RESUMEN
The most critical stage in initiation of melanoma metastasis is the radial to vertical growth transition, yet the triggers of this transition remain elusive. We suggest that the microenvironment drives melanoma metastasis independently of mutation acquisition. Here we examined the changes in microenvironment that occur during melanoma radial growth. We show that direct contact of melanoma cells with the remote epidermal layer triggers vertical invasion via Notch signaling activation, the latter serving to inhibit MITF function. Briefly, within the native Notch ligand-free microenvironment, MITF, the melanocyte lineage master regulator, binds and represses miR-222/221 promoter in an RBPJK-dependent manner. However, when radial growth brings melanoma cells into contact with distal differentiated keratinocytes that express Notch ligands, the activated Notch intracellular domain impairs MITF binding to miR-222/221 promoter. This de-repression of miR-222/221 expression triggers initiation of invasion. Our findings may direct melanoma prevention opportunities via targeting specific microenvironments.
Asunto(s)
Queratinocitos/fisiología , Melanoma Experimental/secundario , Factor de Transcripción Asociado a Microftalmía/metabolismo , Neoplasias Cutáneas/patología , Animales , Secuencia de Bases , Sitios de Unión , Comunicación Celular , Línea Celular Tumoral , Técnicas de Cocultivo , Regulación Neoplásica de la Expresión Génica , Melanoma Experimental/metabolismo , Ratones Endogámicos NOD , Ratones SCID , MicroARNs/genética , MicroARNs/metabolismo , Invasividad Neoplásica , Trasplante de Neoplasias , Regiones Promotoras Genéticas , Interferencia de ARN , Receptores Notch/metabolismo , Transducción de Señal , Neoplasias Cutáneas/metabolismoRESUMEN
Activity-dependent neuroprotective protein (ADNP) is essential for brain formation and function. As such, de novo mutations in ADNP lead to the autistic ADNP syndrome and somatic ADNP mutations may drive Alzheimer's disease (AD) tauopathy. Sirtuin 1 (SIRT1) is positively associated with aging, the major risk for AD. Here, we revealed two key interaction sites for ADNP and SIRT1. One, at the microtubule end-binding protein (EB1 and EB3) Tau level, with EB1/EB3 serving as amplifiers for microtubule dynamics, synapse formation, axonal transport, and protection against tauopathy. Two, on the DNA/chromatin site, with yin yang 1, histone deacetylase 2, and ADNP, sharing a DNA binding motif and regulating SIRT1, ADNP, and EB1 (MAPRE1). This interaction was linked to sex- and age-dependent altered histone modification, associated with ADNP/SIRT1/WD repeat-containing protein 5, which mediates the assembly of histone modification complexes. Single-cell RNA and protein expression analyses as well as gene expression correlations placed SIRT1-ADNP and either MAPRE1 (EB1), MAPRE3 (EB3), or both in the same mouse and human cell; however, while MAPRE1 seemed to be similarly regulated to ADNP and SIRT1, MAPRE3 seemed to deviate. Finally, we demonstrated an extremely tight correlation for the gene transcripts described above, including related gene products. This correlation was specifically abolished in affected postmortem AD and Parkinson's disease brain select areas compared to matched controls, while being maintained in blood samples. Thus, we identified an ADNP-SIRT1 complex that may serve as a new target for the understanding of brain degeneration.
Asunto(s)
Histonas , Sirtuina 1 , Animales , Histonas/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Metilación , Ratones , Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Sirtuina 1/genética , Sirtuina 1/metabolismoRESUMEN
With Alzheimer's disease (AD) exhibiting reduced ability of neural stem cell renewal, we hypothesized that de novo mutations controlling embryonic development, in the form of brain somatic mutations instigate the disease. A leading gene presenting heterozygous dominant de novo autism-intellectual disabilities (ID) causing mutations is activity-dependent neuroprotective protein (ADNP), with intact ADNP protecting against AD-tauopathy. We discovered a genomic autism ADNP mutation (c.2188C>T) in postmortem AD olfactory bulbs and hippocampi. RNA-Seq of olfactory bulbs also identified a novel ADNP hotspot mutation, c.2187_2188insA. Altogether, 665 mutations in 596 genes with 441 mutations in AD patients (389 genes, 38% AD-exclusive mutations) and 104 genes presenting disease-causing mutations (OMIM) were discovered. OMIM AD mutated genes converged on cytoskeletal mechanisms, autism and ID causing mutations (about 40% each). The number and average frequencies of AD-related mutations per subject were higher in AD subjects compared to controls. RNA-seq datamining (hippocampus, dorsolateral prefrontal cortex, fusiform gyrus and superior frontal gyrus-583 subjects) yielded similar results. Overlapping all tested brain areas identified unique and shared mutations, with ADNP singled out as a gene associated with autism/ID/AD and presenting several unique aging/AD mutations. The large fusiform gyrus library (117 subjects) with high sequencing coverage correlated the c.2187_2188insA ADNP mutation frequency to Braak stage (tauopathy) and showed more ADNP mutations in AD specimens. In cell cultures, the ADNP-derived snippet NAP inhibited mutated-ADNP-microtubule (MT) toxicity and enhanced Tau-MT association. We propose a paradigm-shifting concept in the perception of AD whereby accumulating mosaic somatic mutations promote brain pathology.
Asunto(s)
Enfermedad de Alzheimer , Trastorno Autístico , Proteínas de Homeodominio/genética , Discapacidad Intelectual , Proteínas del Tejido Nervioso/genética , Enfermedad de Alzheimer/genética , Trastorno Autístico/genética , Encéfalo/metabolismo , Humanos , MutaciónRESUMEN
Activity-dependent neuroprotective protein (ADNP) and its protein snippet NAP (drug candidate CP201) regulate synapse formation and cognitive as well as behavioral functions, in part, through microtubule interaction. Given potential interactions between the microbiome and brain function, we now investigated the potential effects of the ADNP-deficient genotype, mimicking the ADNP syndrome on microbiota composition in the Adnp+/- mouse model. We have discovered a surprising robust sexually dichotomized Adnp genotype effect and correction by NAP (CP201) as follows. Most of the commensal bacterial microbiota tested were affected by the Adnp genotype and corrected by NAP treatment in a male sex-dependent manner. The following list includes all the bacterial groups tested-labeled in bold are male Adnp-genotype increased and corrected (decreased) by NAP. (1) Eubacteriaceae (EubV3), (2) Enterobacteriaceae (Entero), (3) Enterococcus genus (gEncocc), (4) Lactobacillus group (Lacto), (5) Bifidobacterium genus (BIF), (6) Bacteroides/Prevotella species (Bac), (7) Clostridium coccoides group (Coer), (8) Clostridium leptum group (Cluster IV, sgClep), and (9) Mouse intestinal Bacteroides (MIB). No similarities were found between males and females regarding sex- and genotype-dependent microbiota distributions. Furthermore, a female Adnp+/- genotype associated decrease (contrasting male increase) was observed in the Lactobacillus group (Lacto). Significant correlations were discovered between specific bacterial group loads and open-field behavior as well as social recognition behaviors. In summary, we discovered ADNP deficiency associated changes in commensal gut microbiota compositions, a sex-dependent biomarker for the ADNP syndrome and beyond. Strikingly, we discovered rapidly detected NAP (CP201) treatment-dependent biomarkers within the gut microbiota.
Asunto(s)
Trastorno del Espectro Autista/tratamiento farmacológico , Conducta Animal , Microbioma Gastrointestinal , Naftoquinonas/farmacología , Proteínas del Tejido Nervioso/deficiencia , Animales , Trastorno del Espectro Autista/microbiología , Trastorno del Espectro Autista/fisiopatología , Conducta Animal/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Microbioma Gastrointestinal/efectos de los fármacos , Genotipo , Proteínas de Homeodominio/genética , Masculino , Ratones , Ratones Transgénicos , Naftoquinonas/administración & dosificación , Naftoquinonas/farmacocinética , Proteínas del Tejido Nervioso/genética , Conducta Social , Cognición Social , SíndromeRESUMEN
The activity-dependent neuroprotective protein (ADNP), a double-edged sword, sex-dependently regulates multiple genes and was previously associated with the control of early muscle development and aging. Here we aimed to decipher the involvement of ADNP in versatile muscle gene expression patterns in correlation with motor function throughout life. Using quantitative RT-PCR we showed that Adnp+/- heterozygous deficiency in mice resulted in aberrant gastrocnemius (GC) muscle, tongue and bladder gene expression, which was corrected by the Adnp snippet, drug candidate, NAP (CP201). A significant sexual dichotomy was discovered, coupled to muscle and age-specific gene regulation. As such, Adnp was shown to regulate myosin light chain (Myl) in the gastrocnemius (GC) muscle, the language acquisition gene forkhead box protein P2 (Foxp2) in the tongue and the pituitary-adenylate cyclase activating polypeptide (PACAP) receptor PAC1 mRNA (Adcyap1r1) in the bladder, with PACAP linked to bladder function. A tight age regulation was observed, coupled to an extensive correlation to muscle function (gait analysis), placing ADNP as a muscle-regulating gene/protein.
Asunto(s)
Expresión Génica/genética , Proteínas de Homeodominio/genética , Actividad Motora/genética , Músculo Esquelético/fisiología , Proteínas del Tejido Nervioso/genética , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/genética , Animales , Retroalimentación , Femenino , Factores de Transcripción Forkhead/genética , Regulación de la Expresión Génica/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Cadenas Ligeras de Miosina/genética , ARN Mensajero/genética , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/genética , Lengua/fisiología , Vejiga Urinaria/fisiologíaRESUMEN
BACKGROUND: ADNP is essential for embryonic development. As such, de novo ADNP mutations lead to an intractable autism/intellectual disability syndrome requiring investigation. METHODS: Mimicking humans, CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 editing produced mice carrying heterozygous Adnp p.Tyr718∗ (Tyr), a paralog of the most common ADNP syndrome mutation. Phenotypic rescue was validated by treatment with the microtubule/autophagy-protective ADNP fragment NAPVSIPQ (NAP). RESULTS: RNA sequencing of spleens, representing a peripheral biomarker source, revealed Tyr-specific sex differences (e.g., cell cycle), accentuated in females (with significant effects on antigen processing and cellular senescence) and corrected by NAP. Differentially expressed, NAP-correctable transcripts, including the autophagy and microbiome resilience-linked FOXO3, were also deregulated in human patient-derived ADNP-mutated lymphoblastoid cells. There were also Tyr sex-specific microbiota signatures. Phenotypically, Tyr mice, similar to patients with ADNP syndrome, exhibited delayed development coupled with sex-dependent gait defects. Speech acquisition delays paralleled sex-specific mouse syntax abnormalities. Anatomically, dendritic spine densities/morphologies were decreased with NAP amelioration. These findings were replicated in the Adnp+/- mouse, including Foxo3 deregulation, required for dendritic spine formation. Grooming duration and nociception threshold (autistic traits) were significantly affected only in males. Early-onset tauopathy was accentuated in males (hippocampus and visual cortex), mimicking humans, and was paralleled by impaired visual evoked potentials and correction by acute NAP treatment. CONCLUSIONS: Tyr mice model ADNP syndrome pathology. The newly discovered ADNP/NAP target FOXO3 controls the autophagy initiator LC3 (microtubule-associated protein 1 light chain 3), with known ADNP binding to LC3 augmented by NAP, protecting against tauopathy. NAP amelioration attests to specificity, with potential for drug development targeting accessible biomarkers.
Asunto(s)
Trastorno Autístico , Discapacidad Intelectual , Tauopatías , Animales , Trastorno Autístico/patología , Encéfalo/metabolismo , Potenciales Evocados Visuales , Femenino , Expresión Génica , Proteínas de Homeodominio/genética , Humanos , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Masculino , Ratones , Proteínas del Tejido Nervioso/genética , Tauopatías/metabolismo , Proteínas tauRESUMEN
Almost half of the human microRNAs (miRNAs) are encoded in clusters. Although transcribed as a single unit, the levels of individual mature miRNAs often differ. The mechanisms underlying differential biogenesis of clustered miRNAs and the resulting physiological implications are mostly unknown. In this study, we report that the melanoma master transcription regulator MITF regulates the differential expression of the 99a/let-7c/125b-2 cluster by altering the distribution of RNA polymerase II along the cluster. We discovered that MITF interacts with TRIM28, a known inhibitor of RNA polymerase II transcription elongation, at the mIR-let-7c region, resulting in the pausing of RNA polymerase II activity and causing an elevation in mIR-let-7c expression; low levels of RNA polymerase II occupation over miR-99a and miR-125b-2 regions decreases their biogenesis. Furthermore, we showed that this differential expression affects the phenotypic state of melanoma cells. RNA-sequencing analysis of proliferative melanoma cells that express miR-99a and miR-125b mimics revealed a transcriptomic shift toward an invasive phenotype. Conversely, expression of a mIR-let-7c mimic in invasive melanoma cells induced a shift to a more proliferative state. We confirmed direct target genes of these miRNAs, including FGFR3, BAP1, Bcl2, TGFBR1, and CDKN1A. Our study demonstrates an MITF-governed biogenesis mechanism that results in differential expression of clustered 99a/let-7c/125b-2 miRNAs that control melanoma progression.
Asunto(s)
Adaptación Fisiológica/fisiología , Melanoma/genética , MicroARNs/genética , Animales , Línea Celular Tumoral , Femenino , Células HEK293 , Humanos , Melanoma/fisiopatología , Ratones , Factor de Transcripción Asociado a Microftalmía/fisiología , Transcripción Genética , Proteína 28 que Contiene Motivos Tripartito/fisiologíaRESUMEN
The activity-dependent neuroprotective protein (ADNP) syndrome is an autistic-like disorder, instigated by mutations in ADNP. This syndrome is characterized by developmental delays, impairments in speech, motor function, abnormal hearing, and intellectual disabilities. In the Adnp-haploinsufficient mouse model, many of these impediments are evident, appearing in a sex-dependent manner. In zebra finch songbird (ZF; Taeniopygia guttata), an animal model used for song/language studies, ADNP mRNA most robust expression is observed in the cerebrum of young males, potentially corroborating with male ZF exclusive singing behavior and developed cerebral song system. Herein, we report a similar sex-dependent ADNP expression profile, with the highest expression in the cerebrum (qRT-PCR) in the brain of another songbird, the domesticated canary (Serinus canaria domestica). Additional analyses for the mRNA transcripts of the ADNP regulator, vasoactive intestinal peptide (VIP), sister gene ADNP2, and speech-related Forkhead box protein P2 (FoxP2) revealed multiple sex and brain region-dependent positive correlations between the genes (including ADNP). Parallel transcript expression patterns for FoxP2 and VIP were observed alongside specific FoxP2 increase in males compared with females as well as VIP/ADNP2 correlations. In spatial view, a sexually independent extensive form of expression was found for ADNP in the canary cerebrum (RNA in situ hybridization). The songbird cerebral mesopallium area stood out as a potentially high-expressing ADNP tissue, further strengthening the association of ADNP with sense integration and auditory memory formation, previously implicated in mouse and human.
Asunto(s)
Trastorno Autístico/genética , Encéfalo/metabolismo , Canarios/genética , Vocalización Animal , Animales , Proteínas Aviares/genética , Proteínas Aviares/metabolismo , Encéfalo/fisiología , Canarios/fisiología , Femenino , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Masculino , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Especificidad de Órganos , Factores Sexuales , Péptido Intestinal Vasoactivo/genética , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
Activity-dependent neuroprotective protein (ADNP) mutations are linked with cognitive dysfunctions characterizing the autistic-like ADNP syndrome patients, who also suffer from delayed motor maturation. We thus hypothesized that ADNP is deregulated in versatile myopathies and that local ADNP muscle deficiency results in myopathy, treatable by the ADNP fragment NAP. Here, single-cell transcriptomics identified ADNP as a major constituent of the developing human muscle. ADNP transcript concentrations further predicted multiple human muscle diseases, with concentrations negatively correlated with the ADNP target interacting protein, microtubule end protein 1 (EB1). Reverting back to modeling at the single-cell level of the male mouse transcriptome, Adnp mRNA concentrations age-dependently correlated with motor disease as well as with sexual maturation gene transcripts, while Adnp expressing limb muscle cells significantly decreased with aging. Mouse Adnp heterozygous deficiency exhibited muscle microtubule reduction and myosin light chain (Myl2) deregulation coupled with motor dysfunction. CRISPR knockdown of adult gastrocnemius muscle Adnp in a Cas9 mouse resulted in treadmill (male) and gait (female) dysfunctions that were specifically ameliorated by treatment with the ADNP snippet, microtubule interacting, Myl2-regulating, NAP (CP201). Taken together, our studies provide new hope for personalized diagnosis/therapeutics in versatile myopathies.
Asunto(s)
Técnicas de Silenciamiento del Gen , Proteínas de Homeodominio/metabolismo , Músculos/patología , Enfermedades Musculares/metabolismo , Enfermedades Musculares/patología , Proteínas del Tejido Nervioso/metabolismo , Análisis de la Célula Individual , Síndrome Debilitante/patología , Adulto , Animales , Secuencia de Bases , Conducta Animal , Niño , Femenino , Marcha , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Humanos , Masculino , Ratones , Ratones Noqueados , Actividad Motora , Músculos/metabolismo , Células 3T3 NIH , Naftoquinonas , Proteínas del Tejido Nervioso/genética , Unión Neuromuscular/metabolismo , Unión Neuromuscular/patología , Condicionamiento Físico Animal , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células Madre/metabolismo , Síndrome Debilitante/metabolismoRESUMEN
Activity-dependent neuroprotective protein (ADNP) is essential for embryonic development with ADNP mutations leading to syndromic autism, coupled with intellectual disabilities and motor developmental delays. Here, mining human muscle gene-expression databases, we have investigated the association of ADNP transcripts with muscle aging. We discovered increased ADNP and its paralogue ADNP2 expression in the vastus lateralis muscle of aged compared to young subjects, as well as altered expression of the ADNP and the ADNP2 genes in bicep brachii muscle of elderly people, in a sex-dependent manner. Prolonged exercise resulted in decreased ADNP expression, and increased ADNP2 expression in an age-dependent manner in the vastus lateralis muscle. ADNP expression level was further correlated with 49 genes showing age-dependent changes in muscle transcript expression. A high degree of correlation with ADNP was discovered for 24 genes with the leading gene/protein being NMNAT1 (nicotinamide nucleotide adenylyl transferase 1). Looking at correlations differentiating the young and the old muscles and comparing protein interactions revealed an association of ADNP with the cell division cycle 5-like protein (CDC5L), and an aging-muscle-related interactive pathway in the vastus lateralis. In the bicep brachii, very high correlation was detected with genes associated with immune functions as well as mitochondrial structure and function among others. Taken together, the results suggest a direct association of ADNP with muscle strength and implicate ADNP fortification in the protection against age-associated muscle wasting.
Asunto(s)
Envejecimiento/genética , Envejecimiento/fisiología , Proteínas de Homeodominio/genética , Músculo Esquelético/fisiopatología , Proteínas del Tejido Nervioso/genética , Adulto , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Nicotinamida-Nucleótido Adenililtransferasa/genética , Resistencia Física/genética , Resistencia Física/fisiología , Sarcopenia/genética , Sarcopenia/fisiopatología , Transcripción Genética/genéticaRESUMEN
Focusing on microtubule heterogeneity and brain specificity allowed for initial discoveries of multiple tubulin isotypes four decades ago. Methods evolved from using radioactive labelling and single cell cultures to monoclonal antibodies recognizing discrete forms of tubulin in single neurons. With the advantage of molecular cloning and fluorescent protein tagging, essential components for microtubule dynamics/stability and function were identified, including activity-dependent neuroprotective protein, ADNP and its peptide snippet, NAP (drug candidate, davunetide/CP201). ADNP/NAP through the SxIP motif interact with microtubule end binding proteins EB1 and EB3 to increase microtubule dynamics, axonal transport and dendritic spine formation. Recent transcriptomic analysis of the young mouse brain at the single cell level enabled characterization of cell-type specific cytoskeleton related gene signatures (e.g., tubulin transcripts, microtubule-associated protein Tau, Mapt and microtubule end binding protein, EB3, Mapre3) at unprecedented detail. Here, we review these findings with a methodological perspective to highlight how cutting-edge techniques have allowed us to disentangle cytoskeleton dynamics in health and disease.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Focalización Isoeléctrica , Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , RNA-Seq , Análisis de la Célula Individual , Tubulina (Proteína)/metabolismo , AnimalesRESUMEN
Mutations in ADNP have been recently associated with intellectual disability and autism spectrum disorder. However, the clinical features of patients with this syndrome are not fully identified, and no treatment currently exists for these patients. Here, we extended the ADNP syndrome phenotype describing skin abnormalities in both a patient with ADNP syndrome and an Adnp haploinsufficient mice. The patient displayed thin dermis, hyperkeratotic lesions in periarticular areas and delayed wound healing. Patient-derived skin keratinocytes showed reduced proliferation and increased differentiation. Additionally, detection of cell cycle markers indicated that mutant cells exhibited impaired cell cycle progression. Treatment of ADNP-deficient keratinocytes with the ADNP-derived NAP peptide significantly reduced the expression of differentiation markers. Sonography and immunofluorescence staining of epidermal layers revealed that the dermis was thinner in the patient than in a healthy control. Adnp haploinsufficient mice (Adnp+/-) mimicked the human condition showing reduced dermal thickness. Intranasal administration of NAP significantly increased dermal thickness and normalized the levels of cell cycle and differentiation markers. Our observations provide a novel activity of the autism-linked ADNP in the skin that may serve to define the clinical phenotype of patients with ADNP syndrome and provide an attractive therapeutic option for skin alterations in these patients.
Asunto(s)
Trastorno del Espectro Autista/genética , Proteínas de Homeodominio/genética , Proteínas del Tejido Nervioso/genética , Anomalías Cutáneas/genética , Animales , Trastorno del Espectro Autista/patología , Ciclo Celular/genética , Diferenciación Celular/genética , Modelos Animales de Enfermedad , Haploinsuficiencia/genética , Humanos , Discapacidad Intelectual/genética , Discapacidad Intelectual/patología , Queratinocitos/metabolismo , Ratones , Ratones Transgénicos , Mutación/genética , Anomalías Cutáneas/patología , Cicatrización de Heridas/genéticaRESUMEN
Epidemiological studies suggest a direct link between melanoma and Parkinson's disease (PD); however, the underlying molecular basis is unknown. Since mutations in Parkin are the major driver of early-onset PD and Parkin was recently reported to play a role in cancer development, we hypothesized that Parkin links melanoma and PD. By analyzing whole exome/genome sequencing of Parkin from 246 melanoma patients, we identified five non-synonymous mutations, three synonymous mutations, and one splice region variant in Parkin in 3.6% of the samples. In vitro analysis showed that wild-type Parkin plays a tumor suppressive role in melanoma development resulting in cell-cycle arrest, reduction of metabolic activity, and apoptosis. Using a mass spectrometry-based analysis, we identified potential Parkin substrates in melanoma and generated a functional protein association network. The activity of mutated Parkin was assessed by protein structure modeling and examination of Parkin E3 ligase activity. The Parkin-E28K mutation impairs Parkin ubiquitination activity and abolishes its tumor suppressive effect. Taken together, our analysis of genomic sequence and in vitro data indicate that Parkin is a potential link between melanoma and Parkinson's disease. Our findings suggest new approaches for early diagnosis and treatment against both diseases.