramatically altered in cancer and in neurological and neurodegenerative disorders, emerging as a regulatory molecule with important functions in human disease. Herein, we discover severe neurogenic deficits and hippocampal-based spatial memory defects accompanied by increased neuroblast death and high loss of newly formed neurons in Kidins220 deficient mice. Mechanistically, we demonstrate that Kidins220-dependent activation of AKT in response to EGF restraints GSK3 activity preventing NSCs apoptosis. We also show that NSCs with Kidins220 can survive with lower concentrations of EGF than the ones lacking this molecule. Hence, Kidins220 levels set a molecular threshold for survival in response to mitogens, allowing adult NSCs growth and expansion. Our study identifies Kidins220 as a key player for sensing the availability of growth factors to sustain adult neurogenesis, uncovering a molecular link that may help paving the way towards neurorepair.
Subject(s)
Adult Stem Cells , Neural Stem Cells , Adult , Animals , Humans , Mice , Adult Stem Cells/metabolism , Epidermal Growth Factor/metabolism , Glycogen Synthase Kinase 3/metabolism , Hippocampus/metabolism , Mammals , Neural Stem Cells/metabolism , Neurogenesis/physiology , Neurons/metabolismABSTRACT
While sleeping, many vertebrate groups alternate between at least two sleep stages: rapid eye movement and slow wave sleep1-4, in part characterized by wake-like and synchronous brain activity, respectively. Here we delineate neural and behavioural correlates of two stages of sleep in octopuses, marine invertebrates that evolutionarily diverged from vertebrates roughly 550 million years ago (ref. 5) and have independently evolved large brains and behavioural sophistication. 'Quiet' sleep in octopuses is rhythmically interrupted by approximately 60-s bouts of pronounced body movements and rapid changes in skin patterning and texture6. We show that these bouts are homeostatically regulated, rapidly reversible and come with increased arousal threshold, representing a distinct 'active' sleep stage. Computational analysis of active sleep skin patterning reveals diverse dynamics through a set of patterns conserved across octopuses and strongly resembling those seen while awake. High-density electrophysiological recordings from the central brain reveal that the local field potential (LFP) activity during active sleep resembles that of waking. LFP activity differs across brain regions, with the strongest activity during active sleep seen in the superior frontal and vertical lobes, anatomically connected regions associated with learning and memory function7-10. During quiet sleep, these regions are relatively silent but generate LFP oscillations resembling mammalian sleep spindles11,12 in frequency and duration. The range of similarities with vertebrates indicates that aspects of two-stage sleep in octopuses may represent convergent features of complex cognition.
Subject(s)
Central Nervous System , Integumentary System , Octopodiformes , Sleep , Wakefulness , Animals , Mammals/physiology , Octopodiformes/physiology , Sleep/physiology , Sleep, REM/physiology , Wakefulness/physiology , Integumentary System/innervation , Integumentary System/physiology , Movement/physiology , Time Factors , Local Field Potential Measurement , Learning/physiology , Central Nervous System/anatomy & histology , Central Nervous System/physiology , Arousal/physiologyABSTRACT
Spry2 is a molecular modulator of tyrosine kinase receptor signaling pathways that has cancer-type-specific effects. Mammalian Spry2 protein undergoes tyrosine and serine phosphorylation in response to growth factor stimulation. Spry2 expression is distinctly altered in various cancer types. Inhibition of the proteasome functionality results in reduced intracellular Spry2 degradation. Using in vitro and in vivo assays, we show that protein kinase D (PKD) phosphorylates Spry2 at serine 112 and interacts in vivo with the C-terminal half of this protein. Importantly, missense mutation of Ser112 decreases the rate of Spry2 intracellular protein degradation. Either knocking down the expression of all three mammalian PKD isoforms or blocking their kinase activity with a specific inhibitor contributes to the stabilization of Spry2 wild-type protein. Downregulation of CSN3, a component of the COP9/Signalosome that binds PKD, significantly increases the half-life of Spry2 wild-type protein but does not affect the stability of a Spry2 after mutating Ser112 to the non-phosphorylatable residue alanine. Our data demonstrate that both PKD and the COP9/Signalosome play a significant role in control of Spry2 intracellular stability and support the consideration of the PKD/COP9 complex as a potential therapeutic target in tumors where Spry2 expression is reduced.
ABSTRACT
Aquaporin-4 (AQP4) plays a crucial role in brain water circulation and is considered a therapeutic target in hydrocephalus. Congenital hydrocephalus is associated with a reaction of astrocytes in the periventricular white matter both in experimental models and human cases. A previous report showed that bone marrow-derived mesenchymal stem cells (BM-MSCs) transplanted into the lateral ventricles of hyh mice exhibiting severe congenital hydrocephalus are attracted by the periventricular astrocyte reaction, and the cerebral tissue displays recovery. The present investigation aimed to test the effect of BM-MSC treatment on astrocyte reaction formation. BM-MSCs were injected into the lateral ventricles of four-day-old hyh mice, and the periventricular reaction was detected two weeks later. A protein expression analysis of the cerebral tissue differentiated the BM-MSC-treated mice from the controls and revealed effects on neural development. In in vivo and in vitro experiments, BM-MSCs stimulated the generation of periventricular reactive astrocytes overexpressing AQP4 and its regulatory protein kinase D-interacting substrate of 220 kDa (Kidins220). In the cerebral tissue, mRNA overexpression of nerve growth factor (NGF), vascular endothelial growth factor (VEGF), hypoxia-inducible factor-1 (HIF1α), and transforming growth factor beta 1 (TGFß1) could be related to the regulation of the astrocyte reaction and AQP4 expression. In conclusion, BM-MSC treatment in hydrocephalus can stimulate a key developmental process such as the periventricular astrocyte reaction, where AQP4 overexpression could be implicated in tissue recovery.
Subject(s)
Hydrocephalus , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells , Mice , Humans , Animals , Astrocytes/metabolism , Aquaporin 4/genetics , Aquaporin 4/metabolism , Vascular Endothelial Growth Factor A/metabolism , Mesenchymal Stem Cells/metabolism , Hydrocephalus/therapy , Hydrocephalus/metabolism , Membrane Proteins/metabolism , Nerve Tissue Proteins/metabolismABSTRACT
AIMS: The aim of this study is to extend the analysis of the Lung Cancer Biomarker Testing Registry (LungPath), by analysing the techniques used in the determination of epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), c-ros oncogene 1 (ROS1) and programmed death ligand-1 (PD-L1) for the diagnostic of patients with advanced non-small-cell lung cancer (NSCLC). METHODS: Information of the technique used for the determination of EGFR, ALK, ROS1 and PD-L1 was recorded from March 2018 to January 2019 from 44 centres, but only 34 centres matched with the 38 centres previously analysed, allowing to analyse the techniques used in 8970 matched determinations of EGFR, ALK, ROS1 and PD-L1. Therefore, a by-centre analysis studied the level of implementation of the techniques in the 44 centres, while a by-determination analysis made it possible to assess the overall frequency of the techniques used on the 9134 matched samples. RESULTS: By-centre analysis showed that only 46.5% and 25.6% of the centres used reflex strategies for ALK and ROS1 determination, respectively. By-determination analysis showed that 94.4% of EGFR determinations were performed by PCR, 80.7% of ALK determinations were performed by IHC with clone D5F3, while 55.7% of ROS1 determinations were performed by IHC with clone D4D6. 22C3 were the PD-L1 clone more used (43.5%) followed by SP263 clone (31.1%). CONCLUSIONS: The real-world evidence obtained from LungPath shows the effort of Spanish hospitals in performing biomarker determination in NSCLC with different methodologies despite that next-generation sequencing (NGS) utilisation in the year of the analysis was low. Biomarker determination results could be optimised with the incorporation of sequencing methods such as NGS in pathology departments.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Humans , Carcinoma, Non-Small-Cell Lung/diagnosis , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Lung Neoplasms/diagnosis , Lung Neoplasms/genetics , Lung Neoplasms/pathology , B7-H1 Antigen , Protein-Tyrosine Kinases , Biomarkers, Tumor/genetics , Biomarkers, Tumor/analysis , Prospective Studies , Proto-Oncogene Proteins/metabolism , ErbB Receptors/genetics , Lung/pathology , RegistriesABSTRACT
Mice with insulin receptor (IR)-deficient astrocytes (GFAP-IR knockout [KO] mice) show blunted responses to insulin and reduced brain glucose uptake, whereas IR-deficient astrocytes show disturbed mitochondrial responses to glucose. While exploring the functional impact of disturbed mitochondrial function in astrocytes, we observed that GFAP-IR KO mice show uncoupling of brain blood flow with glucose uptake. Since IR-deficient astrocytes show higher levels of reactive oxidant species (ROS), this leads to stimulation of hypoxia-inducible factor-1α and, consequently, of the vascular endothelial growth factor angiogenic pathway. Indeed, GFAP-IR KO mice show disturbed brain vascularity and blood flow that is normalized by treatment with the antioxidant N-acetylcysteine (NAC). NAC ameliorated high ROS levels, normalized angiogenic signaling and mitochondrial function in IR-deficient astrocytes, and normalized neurovascular coupling in GFAP-IR KO mice. Our results indicate that by modulating glucose uptake and angiogenesis, insulin receptors in astrocytes participate in neurovascular coupling.
Subject(s)
Astrocytes , Brain , Insulin , Neovascularization, Physiologic , Neurovascular Coupling , Animals , Astrocytes/metabolism , Brain/blood supply , Glial Fibrillary Acidic Protein/genetics , Glucose/metabolism , Insulin/metabolism , Mice , Mice, Knockout , Reactive Oxygen Species/metabolism , Receptor, Insulin/genetics , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Coleoid cephalopods camouflage on timescales of seconds to match their visual surroundings. To date, studies of cephalopod camouflage-to-substrate have been focused primarily on benthic cuttlefish and octopus, because they are readily found sitting on the substrate. In contrast to benthic cephalopods, oval squid (Sepioteuthis lessoniana species complex) are semi-pelagic animals that spend most of their time in the water column. In this study, we demonstrate that in captivity, S. lessoniana Sp.2 (Shiro-ika, white-squid) from the Okinawa archipelago, Japan, adapts the coloration of their skin using their chromatophores according to the background substrate. We show that if the animal moves between substrates of different reflectivity, the body patterning is changed to match. Chromatophore matching to substrate has not been reported in any loliginid cephalopod under laboratory conditions. Adaptation of the chromatophore system to the bottom substrate in the laboratory is a novel experimental finding that establishes oval squid as laboratory model animals for further research on camouflage.
Subject(s)
Chromatophores , Octopodiformes , Animals , Decapodiformes , JapanABSTRACT
AIM: The aim of this study was to describe the testing rate and frequency of molecular alterations observed in the Lung Cancer Biomarker Testing Registry (LungPath). METHODS: A descriptive study of NSCLC biomarker determinations collected from March 2018 to January 2019, from 38 Spanish hospitals, was carried out. Only adenocarcinoma and not otherwise specified histologies were included for epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), c-ros oncogene 1 (ROS1) and programmed death ligand-1 (PD-L1) expression. The testing rate and the positivity rate were calculated. Multivariate logistic regression was used to explore the joint relationship between independent explanatory factors and both testing and positivity rates. Two models were adjusted: one with sample type and histology as independent factors, and the other adding the testing rate or the positivity rate of the other biomarkers. RESULTS: 3226 patient samples were analysed, where EGFR, ALK, ROS1 and PD-L1 information was collected (a total of 12 904 determinations). Overall, 9118 (71.4%) determinations were finally assessed. EGFR (91.4%) and ALK (80.1%) were the mainly tested biomarkers. Positivity rates for EGFR, ALK, ROS1 and PD-L1 were 13.6%, 3.4%, 2.0% and 49.2%, respectively. Multivariate models showed a lower testing rate for ALK in surgical pieces, fine-needle aspiration or other types of samples versus biopsies. CONCLUSIONS: Despite the high testing rate in EGFR and ALK in NSCLC, the real-world evidence obtained from the LungPath demonstrates that ROS1 and PD-L1 were not determined in a significant portion of patients. LungPath provides crucial information to improve the coverage in molecular testing in lung cancer, to monitor the positivity rate and the introduction of new biomarker testing in clinical practice.
Subject(s)
Biomarkers, Tumor/analysis , Carcinoma, Non-Small-Cell Lung/diagnosis , Lung Neoplasms/diagnosis , Anaplastic Lymphoma Kinase/analysis , B7-H1 Antigen/analysis , Carcinoma, Non-Small-Cell Lung/pathology , ErbB Receptors/analysis , Humans , Logistic Models , Lung/pathology , Lung Neoplasms/pathology , Molecular Diagnostic Techniques , Prospective Studies , Protein-Tyrosine Kinases/analysis , Proto-Oncogene Proteins/analysis , Registries , SpainABSTRACT
Huntington's disease (HD) is a hereditary neurodegenerative disorder of the basal ganglia for which disease-modifying treatments are not yet available. Although gene-silencing therapies are currently being tested, further molecular mechanisms must be explored to identify druggable targets for HD. Cytoplasmic polyadenylation element binding proteins 1 to 4 (CPEB1 to CPEB4) are RNA binding proteins that repress or activate translation of CPE-containing transcripts by shortening or elongating their poly(A) tail. Here, we found increased CPEB1 and decreased CPEB4 protein in the striatum of patients and mouse models with HD. This correlated with a reprogramming of polyadenylation in 17.3% of the transcriptome, markedly affecting neurodegeneration-associated genes including PSEN1, MAPT, SNCA, LRRK2, PINK1, DJ1, SOD1, TARDBP, FUS, and HTT and suggesting a new molecular mechanism in neurodegenerative disease etiology. We found decreased protein content of top deadenylated transcripts, including striatal atrophylinked genes not previously related to HD, such as KTN1 and the easily druggable SLC19A3 (the ThTr2 thiamine transporter). Mutations in SLC19A3 cause biotin-thiamineresponsive basal ganglia disease (BTBGD), a striatal disorder that can be treated with a combination of biotin and thiamine. Similar to patients with BTBGD, patients with HD demonstrated decreased thiamine in the cerebrospinal fluid. Furthermore, patients and mice with HD showed decreased striatal concentrations of thiamine pyrophosphate (TPP), the metabolically active form of thiamine. High-dose biotin and thiamine treatment prevented TPP deficiency in HD mice and attenuated the radiological, neuropathological, and motor HD-like phenotypes, revealing an easily implementable therapy that might benefit patients with HD.
Subject(s)
Huntington Disease , Polyadenylation , Transcription Factors/genetics , mRNA Cleavage and Polyadenylation Factors/genetics , Humans , Huntington Disease/genetics , Huntington Disease/therapy , Membrane Transport Proteins , TranscriptomeABSTRACT
Allopatry has traditionally been viewed as the primary driver of speciation in marine taxa, but the geography of the marine environment and the larval dispersal capabilities of many marine organisms render this view somewhat questionable. In marine fishes, one of the earliest and most highly cited empirical examples of ecological speciation with gene flow is the slippery dick wrasse, Halichoeres bivittatus. Evidence for this cryptic or incipient speciation event was primarily in the form of a deep divergence in a single mitochondrial locus between the northern and southern Gulf of Mexico, combined with a finding that these two haplotypes were associated with different habitat types ("tropical" vs. "subtropical") in the Florida Keys and Bermuda, where they overlap. Here, we examine habitat assortment in the Florida Keys using a broader sampling of populations and habitat types than were available for the original study. We find no evidence to support the claim that haplotype frequencies differ between habitat types, and little evidence to support any differences between populations in the Keys. These results undermine claims of ecological speciation with gene flow in Halichoeres bivittatus. Future claims of this type should be supported by multiple lines of evidence that illuminate potential mechanisms and allow researchers to rule out alternative explanations for spatial patterns of genetic differences.
ABSTRACT
Several psychiatric, neurologic and neurodegenerative disorders present increased brain ventricles volume, being hydrocephalus the disease with the major manifestation of ventriculomegaly caused by the accumulation of high amounts of cerebrospinal fluid (CSF). The molecules and pathomechanisms underlying cerebral ventricular enlargement are widely unknown. Kinase D interacting substrate of 220 kDa (KIDINS220) gene has been recently associated with schizophrenia and with a novel syndrome characterized by spastic paraplegia, intellectual disability, nystagmus and obesity (SINO syndrome), diseases frequently occurring with ventriculomegaly. Here we show that Kidins220, a transmembrane protein effector of various key neuronal signalling pathways, is a critical regulator of CSF homeostasis. We observe that both KIDINS220 and the water channel aquaporin-4 (AQP4) are markedly downregulated at the ventricular ependymal lining of idiopathic normal pressure hydrocephalus (iNPH) patients. We also find that Kidins220 deficient mice develop ventriculomegaly accompanied by water dyshomeostasis and loss of AQP4 in the brain ventricular ependymal layer and astrocytes. Kidins220 is a known cargo of the SNX27-retromer, a complex that redirects endocytosed plasma membrane proteins (cargos) back to the cell surface, thus avoiding their targeting to lysosomes for degradation. Mechanistically, we show that AQP4 is a novel cargo of the SNX27-retromer and that Kidins220 deficiency promotes a striking and unexpected downregulation of the SNX27-retromer that results in AQP4 lysosomal degradation. Accordingly, SNX27 silencing decreases AQP4 levels in wild-type astrocytes whereas SNX27 overexpression restores AQP4 content in Kidins220 deficient astrocytes. Together our data suggest that the KIDINS220-SNX27-retromer-AQP4 pathway is involved in human ventriculomegaly and open novel therapeutic perspectives.
Subject(s)
Hydrocephalus , Animals , Aquaporin 4/genetics , Aquaporin 4/metabolism , Ependyma/metabolism , Humans , Hydrocephalus/genetics , Hydrocephalus/metabolism , Lysosomes/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Sorting Nexins/geneticsABSTRACT
Hemidactylus mabouia is one of the most successful, widespread invasive reptile species and has become ubiquitous across tropical urban settings in the Western Hemisphere. Its ability to thrive in close proximity to humans has been linked to the rapid disappearance of native geckos. However, aspects of Hemidactylus mabouia natural history and ecomorphology, often assumed to be linked with this effect on native populations, remain understudied or untested. Here, we combine data from ∂15N and ∂13C stable isotopes, stomach contents, and morphometric analyses of traits associated with feeding and locomotion to test alternate hypotheses of displacement between H. mabouia and a native gecko, Phyllodactylus martini, on the island of Curaçao. We demonstrate substantial overlap of invertebrate prey resources between the species, with H. mabouia stomachs containing larger arthropod prey as well as vertebrate prey. We additionally show that H. mabouia possesses several morphological advantages, including larger sizes in feeding-associated traits and limb proportions that could offer a propulsive locomotor advantage on vertical surfaces. Together, these findings provide the first support for the hypotheses that invasive H. mabouia and native P. martini overlap in prey resources and that H. mabouia possess ecomorphological advantages over P. martini. This work provides critical context for follow-up studies of H. mabouia and P. martini natural history and direct behavioral experiments that may ultimately illuminate the mechanisms underlying displacement on this island and act as a potential model for other systems with Hemidactylus mabouia invasions.
ABSTRACT
Metastases, the major cause of death from cancer, require cells' acquisition of the ability to migrate and involve multiple steps, including local tumor cell invasion and basement membrane penetration. Certain lymphoid tumors are highly metastatic, but the mechanisms of invasion by lymphoma cells are poorly understood. We recently showed that CDCA7, a protein induced by MYC, is overexpressed in lymphoid tumors and that its knockdown decreases lymphoid tumor growth without inhibiting the proliferation of normal cells. Here we show that CDCA7 is critical for invasion and migration of lymphoma cells. Indeed, CDCA7 knockdown in lymphoma cells limited tumor cell invasion in matrigel-coated transwell plates and tumor invasion of neighboring tissues in a mouse xenograft model and in a zebrafish model of cell invasion. CDCA7 silencing markedly inhibited lymphoma cell migration on fibronectin without modifying cell adhesion to this protein. Instead, CDCA7 knockdown markedly disrupted the precise dynamic reorganization of actomyosin and tubulin cytoskeletons required for efficient migration. In particular, CDCA7 silencing impaired tubulin and actomyosin cytoskeleton polarization, increased filamentous actin formation, and induced myosin activation. Of note, inhibitors of actin polymerization, myosin II, or ROCK reestablished the migration capacity of CDCA7-silenced lymphoma cells. Given the critical role of CDCA7 in lymphoma-genesis and invasion, therapies aimed at inhibiting its expression or activity might provide significant control of lymphoma growth, invasion, and metastatic dissemination.
Subject(s)
Lymphoma , Zebrafish , Animals , Carcinogenesis , Cell Line, Tumor , Cell Movement , Cytoskeleton , Lymphoma/genetics , Mice , Neoplasm InvasivenessABSTRACT
Huntington's disease (HD) is an inherited progressive neurodegenerative disease characterized by brain atrophy particularly in the striatum that produces motor impairment, and cognitive and psychiatric disturbances. Multiple pathogenic mechanisms have been proposed including dysfunctions in neurotrophic support and calpain-overactivation, among others. Kinase D-interacting substrate of 220 kDa (Kidins220), also known as ankyrin repeat-rich membrane spanning (ARMS), is an essential mediator of neurotrophin signaling. In adult brain, Kidins220 presents two main isoforms that differ in their carboxy-terminal length and critical protein-protein interaction domains. These variants are generated through alternative terminal exon splicing of the conventional exon 32 (Kidins220-C32) and the recently identified exon 33 (Kidins220-C33). The lack of domains encoded by exon 32 involved in key neuronal functions, including those controlling neurotrophin pathways, pointed to Kidins220-C33 as a form detrimental for neurons. However, the functional role of Kidins220-C33 in neurodegeneration or other pathologies, including HD, has not been explored. In the present work, we discover an unexpected selective downregulation of Kidins220-C33, in the striatum of HD patients, as well as in the R6/1 HD mouse model starting at early symptomatic stages. These changes are C33-specific as Kidins220-C32 variant remains unchanged. We also find the early decrease in Kidins220-C33 levels takes place in neurons, suggesting an unanticipated neuroprotective role for this isoform. Finally, using ex vivo assays and primary neurons, we demonstrate that Kidins220-C33 is downregulated by mechanisms that depend on the activation of the protease calpain. Altogether, these results strongly suggest that calpain-mediated Kidins220-C33 proteolysis modulates onset and/or progression of HD.
Subject(s)
Huntington Disease/genetics , Huntington Disease/metabolism , Membrane Proteins/genetics , Nerve Tissue Proteins/genetics , Adult , Aged , Alternative Splicing , Animals , Corpus Striatum/metabolism , Disease Models, Animal , Exons/genetics , Female , Hippocampus/metabolism , Humans , Huntington Disease/pathology , Male , Membrane Proteins/metabolism , Mice , Middle Aged , Nerve Tissue Proteins/metabolism , Neurodegenerative Diseases/metabolism , Neurodegenerative Diseases/pathology , Neurons/metabolism , Neurons/pathology , Protein Binding , Protein Isoforms/genetics , Signal TransductionABSTRACT
Excitotoxic neuronal death induced by high concentrations of glutamate is a pathological event common to multiple acute or chronic neurodegenerative diseases. Excitotoxicity is mediated through overactivation of the N-Methyl-D-aspartate type of ionotropic glutamate receptors (NMDARs). Physiological stimulation of NMDARs triggers their endocytosis from the neuronal surface, inducing synaptic activity and survival. However almost nothing is known about the internalization of overactivated NMDARs and their interacting proteins, and how this endocytic process is connected with neuronal death has been poorly explored. Kinase D-interacting substrate of 220 kDa (Kidins220), also known as ankyrin repeat-rich membrane spanning (ARMS), is a component of NMDAR complexes essential for neuronal viability by the control of ERK activation. Here we have investigated Kidins220 endocytosis induced by NMDAR overstimulation and the participation of this internalization step in the molecular mechanisms of excitotoxicity. We show that excitotoxicity induces Kidins220 and GluN1 traffic to the Golgi apparatus (GA) before Kidins220 is degraded by the protease calpain. We also find that excitotoxicity triggers an early activation of Rap1-GTPase followed by its inactivation. Kidins220 excitotoxic endocytosis and subsequent calpain-mediated downregulation governs this late inactivation of Rap1 that is associated to decreases in ERK activity preceding neuronal death. Furthermore, we identify the molecular mechanisms involved in the excitotoxic shutoff of Kidins220/Rap1/ERK prosurvival cascade that depends on calpain processing of Rap1-activation complexes. Our data fit in a model where Kidins220 targeting to the GA during early excitotoxicity would facilitate Rap1 activation and subsequent stimulation of ERK. At later times, activation of Golgi-associated calpain, would promote the degradation of GA-targeted Kidins220 and two additional components of the specific Rap1 activation complex, PDZ-GEF1, and S-SCAM. In this way, late excitotoxicity would turn off Rap1/ERK cascade and compromise neuronal survival.
Subject(s)
Calpain/metabolism , Golgi Apparatus/metabolism , Membrane Proteins/metabolism , Neurons/metabolism , Phosphoproteins/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , rap1 GTP-Binding Proteins/metabolism , Animals , Calpain/antagonists & inhibitors , Cell Death/drug effects , Cell Death/genetics , Cells, Cultured , Endocytosis/drug effects , Endocytosis/genetics , Endosomes/metabolism , Glutamic Acid/metabolism , Golgi Apparatus/drug effects , Membrane Proteins/genetics , Mitogen-Activated Protein Kinase 3/metabolism , Neurons/drug effects , Neurons/enzymology , Neurons/ultrastructure , Phosphoproteins/genetics , Rats , Rats, Wistar , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Receptors, N-Methyl-D-Aspartate/genetics , Signal Transduction/genetics , rab5 GTP-Binding Proteins/metabolism , rap1 GTP-Binding Proteins/antagonists & inhibitors , rap1 GTP-Binding Proteins/geneticsABSTRACT
The nematode Caenorhabditis elegans (C. elegans) is a powerful model organism to study lifespan and aging, protein aggregation, and neurodegeneration, as well as to carry out drug screenings. The C. elegans strain aex-3/T337 expresses human pathogenic V337M mutant tau under a pan-neuronal promoter and presents uncoordinated locomotion, accumulation of phosphorylated insoluble tau, and shortened lifespan. Herein we have used this strain to assay two compounds that could affect tau aggregation and/or phosphorylation, and looked for phenotypic changes in their lifespan and motility. The first compound is Thioflavin T (ThT), a member of the tetracycline family with protein antiaggregant properties, yet to be tested in a tauopathy model. The second is a novel small molecule, NP103, a highly selective inhibitor of glycogen synthase kinase-3 (GSK-3), the main kinase contributing to pathogenic tau hyperphosphorylation. Importantly, we find that ThT extends lifespan of aex-3/T337 worms as it does with control N2 animals, showing both strains similar locomotion features under this treatment. By contrast, NP103 improves the paralysis phenotype of aex-3/T337 mutants but not their lifespan. Our results show that both treatments present beneficial effects for this model of tauopathy and encourage pursuing further investigations on their therapeutic potential for AD and other tauopathies.
ABSTRACT
Sleep is a state of immobility characterized by three key criteria: an increased threshold of arousal, rapid reversal to an alert state and evidence of homeostatic 'rebound sleep' in which there is an increase in the time spent in this quiescent state following sleep deprivation. Common European cuttlefish, Sepia officinalis, show states of quiescence during which they meet the last two of these three criteria, yet also show spontaneous bursts of arm and eye movements that accompany rapid changes in chromatophore patterns in the skin. Here, we report that this rapid eye movement sleep-like (REMS-like) state is cyclic in nature. Iterations of the REMS-like state last 2.42±0.22â min (mean±s.e.m.) and alternate with 34.01±1.49â min of the quiescent sleep-like state for durations lasting 176.89±36.71â min. We found clear evidence that this REMS-like state (i) occurs in animals younger than previously reported; (ii) follows an ultradian pattern; (iii) includes intermittent dynamic chromatophore patterning, representing fragments of normal patterning seen in the waking state for a wide range of signaling and camouflage; and (iv) shows variability in the intensity of expression of these skin patterns between and within individuals. These data suggest that cephalopods, which are mollusks with an elaborate brain and complex behavior, possess a sleep-like state that resembles behaviorally the vertebrate REM sleep state, although the exact nature and mechanism of this form of sleep may differ from that of vertebrates.