RESUMEN

The vast majority of gene mutations and/or gene knockouts result in either no observable changes, or significant deficits in molecular, cellular, or organismal function. However, in a small number of cases, mutant animal models display enhancements in specific behaviors such as learning and memory. To date, most gene deletions shown to enhance cognitive ability generally affect a limited number of pathways such as NMDA receptor- and translation-dependent plasticity, or GABA receptor- and potassium channel-mediated inhibition. While endolysosomal trafficking of AMPA receptors is a critical mediator of synaptic plasticity, mutations in genes that affect AMPAR trafficking either have no effect or are deleterious for synaptic plasticity, learning and memory. NSG2 is one of the three-member family of Neuron-specific genes (NSG1-3), which have been shown to regulate endolysosomal trafficking of a number of proteins critical for neuronal function, including AMPAR subunits (GluA1-2). Based on these findings and the largely universal expression throughout mammalian brain, we predicted that genetic knockout of NSG2 would result in significant impairments across multiple behavioral modalities including motor, affective, and learning/memory paradigms. However, in the current study we show that loss of NSG2 had highly selective effects on associative learning and memory, leaving motor and affective behaviors intact. For instance, NSG2 KO animals performed equivalent to wild-type C57Bl/6n mice on rotarod and Catwalk motor tasks, and did not display alterations in anxiety-like behavior on open field and elevated zero maze tasks. However, NSG2 KO animals demonstrated enhanced recall in the Morris water maze, accelerated reversal learning in a touch-screen task, and accelerated acquisition and enhanced recall on a Trace Fear Conditioning task. Together, these data point to a specific involvement of NSG2 on multiple types of associative learning, and expand the repertoire of pathways that can be targeted for cognitive enhancement.

RESUMEN

Increasing evidence suggests that aberrant regulation of sortilin ectodomain shedding can contribute to amyloid-ß pathology and frontotemporal dementia, although the mechanism by which this occurs has not been elucidated. Here, we probed for novel binding partners of sortilin using multiple and complementary approaches and identified two proteins of the neuron-specific gene (NSG) family, NSG1 and NSG2, that physically interact and colocalize with sortilin. We show both NSG1 and NSG2 induce subcellular redistribution of sortilin to NSG1- and NSG2-enriched compartments. However, using cell surface biotinylation, we found only NSG1 reduced sortilin cell surface expression, which caused significant reductions in uptake of progranulin, a molecular determinant for frontotemporal dementia. In contrast, we demonstrate NSG2 has no effect on sortilin cell surface abundance or progranulin uptake, suggesting specificity for NSG1 in the regulation of sortilin cell surface expression. Using metalloproteinase inhibitors and A disintegrin and metalloproteinase 10 KO cells, we further show that NSG1-dependent reduction of cell surface sortilin occurred via proteolytic processing by A disintegrin and metalloproteinase 10 with a concomitant increase in shedding of sortilin ectodomain to the extracellular space. This represents a novel regulatory mechanism for sortilin ectodomain shedding that is regulated in a neuron-specific manner. Furthermore, this finding has implications for the development of strategies for brain-specific regulation of sortilin and possibly sortilin-driven pathologies.

Asunto(s)

Proteínas Adaptadoras del Transporte Vesicular , Proteínas Portadoras , Metaloproteasas , Proteínas del Tejido Nervioso , Neuronas , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Biotinilación , Encéfalo/citología , Encéfalo/metabolismo , Encéfalo/patología , Proteínas Portadoras/metabolismo , Desintegrinas/deficiencia , Desintegrinas/genética , Desintegrinas/metabolismo , Demencia Frontotemporal/metabolismo , Metaloproteasas/antagonistas & inhibidores , Metaloproteasas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Progranulinas/metabolismo , Unión Proteica , Proteolisis , Membrana Celular/metabolismo , Péptidos beta-Amiloides/metabolismo

RESUMEN

Due to increasing advances in their manufacture and functionalization, nanoparticle-based systems have become a popular tool for in vivo drug delivery and biodetection. Recently, scintillating nanoparticles such as yttrium orthosilicate doped with cerium (Y2(SiO4)O:Ce) have come under study for their potential utility in optogenetic applications, as they emit photons upon low levels of stimulation from remote x-ray sources. The utility of such nanoparticles in vivo is hampered by rapid clearance from circulation by the mononuclear phagocytic system, which heavily restricts nanoparticle accumulation at target tissues. Local transcranial injection of nanoparticles may deliver scintillating nanoparticles to highly specific brain regions by circumventing the blood-brain barrier and avoiding phagocytic clearance. Few studies to date have examined the distribution and response to nanoparticles following localized delivery to cerebral cortex, a crucial step in understanding the therapeutic potential of nanoparticle-based biodetection in the brain. Following the synthesis and surface modification of these nanoparticles, two doses (1 and 3 mg/ml) were introduced into mouse secondary motor cortex (M2). This region was chosen as the site for RLP delivery, as it represents a common target for optogenetic manipulations of mouse behavior, and RLPs could eventually serve as an injectable x-ray inducible light delivery system. The spread of particles through the target tissue was assessed 24 hours, 72 hours, and 9 days post-injection. Y2(SiO4)O:Ce nanoparticles were found to be detectable in the brain for up to 9 days, initially diffusing through the tissue until 72 hours before achieving partial clearance by the final endpoint. Small transient increases in the presence of IBA-1+ microglia and GFAP+ astrocytic cell populations were detected near nanoparticle injection sites of both doses tested 24 hours after surgery. Taken together, these data provide evidence that Y2(SiO4)O:Ce nanoparticles coated with BSA can be injected directly into mouse cortex in vivo, where they persist for days and are broadly tolerated, such that they may be potentially utilized for remote x-ray activated stimulation and photon emission for optogenetic experiments in the near future.

Asunto(s)

Encéfalo , Nanopartículas , Ratones , Animales , Barrera Hematoencefálica , Sistemas de Liberación de Medicamentos , Transporte Biológico

RESUMEN

The Neuron-specific gene family (NSG1-3) consists of small endolysosomal proteins that are critical for trafficking multiple receptors and signaling molecules in neurons. NSG1 has been shown to play a critical role in AMPAR recycling from endosomes to plasma membrane during synaptic plasticity. However, to date nothing is known about whether NSG1 is required for normal behavior at an organismal level. Here we performed a battery of behavioral tests to determine whether loss of NSG1 would affect motor, cognitive, and/or affective behaviors, as well as circadian-related activity. Consistent with unique cerebellar expression of NSG1 among family members, we found that NSG1 was obligatory for motor coordination but not for gross motor function or learning. NSG1 knockout (KO) also altered performance across other behavioral modalities including anxiety-related and diurnal activity paradigms. Surprisingly, NSG1 KO did not cause significant impairments across all tasks within a given modality, but had specific effects within each modality. For instance, we found increases in anxiety-related behaviors in tasks with multiple stressors (e.g., elevation and exposure), but not those with a single main stressor (e.g., exposure). Interestingly, NSG1 KO animals displayed a significant increase in locomotor activity during subjective daytime, suggesting a possible impact on diurnal activity rhythms or vigilance. Surprisingly, loss of NSG1 had no effect on hippocampal-dependent learning despite previous studies showing deficits in CA1 long-term potentiation. Together, these findings do not support a role of NSG1 in hippocampal-dependent learning, but support a role in mediating proper neuronal function across amygdalar and cerebellar circuits.

Asunto(s)

Hipocampo , Neuronas , Animales , Ansiedad/genética , Endosomas/metabolismo , Hipocampo/metabolismo , Masculino , Ratones , Ratones Noqueados , Plasticidad Neuronal/fisiología , Neuronas/metabolismo

RESUMEN

With increased research funding for Alzheimer's disease (AD) and related disorders across the globe, large amounts of data are being generated. Several studies employed machine learning methods to understand the ever-growing omics data to enhance early diagnosis, map complex disease networks, or uncover potential drug targets. We describe results based on a Target Central Resource Database protein knowledge graph and evidence paths transformed into vectors by metapath matching. We extracted features between specific genes and diseases, then trained and optimized our model using XGBoost, termed MPxgb(AD). To determine our MPxgb(AD) prediction performance, we examined the top twenty predicted genes through an experimental screening pipeline. Our analysis identified potential AD risk genes: FRRS1, CTRAM, SCGB3A1, FAM92B/CIBAR2, and TMEFF2. FRRS1 and FAM92B are considered dark genes, while CTRAM, SCGB3A1, and TMEFF2 are connected to TREM2-TYROBP, IL-1ß-TNFα, and MTOR-APP AD-risk nodes, suggesting relevance to the pathogenesis of AD.

Asunto(s)

Enfermedad de Alzheimer , Enfermedad de Alzheimer/diagnóstico , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Diagnóstico Precoz , Humanos , Aprendizaje Automático , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias

RESUMEN

Pathological hyperphosphorylation and aggregation of tau (pTau) and neuroinflammation, driven by interleukin-1ß (IL-1ß), are the major hallmarks of tauopathies. Here, we show that pTau primes and activates IL-1ß. First, RNA-sequence analysis suggests paired-helical filaments (PHFs) from human tauopathy brain primes nuclear factor κB (NF-κB), chemokine, and IL-1ß signaling clusters in human primary microglia. Treating microglia with pTau-containing neuronal media, exosomes, or PHFs causes IL-1ß activation, which is NLRP3, ASC, and caspase-1 dependent. Suppression of pTau or ASC reduces tau pathology and inflammasome activation in rTg4510 and hTau mice, respectively. Although the deletion of MyD88 prevents both IL-1ß expression and activation in the hTau mouse model of tauopathy, ASC deficiency in myeloid cells reduces pTau-induced IL-1ß activation and improves cognitive function in hTau mice. Finally, pTau burden co-exists with elevated IL-1ß and ASC in autopsy brains of human tauopathies. Together, our results suggest pTau activates IL-1ß via MyD88- and NLRP3-ASC-dependent pathways in myeloid cells, including microglia.

Asunto(s)

Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Transducción de Señal , Tauopatías/patología , Proteínas tau/metabolismo , Animales , Proteínas Adaptadoras de Señalización CARD/genética , Proteínas Adaptadoras de Señalización CARD/metabolismo , Caspasa 1/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Doxorrubicina/farmacología , Humanos , Interleucina-1beta/genética , Ratones , Ratones Endogámicos C57BL , Microglía/citología , Microglía/metabolismo , Células Mieloides/citología , Células Mieloides/metabolismo , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , FN-kappa B/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Tauopatías/metabolismo , Proteínas tau/genética

RESUMEN

Objective.Non-invasive light delivery into the brain is needed forin vivooptogenetics to avoid physical damage. An innovative strategy could employ x-ray activation of radioluminescent particles (RLPs) to emit localized light. However, modulation of neuronal or synaptic function by x-ray induced radioluminescence from RLPs has not yet been demonstrated.Approach.Molecular and electrophysiological approaches were used to determine if x-ray dependent radioluminescence emitted from RLPs can activate light sensitive proteins. RLPs composed of cerium doped lutetium oxyorthosilicate (LSO:Ce), an inorganic scintillator that emits blue light, were used as they are biocompatible with neuronal function and synaptic transmission.Main results.We show that 30 min of x-ray exposure at a rate of 0.042 Gy s-1caused no change in the strength of basal glutamatergic transmission during extracellular field recordings in mouse hippocampal slices. Additionally, long-term potentiation, a robust measure of synaptic integrity, was induced after x-ray exposure and expressed at a magnitude not different from control conditions (absence of x-rays). We found that x-ray stimulation of RLPs elevated cAMP levels in HEK293T cells expressing OptoXR, a chimeric opsin receptor that combines the extracellular light-sensitive domain of rhodopsin with an intracellular second messenger signaling cascade. This demonstrates that x-ray radioluminescence from LSO:Ce particles can activate OptoXR. Next, we tested whether x-ray activation of the RLPs can enhance synaptic activity in whole-cell recordings from hippocampal neurons expressing channelrhodopsin-2, both in cell culture and acute hippocampal slices. Importantly, x-ray radioluminescence caused an increase in the frequency of spontaneous excitatory postsynaptic currents in both systems, indicating activation of channelrhodopsin-2 and excitation of neurons.Significance.Together, our results show that x-ray activation of LSO:Ce particles can heighten cellular and synaptic function. The combination of LSO:Ce inorganic scintillators and x-rays is therefore a viable method for optogenetics as an alternative to more invasive light delivery methods.

Asunto(s)

Cerio , Optogenética , Animales , Estudios de Factibilidad , Células HEK293 , Humanos , Ratones , Rayos X

RESUMEN

BACKGROUND: Fetal alcohol syndrome (FAS) due to gestational alcohol exposure represents one of the most common causes of nonheritable lifelong disability worldwide. In vitro and in vivo models have successfully recapitulated multiple facets of the disorder, including morphological and behavioral deficits, but far less is understood regarding the molecular and genetic mechanisms underlying FAS. METHODS: In this study, we utilized an in vitro human pluripotent stem cell-based (hPSC) model of corticogenesis to probe the effects of early, chronic intermittent alcohol exposure on the transcriptome of first trimester-equivalent cortical neurons. RESULTS: We used RNA sequencing of developing hPSC-derived neurons treated for 50 days with 50 mM ethanol and identified a relatively small number of biological pathways significantly altered by alcohol exposure. These included cell-type specification, axon guidance, synaptic function, and regional patterning, with a notable upregulation of WNT signaling-associated transcripts observed in alcohol-exposed cultures relative to alcohol-naïve controls. Importantly, this effect paralleled a shift in gene expression of transcripts associated with regional patterning, such that caudal forebrain-related transcripts were upregulated at the expense of more anterior ones. Results from H9 embryonic stem cells were largely replicated in an induced pluripotent stem cell line (IMR90-4), indicating that these patterning alterations are not cell line-specific. CONCLUSIONS: We found that a major effect of chronic intermittent alcohol on the developing cerebral cortex is an overall imbalance in regionalization, with enrichment of gene expression related to the production of posterodorsal progenitors and a diminution of anteroventral progenitors. This finding parallels behavioral and morphological phenotypes observed in animal models of high-dose prenatal alcohol exposure, as well as patients with FAS.

Asunto(s)

Diferenciación Celular/efectos de los fármacos , Depresores del Sistema Nervioso Central/farmacología , Corteza Cerebral/efectos de los fármacos , Etanol/farmacología , Trastornos del Espectro Alcohólico Fetal/genética , Expresión Génica/efectos de los fármacos , Transcriptoma/efectos de los fármacos , Vía de Señalización Wnt/efectos de los fármacos , Orientación del Axón/efectos de los fármacos , Orientación del Axón/genética , Diferenciación Celular/genética , Corteza Cerebral/embriología , Corteza Cerebral/metabolismo , Humanos , Técnicas In Vitro , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Técnicas de Placa-Clamp , Células Madre Pluripotentes , Prosencéfalo/efectos de los fármacos , Prosencéfalo/embriología , Prosencéfalo/metabolismo , RNA-Seq , Vía de Señalización Wnt/genética

RESUMEN

Pathological accumulation of microtubule associated protein tau in neurons is a major neuropathological hallmark of Alzheimer's disease (AD) and related tauopathies. Several attempts have been made to promote clearance of pathological tau (p-Tau) from neurons. Transcription factor EB (TFEB) has shown to clear p-Tau from neurons via autophagy. However, sustained TFEB activation and autophagy can create burden on cellular bioenergetics and can be deleterious. Here, we modified previously described two-plasmid systems of Light Activated Protein (LAP) from bacterial transcription factor-EL222 and Light Responsive Element (LRE) to encode TFEB. Upon blue-light (465 nm) illumination, the conformation changes in LAP induced LRE-driven expression of TFEB, its nuclear entry, TFEB-mediated expression of autophagy-lysosomal genes and clearance of p-Tau from neuronal cells and AD patient-derived human iPSC-neurons. Turning the blue-light off reversed the expression of TFEB-target genes and attenuated p-Tau clearance. Together, these results suggest that optically regulated TFEB expression unlocks the potential of opto-therapeutics to treat AD and other dementias.

Asunto(s)

Autofagia , Luz , Neuronas/patología , Neuronas/efectos de la radiación , Proteínas tau/metabolismo , Células HEK293 , Humanos , Neuronas/metabolismo , Señales de Localización Nuclear/metabolismo , Tauopatías/metabolismo , Tauopatías/patología , Proteínas tau/química

RESUMEN

Although circular RNAs (circRNAs) are enriched in the mammalian brain, very little is known about their potential involvement in brain function and psychiatric disease. Here, we show that circHomer1a, a neuronal-enriched circRNA abundantly expressed in the frontal cortex, derived from Homer protein homolog 1 (HOMER1), is significantly reduced in both the prefrontal cortex (PFC) and induced pluripotent stem cell-derived neuronal cultures from patients with schizophrenia (SCZ) and bipolar disorder (BD). Moreover, alterations in circHomer1a were positively associated with the age of onset of SCZ in both the dorsolateral prefrontal cortex (DLPFC) and orbitofrontal cortex (OFC). No correlations between the age of onset of SCZ and linear HOMER1 mRNA were observed, whose expression was mostly unaltered in BD and SCZ postmortem brain. Using in vivo circRNA-specific knockdown of circHomer1a in mouse PFC, we show that it modulates the expression of numerous alternative mRNA transcripts from genes involved in synaptic plasticity and psychiatric disease. Intriguingly, in vivo circHomer1a knockdown in mouse OFC resulted in specific deficits in OFC-mediated cognitive flexibility. Lastly, we demonstrate that the neuronal RNA-binding protein HuD binds to circHomer1a and can influence its synaptic expression in the frontal cortex. Collectively, our data uncover a novel psychiatric disease-associated circRNA that regulates synaptic gene expression and cognitive flexibility.

Asunto(s)

Trastorno Bipolar/genética , Cognición , Regulación de la Expresión Génica , ARN Circular/genética , Esquizofrenia/genética , Sinapsis/metabolismo , Adulto , Animales , Femenino , Proteínas de Andamiaje Homer/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Corteza Prefrontal/metabolismo

RESUMEN

The ability of small secretory microvesicles known as exosomes to influence neuronal and glial function via their microRNA (miRNA) cargo has positioned them as a novel and effective method of cell-to-cell communication. However, little is known about the role of exosome-secreted miRNAs in the regulation of glutamate receptor gene expression and their relevance for schizophrenia (SCZ) and bipolar disorder (BD). Using mature miRNA profiling and quantitative real-time PCR (qRT-PCR) in the orbitofrontal cortex (OFC) of SCZ (N = 29; 20 male and 9 female), BD (N = 26; 12 male and 14 female), and unaffected control (N = 25; 21 male and 4 female) subjects, we uncovered that miR-223, an exosome-secreted miRNA that targets glutamate receptors, was increased at the mature miRNA level in the OFC of SCZ and BD patients with positive history of psychosis at the time of death and was inversely associated with deficits in the expression of its targets glutamate ionotropic receptor NMDA-type subunit 2B (GRIN2B) and glutamate ionotropic receptor AMPA-type subunit 2 (GRIA2). Furthermore, changes in miR-223 levels in the OFC were positively and negatively correlated with inflammatory and GABAergic gene expression, respectively. Moreover, miR-223 was found to be enriched in astrocytes and secreted via exosomes, and antipsychotics were shown to control its cellular and exosomal localization in a cell-specific manner. Furthermore, addition of astrocytic exosomes in neuronal cultures resulted in a significant increase in miR-223 expression and a notable reduction in Grin2b and Gria2 mRNA levels, which was strongly inversely associated with miR-223 expression. Lastly, inhibition of astrocytic miR-223 abrogated the exosomal-mediated reduction in neuronal Grin2b expression. Taken together, our results demonstrate that the exosomal secretion of a psychosis-altered and glial-enriched miRNA that controls neuronal gene expression is regulated by antipsychotics.

Asunto(s)

Antipsicóticos/farmacología , Exosomas/efectos de los fármacos , Exosomas/metabolismo , MicroARNs/biosíntesis , Receptores de N-Metil-D-Aspartato/biosíntesis , Esquizofrenia/metabolismo , Animales , Antipsicóticos/uso terapéutico , Células Cultivadas , Exosomas/genética , Femenino , Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/genética , Esquizofrenia/tratamiento farmacológico , Esquizofrenia/genética

RESUMEN

Neurons have evolved a number of unique protein-coding genes that regulate trafficking of protein complexes within small organelles throughout dendrites and axons. Neuron-specific gene 2 (NSG2) encodes for one of the most abundant proteins in the nervous system during perinatal development. NSG2 belongs to a family of small neuronal endosomal proteins but its function has remained uncharacterized to date. Here, we show that NSG2 is found in discrete punctae restricted to the somatodendritic arbors of developing mouse and human neurons, and a significant proportion of NSG2 punctae colocalize with postsynaptic HOMER1 and surface-expressed AMPA-type glutamate receptors (AMPARs) at excitatory synapses. Immunoprecipitation revealed that NSG2 physically interacts with both the GluA1 and GluA2 AMPAR subunits in mouse brain. Knock-out of NSG2 in mouse hippocampal neurons selectively impaired the frequency of miniature EPSCs (mEPSCs) and caused alterations in PSD95 expression at postsynaptic densities (PSDs). In contrast, NSG2 overexpression caused a significant increase in the amplitude of mEPSCs as well as GluA2 surface expression. Thus, NSG2 functions as an AMPAR-binding protein that is required for normal synapse formation and/or maintenance, and has unique functions compared with other NSG family members.

Asunto(s)

Proteínas Portadoras/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Receptores AMPA/metabolismo , Transmisión Sináptica/fisiología , Animales , Proteínas Portadoras/genética , Línea Celular Tumoral , Expresión Génica , Técnicas de Inactivación de Genes , Células HEK293 , Hipocampo/citología , Hipocampo/metabolismo , Proteínas de Andamiaje Homer/metabolismo , Humanos , Proteínas de la Membrana , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/genética , Neuronas/citología , Técnicas de Placa-Clamp , Sinapsis/metabolismo

RESUMEN

Adult neurogenesis is necessary for proper cognition and behavior, however, the mechanisms that underlie the integration and maturation of newborn neurons into the pre-existing hippocampal circuit are not entirely known. In this study, we sought to determine the role of action potential (AP)-dependent synaptic transmission by adult-generated dentate granule cells (DGCs) in their survival and function within the existing circuitry. We used a triple transgenic mouse (NestinCreERT2 :Snap25fl/fl : tdTomato) to inducibly inactivate AP-dependent synaptic transmission within adult hippocampal progenitors and their progeny. Behavioral testing in a hippocampal-dependent A/B contextual fear-discrimination task revealed impaired discrimination learning in mice harboring SNAP-25-deficient adult-generated dentate granule cells (DGCs). Despite poor performance on this neurogenesis-dependent task, the production and survival of newborn DGCs was quantitatively unaltered in tamoxifen-treated NestinCreERT2 :Snap25fl/fl : tdTomato SNAP compared to tamoxifen-treated NestinCreERT2 :Snap25wt/wt : tdTomato control mice. Although SNAP-25-deficient adult DGCs displayed a small but statistically significant enhancement in proximal dendritic branching, their overall dendritic length and distal branching complexity was unchanged. SNAP-25-deficient newborn DGCs also displayed robust efferent mossy fiber output to CA3, with normal linear density of large mossy fiber terminals (LMTs). These studies suggest that AP-dependent neurotransmitter release by newborn DGCs is not essential for their survival or rudimentary structural maturation within the adult hippocampus.

Asunto(s)

Hipocampo/citología , Hipocampo/crecimiento & desarrollo , Discapacidades para el Aprendizaje/genética , Neurogénesis/fisiología , Neuronas/fisiología , Proteína 25 Asociada a Sinaptosomas/deficiencia , Animales , Animales Recién Nacidos , Células Cultivadas , Aprendizaje Discriminativo/efectos de los fármacos , Aprendizaje Discriminativo/fisiología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/genética , Miedo/fisiología , Ácido Glutámico/farmacología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Discapacidades para el Aprendizaje/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Nestina/genética , Nestina/metabolismo , Neuronas/efectos de los fármacos , Fosfopiruvato Hidratasa/metabolismo , ARN Mensajero/metabolismo , Proteína 25 Asociada a Sinaptosomas/genética , Transfección

RESUMEN

Down syndrome (DS) is a genetic disorder that causes cognitive impairment. The staggering effects associated with an extra copy of human chromosome 21 (HSA21) complicates mechanistic understanding of DS pathophysiology. We examined the neuron-astrocyte interplay in a fully recapitulated HSA21 trisomy cellular model differentiated from DS-patient-derived induced pluripotent stem cells (iPSCs). By combining calcium imaging with genetic approaches, we discovered the functional defects of DS astroglia and their effects on neuronal excitability. Compared with control isogenic astroglia, DS astroglia exhibited more-frequent spontaneous calcium fluctuations, which reduced the excitability of co-cultured neurons. Furthermore, suppressed neuronal activity could be rescued by abolishing astrocytic spontaneous calcium activity either chemically by blocking adenosine-mediated signaling or genetically by knockdown of inositol triphosphate (IP3) receptors or S100B, a calcium binding protein coded on HSA21. Our results suggest a mechanism by which DS alters the function of astrocytes, which subsequently disturbs neuronal excitability.

Asunto(s)

Astrocitos/metabolismo , Señalización del Calcio , Síndrome de Down/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Modelos Biológicos , Neuronas/metabolismo , Animales , Astrocitos/patología , Calcio/metabolismo , Diferenciación Celular , Síndrome de Down/patología , Retículo Endoplásmico/metabolismo , Humanos , Imagenología Tridimensional , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Neuronas/patología , Proteínas S100/metabolismo , Sinapsis/metabolismo

RESUMEN

Default differentiation of human pluripotent stem cells has been promoted as a model of cortical development. In this study, a developmental transcriptome analysis of default-differentiated hPSNs revealed a gene expression program resembling in vivo CGE/LGE subpallial domains and GABAergic signaling. A combination of bioinformatic, functional, and immunocytochemical analysis further revealed that hPSNs consist of both cortical glutamatergic and CGE-like GABAergic neurons. This study provides a comprehensive characterization of the heterogeneous group of neurons produced by default differentiation and insight into future directed differentiation strategies.

Asunto(s)

Neuronas GABAérgicas/citología , Regulación del Desarrollo de la Expresión Génica , Neurogénesis , Células Madre Pluripotentes/citología , Factor de Transcripción COUP II/genética , Factor de Transcripción COUP II/metabolismo , Calbindina 2/genética , Calbindina 2/metabolismo , Células Cultivadas , Neuronas GABAérgicas/metabolismo , Ácido Glutámico/metabolismo , Humanos , Células Madre Pluripotentes/metabolismo , Transcriptoma

RESUMEN

BACKGROUND: Prenatal alcohol exposure (PAE) in animal models results in excitatory-inhibitory (E/I) imbalance in neocortex due to alterations in the GABAergic interneuron (IN) differentiation and migration. Thus, E/I imbalance is a potential cause for intellectual disability in individuals with fetal alcohol spectrum disorder (FASD), but whether ethanol (EtOH) changes glutamatergic and GABAergic IN specification during human development remains unknown. Here, we created a human cellular model of PAE/FASD and tested the hypothesis that EtOH exposure during differentiation of human pluripotent stem cell-derived neurons (hPSNs) would cause the aberrant production of glutamatergic and GABAergic neurons, resulting in E/I imbalance. METHODS: We applied 50 mM EtOH daily to differentiating hPSNs for 50 days to model chronic first-trimester exposure. We used quantitative polymerase chain reaction, immunocytochemical, and electrophysiological analysis to examine the effects of EtOH on hPSN specification and functional E/I balance. RESULTS: We found that EtOH did not alter neural induction nor general forebrain patterning and had no effect on the expression of markers of excitatory cortical pyramidal neurons. In contrast, our data revealed highly significant changes to levels of transcripts involved with IN precursor development (e.g., GSX2, DLX1/2/5/6, NR2F2) as well as mature IN specification (e.g., SST, NPY). Interestingly, EtOH did not affect the number of GABAergic neurons generated nor the frequency or amplitude of miniature excitatory and inhibitory postsynaptic currents. CONCLUSIONS: Similar to in vivo rodent studies, EtOH significantly and specifically altered the expression of genes involved with IN specification from hPSNs, but did not cause imbalances of synaptic excitation-inhibition. Thus, our findings corroborate previous studies pointing to aberrant neuronal differentiation as an underlying mechanism of intellectual disability in FASD. However, in contrast to rodent binge models, our chronic exposure model suggests possible compensatory mechanisms that may cause more subtle defects of network processing rather than gross alterations in total E/I balance.

Asunto(s)

Depresores del Sistema Nervioso Central/efectos adversos , Etanol/efectos adversos , Neuronas GABAérgicas/efectos de los fármacos , Neurogénesis/efectos de los fármacos , Células Madre Pluripotentes/efectos de los fármacos , Excitabilidad Cortical/efectos de los fármacos , Humanos , Potenciales de la Membrana/efectos de los fármacos

RESUMEN

The process of neuroepithelial differentiation from human pluripotent stem cells (PSCs) resembles in vivo neuroectoderm induction in the temporal course, morphogenesis, and biochemical changes. This in vitro model is therefore well-suited to reveal previously unknown molecular mechanisms underlying neural induction in humans. By transcriptome analysis of cells along PSC differentiation to early neuroepithelia at day 6 and definitive neuroepithelia at day 10, we found downregulation of genes that are associated with TGF-ß and canonical WNT/ß-CATENIN signaling, confirming the roles of classical signaling in human neural induction. Interestingly, WNT/Ca(2+) signaling was upregulated. Pharmacological inhibition of the downstream effector of WNT/Ca(2+) pathway, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), led to an inhibition of the neural marker PAX6 and upregulation of epidermal marker K18, suggesting that Ca(2+)/CaMKII signaling promotes neural induction by preventing the alternative epidermal fate. In addition, our analyses revealed known and novel expression patterns of genes that are involved in DNA methylation, histone modification, as well as epithelial-mesenchymal transition, highlighting potential roles of those genes and signaling pathways during neural differentiation.

Asunto(s)

Perfilación de la Expresión Génica , Células Neuroepiteliales/metabolismo , Vía de Señalización Wnt/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Diferenciación Celular/genética , Ectodermo/embriología , Ectodermo/metabolismo , Epigénesis Genética , Transición Epitelial-Mesenquimal/genética , Espacio Extracelular/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Humanos , Células Neuroepiteliales/citología , Neurogénesis/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación , Prosencéfalo/embriología , Prosencéfalo/metabolismo , Transducción de Señal/genética , Factores de Tiempo

RESUMEN

Stem cell-derived neurons from various source materials present unique model systems to examine the fundamental properties of central nervous system (CNS) development as well as the molecular underpinnings of disease phenotypes. In order to more accurately assess potential therapies for neurological disorders, multiple strategies have been employed in recent years to produce neuronal populations that accurately represent in vivo regional and transmitter phenotypes. These include new technologies such as direct conversion of somatic cell types into neurons and glia which may accelerate maturation and retain genetic hallmarks of aging. In addition, novel forms of genetic manipulations have brought human stem cells nearly on par with those of rodent with respect to gene targeting. For neurons of the CNS, the ultimate phenotypic characterization lies with their ability to recapitulate functional properties such as passive and active membrane characteristics, synaptic activity, and plasticity. These features critically depend on the coordinated expression and localization of hundreds of ion channels and receptors, as well as scaffolding and signaling molecules. In this review I will highlight the current state of knowledge regarding functional properties of human stem cell-derived neurons, with a primary focus on pluripotent stem cells. While significant advances have been made, critical hurdles must be overcome in order for this technology to support progression toward clinical applications.

RESUMEN

The cause of Alzheimer disease (AD) is not well understood and there is no cure. Our ability to understand the early events in the course of AD is severely limited by the difficulty of identifying individuals who are in the early, preclinical stage of this disease. Most individuals with Down's syndrome (DS, trisomy 21) will predictably develop AD and that they will do so at a young age makes them an ideal population in which to study the early stages of AD. Several recent studies have exploited induced pluripotent stem cells (iPSCs) generated from individuals with familial AD, spontaneous AD and DS to attempt to identify early events and discover novel biomarkers of disease progression in AD. Here, we summarize the progress and limitations of these iPSC studies with a focus on iPSC-derived neurons. Further, we outline the methodology and results for comparing gene expression between AD and DS iPSC-derived neurons. We highlight differences and commonalities in these data that may implicate underlying genes and pathways that are causative for AD.

Asunto(s)

Enfermedad de Alzheimer/complicaciones , Síndrome de Down/genética , Síndrome de Down/patología , Perfilación de la Expresión Génica/métodos , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas/metabolismo , Animales , Síndrome de Down/complicaciones , Humanos