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INTRODUCTION: Triggering receptor expressed on myeloid cells 2 (TREM2) agonists are being clinically evaluated as disease-modifying therapeutics for Alzheimer's disease. Clinically translatable pharmacodynamic (PD) biomarkers are needed to confirm drug activity and select the appropriate therapeutic dose in clinical trials. METHODS: We conducted multi-omic analyses on paired non-human primate brain and cerebrospinal fluid (CSF), and stimulation of human induced pluripotent stem cell-derived microglia cultures after TREM2 agonist treatment, followed by validation of candidate fluid PD biomarkers using immunoassays. We immunostained microglia to characterize proliferation and clustering. RESULTS: We report CSF soluble TREM2 (sTREM2) and CSF chitinase-3-like protein 1 (CHI3L1/YKL-40) as PD biomarkers for the TREM2 agonist hPara.09. The respective reduction of sTREM2 and elevation of CHI3L1 in brain and CSF after TREM2 agonist treatment correlated with transient microglia proliferation and clustering. DISCUSSION: CSF CHI3L1 and sTREM2 reflect microglial TREM2 agonism and can be used as clinical PD biomarkers to monitor TREM2 activity in the brain. HIGHLIGHTS: CSF soluble triggering receptor expressed on myeloid cells 2 (sTREM2) reflects brain target engagement for a novel TREM2 agonist, hPara.09. CSF chitinase-3-like protein 1 reflects microglial TREM2 agonism. Both can be used as clinical fluid biomarkers to monitor TREM2 activity in brain.
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Human genetics and preclinical studies have identified key contributions of TREM2 to several neurodegenerative conditions, inspiring efforts to modulate TREM2 therapeutically. Here, we characterize the activities of three TREM2 agonist antibodies in multiple mixed-sex mouse models of Alzheimer's disease (AD) pathology and remyelination. Receptor activation and downstream signaling are explored in vitro, and active dose ranges are determined in vivo based on pharmacodynamic responses from microglia. For mice bearing amyloid-ß (Aß) pathology (PS2APP) or combined Aß and tau pathology (TauPS2APP), chronic TREM2 agonist antibody treatment had limited impact on microglia engagement with pathology, overall pathology burden, or downstream neuronal damage. For mice with demyelinating injuries triggered acutely with lysolecithin, TREM2 agonist antibodies unexpectedly disrupted injury resolution. Likewise, TREM2 agonist antibodies limited myelin recovery for mice experiencing chronic demyelination from cuprizone. We highlight the contributions of dose timing and frequency across models. These results introduce important considerations for future TREM2-targeting approaches.
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Enfermedad de Alzheimer , Glicoproteínas de Membrana , Microglía , Esclerosis Múltiple , Receptores Inmunológicos , Animales , Receptores Inmunológicos/agonistas , Receptores Inmunológicos/metabolismo , Receptores Inmunológicos/genética , Glicoproteínas de Membrana/agonistas , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Ratones , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/inmunología , Femenino , Masculino , Microglía/efectos de los fármacos , Microglía/metabolismo , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Ratones Transgénicos , Anticuerpos/farmacología , Humanos , Péptidos beta-Amiloides/metabolismo , Proteínas tau/metabolismoRESUMEN
Tumor progression locus 2 (TPL2) (MAP3K8) is a central signaling node in the inflammatory response of peripheral immune cells. We find that TPL2 kinase activity modulates microglial cytokine release and is required for microglia-mediated neuron death in vitro. In acute in vivo neuroinflammation settings, TPL2 kinase activity regulates microglia activation states and brain cytokine levels. In a tauopathy model of chronic neurodegeneration, loss of TPL2 kinase activity reduces neuroinflammation and rescues synapse loss, brain volume loss, and behavioral deficits. Single-cell RNA sequencing analysis indicates that protection in the tauopathy model was associated with reductions in activated microglia subpopulations as well as infiltrating peripheral immune cells. Overall, using various models, we find that TPL2 kinase activity can promote multiple harmful consequences of microglial activation in the brain including cytokine release, iNOS (inducible nitric oxide synthase) induction, astrocyte activation, and immune cell infiltration. Consequently, inhibiting TPL2 kinase activity could represent a potential therapeutic strategy in neurodegenerative conditions.
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Quinasas Quinasa Quinasa PAM , Tauopatías , Animales , Humanos , Ratones , Encéfalo/patología , Células Cultivadas , Espinas Dendríticas/patología , Lipopolisacáridos , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Ratones Noqueados , Microglía/metabolismo , Enfermedades Neuroinflamatorias/patología , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Proteínas tau/genética , Proteínas tau/metabolismo , Tauopatías/metabolismo , Tauopatías/patología , Tauopatías/fisiopatologíaRESUMEN
Loss-of-function mutations in Nav1.7, a voltage-gated sodium channel, cause congenital insensitivity to pain (CIP) in humans, demonstrating that Nav1.7 is essential for the perception of pain. However, the mechanism by which loss of Nav1.7 results in insensitivity to pain is not entirely clear. It has been suggested that loss of Nav1.7 induces overexpression of enkephalin, an endogenous opioid receptor agonist, leading to opioid-dependent analgesia. Using behavioral pharmacology and single-cell RNA-seq analysis, we find that overexpression of enkephalin occurs only in cLTMR neurons, a subclass of sensory neurons involved in low-threshold touch detection, and that this overexpression does not play a role in the analgesia observed following genetic removal of Nav1.7. Furthermore, we demonstrate using laser speckle contrast imaging (LSCI) and in vivo electrophysiology that Nav1.7 function is required for the initiation of C-fiber action potentials (APs), which explains the observed insensitivity to pain following genetic removal or inhibition of Nav1.7.
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Analgésicos Opioides , Nociceptores , Ratones , Humanos , Animales , Analgésicos Opioides/farmacología , Potenciales de Acción , Canal de Sodio Activado por Voltaje NAV1.7/genética , Dolor/genética , Células Receptoras Sensoriales , Péptidos Opioides , Encefalinas , Ganglios EspinalesRESUMEN
Parkinson's disease (PD), a neurodegenerative disease affecting dopaminergic (DA) neurons, is characterized by decline of motor function and cognition. Dopaminergic cell loss is associated with accumulation of toxic alpha synuclein aggregates. As DA neuron death occurs late in the disease, therapeutics that block the spread of alpha synuclein may offer functional benefit and delay disease progression. To test this hypothesis, we generated antibodies to the C terminal region of synuclein with high nanomolar affinity and characterized them in in vitro and in vivo models of spread. Interestingly, we found that only antibodies with high affinity to the distal most portion of the C-terminus robustly reduced uptake of alpha synuclein preformed fibrils (PFF) and accumulation of phospho (S129) alpha synuclein in cell culture. Additionally, the antibody treatment blocked the spread of phospho (S129) alpha synuclein associated-pathology in a mouse model of synucleinopathy. Blockade of neuronal PFF uptake by different antibodies was more predictive of in vivo activity than their binding potency to monomeric or oligomeric forms of alpha synuclein. These data demonstrate that antibodies directed to the C-terminus of the alpha synuclein have differential effects on target engagement and efficacy. Furthermore, our data provides additional support for the development of alpha synuclein antibodies as a therapeutic strategy for PD patients.
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Enfermedades Neurodegenerativas , Enfermedad de Parkinson , Sinucleinopatías , Ratones , Animales , alfa-Sinucleína/metabolismo , Enfermedad de Parkinson/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Sinucleinopatías/patología , Neuronas Dopaminérgicas/metabolismoRESUMEN
The gene GPNMB is known to play roles in phagocytosis and tissue repair, and is upregulated in microglia in many mouse models of neurodegenerative disease as well as in human patients. Nearby genomic variants are associated with both elevated Parkinson's disease (PD) risk and higher expression of this gene, suggesting that inhibiting GPNMB activity might be protective in Parkinson's disease. We tested this hypothesis in three different mouse models of neurological diseases: a remyelination model and two models of alpha-synuclein pathology. We found that Gpnmb deletion had no effect on histological, cellular, behavioral, neurochemical or gene expression phenotypes in any of these models. These data suggest that Gpnmb does not play a major role in the development of pathology or functional defects in these models and that further work is necessary to study its role in the development or progression of Parkinson's disease.
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Proteínas del Ojo/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Enfermedad de Parkinson/metabolismo , Remielinización/genética , Sustancia Negra/metabolismo , Sinucleinopatías/genética , Anciano , Anciano de 80 o más Años , Animales , Encéfalo/metabolismo , Encéfalo/patología , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Enfermedad de Parkinson/patología , Sustancia Negra/patología , Sinucleinopatías/metabolismo , Sinucleinopatías/patologíaRESUMEN
RIP1 kinase-mediated inflammatory and cell death pathways have been implicated in the pathology of acute and chronic disorders of the nervous system. Here, we describe a novel animal model of RIP1 kinase deficiency, generated by knock-in of the kinase-inactivating RIP1(D138N) mutation in rats. Homozygous RIP1 kinase-dead (KD) rats had normal development, reproduction and did not show any gross phenotypes at baseline. However, cells derived from RIP1 KD rats displayed resistance to necroptotic cell death. In addition, RIP1 KD rats were resistant to TNF-induced systemic shock. We studied the utility of RIP1 KD rats for neurological disorders by testing the efficacy of the genetic inactivation in the transient middle cerebral artery occlusion/reperfusion model of brain injury. RIP1 KD rats were protected in this model in a battery of behavioral, imaging, and histopathological endpoints. In addition, RIP1 KD rats had reduced inflammation and accumulation of neuronal injury biomarkers. Unbiased proteomics in the plasma identified additional changes that were ameliorated by RIP1 genetic inactivation. Together these data highlight the utility of the RIP1 KD rats for target validation and biomarker studies for neurological disorders.
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Lesiones Encefálicas/genética , Muerte Celular/genética , Isquemia/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Modelos Animales de Enfermedad , Masculino , Ratas , Ratas Sprague-Dawley , Proteína Serina-Treonina Quinasas de Interacción con ReceptoresRESUMEN
Loss-of-function TREM2 mutations strongly increase Alzheimer's disease (AD) risk. Trem2 deletion has revealed protective Trem2 functions in preclinical models of ß-amyloidosis, a prominent feature of pre-diagnosis AD stages. How TREM2 influences later AD stages characterized by tau-mediated neurodegeneration is unclear. To understand Trem2 function in the context of both ß-amyloid and tau pathologies, we examined Trem2 deficiency in the pR5-183 mouse model expressing mutant tau alone or in TauPS2APP mice, in which ß-amyloid pathology exacerbates tau pathology and neurodegeneration. Single-cell RNA sequencing in these models revealed robust disease-associated microglia (DAM) activation in TauPS2APP mice that was amyloid-dependent and Trem2-dependent. In the presence of ß-amyloid pathology, Trem2 deletion further exacerbated tau accumulation and spreading and promoted brain atrophy. Without ß-amyloid pathology, Trem2 deletion did not affect these processes. Therefore, TREM2 may slow AD progression and reduce tau-driven neurodegeneration by restricting the degree to which ß-amyloid facilitates the spreading of pathogenic tau.
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Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Amiloide/metabolismo , Encéfalo/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores Inmunológicos/metabolismo , Proteínas tau/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animales , Atrofia/genética , Atrofia/metabolismo , Atrofia/patología , Encéfalo/patología , Modelos Animales de Enfermedad , Glicoproteínas de Membrana/genética , Ratones , Ratones Transgénicos , Receptores Inmunológicos/genética , Proteínas tau/genéticaRESUMEN
The transient receptor potential (TRP) superfamily of ion channels has garnered significant attention by the pharmaceutical industry. In particular, TRP channels showing high levels of expression in sensory neurons such as TRPV1, TRPA1, and TRPM8, have been considered as targets for indications where sensory neurons play a fundamental role, such as pain, itch, and asthma. Modeling these indications in rodents is challenging, especially in mice. The rat is the preferred species for pharmacological studies in pain, itch, and asthma, but until recently, genetic manipulation of the rat has been technically challenging. Here, using CRISPR technology, we have generated a TRPA1 KO rat to enable more sophisticated modeling of pain, itch, and asthma. We present a detailed phenotyping of the TRPA1 KO rat in models of pain, itch, and asthma that have previously only been investigated in the mouse. With the exception of nociception induced by direct TRPA1 activation, we have found that the TRPA1 KO rat shows apparently normal behavioral responses in multiple models of pain and itch. Immune cell infiltration into the lung in the rat OVA model of asthma, on the other hand, appears to be dependent on TRPA1, similar to was has been observed in TRPA1 KO mice. Our hope is that the TRPA1 KO rat will become a useful tool in further studies of TRPA1 as a drug target.
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Asma/genética , Conducta Animal/fisiología , Dolor/genética , Prurito/genética , Canal Catiónico TRPA1/genética , Animales , Asma/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Dolor/metabolismo , Fenotipo , Prurito/metabolismo , Ratas , Ratas Transgénicas , Canal Catiónico TRPA1/metabolismoRESUMEN
TREM2 is an Alzheimer's disease (AD) risk gene expressed in microglia. To study the role of Trem2 in a mouse model of ß-amyloidosis, we compared PS2APP transgenic mice versus PS2APP mice lacking Trem2 (PS2APP;Trem2ko) at ages ranging from 4 to 22 months. Microgliosis was impaired in PS2APP;Trem2ko mice, with Trem2-deficient microglia showing compromised expression of proliferation/Wnt-related genes and marked accumulation of ApoE. Plaque abundance was elevated in PS2APP;Trem2ko females at 6-7 months; but by 12 or 19-22 months of age, it was notably diminished in female and male PS2APP;Trem2ko mice, respectively. Across all ages, plaque morphology was more diffuse in PS2APP;Trem2ko brains, and the Aß42:Aß40 ratio was elevated. The amount of soluble, fibrillar Aß oligomers also increased in PS2APP;Trem2ko hippocampi. Associated with these changes, axonal dystrophy was exacerbated from 6 to 7 months onward in PS2APP;Trem2ko mice, notwithstanding the reduced plaque load at later ages. PS2APP;Trem2ko mice also exhibited more dendritic spine loss around plaque and more neurofilament light chain in CSF. Thus, aggravated neuritic dystrophy is a more consistent outcome of Trem2 deficiency than amyloid plaque load, suggesting that the microglial packing of Aß into dense plaque is an important neuroprotective activity.SIGNIFICANCE STATEMENT Genetic studies indicate that TREM2 gene mutations confer increased Alzheimer's disease (AD) risk. We studied the effects of Trem2 deletion in the PS2APP mouse AD model, in which overproduction of Aß peptide leads to amyloid plaque formation and associated neuritic dystrophy. Interestingly, neuritic dystrophies were intensified in the brains of Trem2-deficient mice, despite these mice displaying reduced plaque accumulation at later ages (12-22 months). Microglial clustering around plaques was impaired, plaques were more diffuse, and the Aß42:Aß40 ratio and amount of soluble, fibrillar Aß oligomers were elevated in Trem2-deficient brains. These results suggest that the Trem2-dependent compaction of Aß into dense plaques is a protective microglial activity, limiting the exposure of neurons to toxic Aß species.
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Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Axones/patología , Espinas Dendríticas/patología , Glicoproteínas de Membrana/genética , Fragmentos de Péptidos/metabolismo , Placa Amiloide/genética , Receptores Inmunológicos/genética , Factor Trefoil-1/metabolismo , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/patología , Neuritas/patología , Proteínas de Neurofilamentos/líquido cefalorraquídeo , Placa Amiloide/patologíaRESUMEN
Dual leucine zipper kinase (DLK, MAP3K12) was recently identified as an essential regulator of neuronal degeneration in multiple contexts. Here we describe the generation of potent and selective DLK inhibitors starting from a high-throughput screening hit. Using proposed hinge-binding interactions to infer a binding mode and specific design parameters to optimize for CNS druglike molecules, we came to focus on the di(pyridin-2-yl)amines because of their combination of desirable potency and good brain penetration following oral dosing. Our lead inhibitor GNE-3511 (26) displayed concentration-dependent protection of neurons from degeneration in vitro and demonstrated dose-dependent activity in two different animal models of disease. These results suggest that specific pharmacological inhibition of DLK may have therapeutic potential in multiple indications.
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Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Degeneración Nerviosa/prevención & control , Enfermedades Neurodegenerativas/prevención & control , Inhibidores de Proteínas Quinasas/farmacología , Animales , Modelos Animales de Enfermedad , Perros , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Células HEK293 , Humanos , Células de Riñón Canino Madin Darby , Ratones Endogámicos C57BL , Modelos Químicos , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , RatasRESUMEN
We used a mouse model of the schizophrenia-predisposing 22q11.2 microdeletion to evaluate how this genetic lesion affects cortical neural circuits at the synaptic, cellular, and molecular levels. Guided by cognitive deficits, we demonstrated that mutant mice display robust deficits in high-frequency synaptic transmission and short-term plasticity (synaptic depression and potentiation), as well as alterations in long-term plasticity and dendritic spine stability. Apart from previously reported reduction in dendritic complexity of layer 5 pyramidal neurons, altered synaptic plasticity occurs in the context of relatively circumscribed and often subtle cytoarchitectural changes in neuronal density and inhibitory neuron numbers. We confirmed the pronounced DiGeorge critical region 8 (Dgcr8)-dependent deficits in primary micro-RNA processing and identified additional changes in gene expression and RNA splicing that may underlie the effects of this mutation. Reduction in Dgcr8 levels appears to be a major driver of altered short-term synaptic plasticity in prefrontal cortex and working memory but not of long-term plasticity and cytoarchitecture. Our findings inform the cortical synaptic and neuronal mechanisms of working memory impairment in the context of psychiatric disorders. They also provide insight into the link between micro-RNA dysregulation and genetic liability to schizophrenia and cognitive dysfunction.
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Síndrome de DiGeorge/patología , Potenciación a Largo Plazo/genética , Depresión Sináptica a Largo Plazo/genética , Neuronas/fisiología , Corteza Prefrontal/patología , Animales , Trastornos del Conocimiento/etiología , Trastornos del Conocimiento/genética , Espinas Dendríticas/patología , Espinas Dendríticas/ultraestructura , Síndrome de DiGeorge/complicaciones , Síndrome de DiGeorge/genética , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/genética , Redes Reguladoras de Genes/genética , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/patología , Fosfopiruvato Hidratasa/metabolismo , Proteínas/genética , Proteínas de Unión al ARN , Reconocimiento en Psicología/fisiología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismoRESUMEN
22q11.2 microdeletions result in specific cognitive deficits and schizophrenia. Analysis of Df(16)A(+/-) mice, which model this microdeletion, revealed abnormalities in the formation of neuronal dendrites and spines, as well as altered brain microRNAs. Here, we show a drastic reduction of miR-185, which resides within the 22q11.2 locus, to levels more than expected by a hemizygous deletion, and we demonstrate that this reduction alters dendritic and spine development. miR-185 represses, through an evolutionarily conserved target site, a previously unknown inhibitor of these processes that resides in the Golgi apparatus and shows higher prenatal brain expression. Sustained derepression of this inhibitor after birth represents the most robust transcriptional disturbance in the brains of Df(16)A(+/-) mice and results in structural alterations in the hippocampus. Reduction of miR-185 also has milder age- and region-specific effects on the expression of some Golgi-related genes. Our findings illuminate the contribution of microRNAs in psychiatric disorders and cognitive dysfunction.
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Encéfalo/metabolismo , Embrión de Mamíferos/metabolismo , MicroARNs/metabolismo , Secuencia de Aminoácidos , Animales , Encéfalo/embriología , Deleción Cromosómica , Cromosomas Humanos Par 22/genética , Modelos Animales de Enfermedad , Aparato de Golgi/metabolismo , Hipocampo/metabolismo , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , MicroARNs/genética , Datos de Secuencia Molecular , Plasticidad Neuronal , Neuronas/metabolismo , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Proteínas de Unión al ARNRESUMEN
22q11.2 chromosomal deletions are recurrent copy number mutations that increase the risk of schizophrenia around thirty-fold. Deletion of the orthologous chromosomal region in mice offers an opportunity to characterize changes to neuronal structure and function that may account for the development of this disease. The hippocampus has been implicated in schizophrenia pathogenesis, is reduced in volume in 22q11.2 deletion carriers and displays altered neuronal structure in a mouse model of the mutation (Df(16)A(+/-) mice). Here we investigate hippocampal CA1 physiology, hippocampal-dependent spatial memory and novelty-induced hippocampal activation in Df(16)A(+/-) mice. We found normal spatial reference memory (as assayed by the Morris water maze test) as well as modest but potentially important deficits in physiology. In particular, a reduction in the level of inhibition of CA1 pyramidal neurons was observed, implying a decrease in interneuron activity. Additionally, deficits in LTP were observed using certain induction protocols. Induction of c-Fos expression by exploration of a novel environment suggested a relative sparing of CA1 and dentate gyrus function but showed a robust decrease in the number of activated CA3 pyramidal neurons in Df(16)A(+/-) mice. Overall, experiments performed in this 22q11.2 deletion model demonstrated deficits of various degrees across different regions of the hippocampus, which together may contribute to the increased risk of developing schizophrenia.
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Deleción Cromosómica , Hipocampo/fisiología , Modelos Animales , Potenciales de Acción/fisiología , Animales , Cromosomas Humanos Par 22 , Humanos , Interneuronas/metabolismo , Aprendizaje por Laberinto/fisiología , Memoria/fisiología , Ratones , Ratones Endogámicos C57BL , Plasticidad Neuronal/fisiología , Neuronas/citología , Neuronas/fisiología , Técnicas de Placa-Clamp , Proteínas Proto-Oncogénicas c-fos/metabolismo , Factores de Riesgo , Esquizofrenia/genéticaRESUMEN
Over the last fifteen years it has become established that 22q11.2 deletion syndrome (22q11DS) is a true genetic risk factor for schizophrenia. Carriers of deletions in chromosome 22q11.2 develop schizophrenia at rate of 25-30% and such deletions account for as many as 1-2% of cases of sporadic schizophrenia in the general population. Access to a relatively homogeneous population of individuals that suffer from schizophrenia as the result of a shared etiological factor and the potential to generate etiologically valid mouse models provides an immense opportunity to better understand the pathobiology of this disease. In this review we survey the clinical literature associated with the 22q11.2 microdeletions with a focus on neuroanatomical changes. Then, we highlight results from work modeling this structural mutation in animals. The key biological pathways disrupted by the mutation are discussed and how these changes impact the structure and function of neural circuits is described.
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Deleción Cromosómica , Cromosomas Humanos Par 22/genética , Predisposición Genética a la Enfermedad , Trastornos Mentales/genética , Trastornos Mentales/patología , Animales , Encéfalo/anomalías , Encéfalo/fisiología , Encéfalo/fisiopatología , Catecol O-Metiltransferasa/genética , Catecol O-Metiltransferasa/metabolismo , Modelos Animales de Enfermedad , Epistasis Genética , Humanos , Células Madre Pluripotentes Inducidas/fisiología , MicroARNs/metabolismo , Modelos Genéticos , Prolina Oxidasa/genética , Prolina Oxidasa/metabolismo , Esquizofrenia/genética , SíndromeRESUMEN
Abnormalities in functional connectivity between brain areas have been postulated as an important pathophysiological mechanism underlying schizophrenia. In particular, macroscopic measurements of brain activity in patients suggest that functional connectivity between the frontal and temporal lobes may be altered. However, it remains unclear whether such dysconnectivity relates to the aetiology of the illness, and how it is manifested in the activity of neural circuits. Because schizophrenia has a strong genetic component, animal models of genetic risk factors are likely to aid our understanding of the pathogenesis and pathophysiology of the disease. Here we study Df(16)A(+/-) mice, which model a microdeletion on human chromosome 22 (22q11.2) that constitutes one of the largest known genetic risk factors for schizophrenia. To examine functional connectivity in these mice, we measured the synchronization of neural activity between the hippocampus and the prefrontal cortex during the performance of a task requiring working memory, which is one of the cognitive functions disrupted in the disease. In wild-type mice, hippocampal-prefrontal synchrony increased during working memory performance, consistent with previous reports in rats. Df(16)A(+/-) mice, which are impaired in the acquisition of the task, showed drastically reduced synchrony, measured both by phase-locking of prefrontal cells to hippocampal theta oscillations and by coherence of prefrontal and hippocampal local field potentials. Furthermore, the magnitude of hippocampal-prefrontal coherence at the onset of training could be used to predict the time it took the Df(16)A(+/-) mice to learn the task and increased more slowly during task acquisition. These data suggest how the deficits in functional connectivity observed in patients with schizophrenia may be realized at the single-neuron level. Our findings further suggest that impaired long-range synchrony of neural activity is one consequence of the 22q11.2 deletion and may be a fundamental component of the pathophysiology underlying schizophrenia.
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Cromosomas de los Mamíferos/genética , Modelos Animales de Enfermedad , Hipocampo/fisiopatología , Corteza Prefrontal/fisiopatología , Esquizofrenia/genética , Esquizofrenia/fisiopatología , Potenciales de Acción/fisiología , Alelos , Animales , Conducta Animal/fisiología , Cromosomas Humanos Par 22/genética , Femenino , Predisposición Genética a la Enfermedad/genética , Humanos , Masculino , Memoria/fisiología , Ratones , Ratones Endogámicos C57BL , Modelos Genéticos , Modelos NeurológicosRESUMEN
Animal models have been useful in elucidating the genetic basis of the cognitive and behavioural phenotypes associated with the 22q11.2 microdeletions. Loss-of-function models have implicated a number of genes as playing a role in prepulse inhibition (PPI) of the startle response. Here, we report the generation and initial analysis of bacterial artificial chromosome (BAC) transgenic (Tg) mice, overexpressing genes from within the 22q11.2 locus. We used engineered BAC constructs to generate Tg lines and quantitative RT-PCR to assess levels of gene expression in each line. We assessed PPI and open-field activity in mice from two low copy number lines. In Tg-1, a line overexpressing Prodh and Vpreb2, PPI was significantly increased at prepulse levels of 78 dB and 82 dB while no differences were found in activity measures. By contrast, no significant differences were found in PPI testing of the Tg-2 line overexpressing Zdhhc8, Ranbp1, Htf9c, T10, Arvcf and Comt. Taken together with previous loss-of-function reports, these findings suggest that Prodh has a key role in modulating the degree of sensorimotor gating in mice and possibly in humans and provide additional support for an important role of this pathway in modulating behavioural deficits associated with genomic gains or losses at 22q11.2.
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Conducta Animal , Cromosomas de los Mamíferos , Reflejo de Sobresalto/genética , Estimulación Acústica , Animales , Cromosomas Artificiales Bacterianos , Genes Reporteros , Genotipo , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Inmunoglobulina de Cadenas Ligeras Subrogadas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Actividad Motora/genética , Fenotipo , Prolina Oxidasa/genética , Filtrado Sensorial/genéticaRESUMEN
Individuals with 22q11.2 microdeletions have cognitive deficits and a high risk of developing schizophrenia. Here we provide evidence that primary hippocampal neurons from a mouse model of 22q11.2 deletion (Df(16)A(+/-) mice) have decreased density of dendritic spines and glutamatergic synapses, as well as impaired dendritic growth. These deficits were prevented by introduction of the enzymatically active ZDHHC8 palmitoyltransferase encoded by a gene in the 22q11.2 locus, and they were also observed in primary cultures from Zdhhc8-deficient mice. Many of these deficits were also present in the hippocampi of adult Df(16)A(+/-) and Zdhhc8-deficient mice. Finally, we provide evidence that PSD95 is one of the substrates of ZDHHC8. Our analysis reveals that 22q11.2 microdeletion results in deficits in neuronal development and suggests that impaired neuronal protein palmitoylation contributes to many of these deficits.
Asunto(s)
Aciltransferasas/genética , Encefalopatías/patología , Aberraciones Cromosómicas , Deleción Cromosómica , Cromosomas Humanos Par 22/genética , Neuronas/patología , Aciltransferasas/química , Animales , Células Cultivadas , Dendritas/patología , Espinas Dendríticas/patología , Diagnóstico por Imagen/métodos , Modelos Animales de Enfermedad , Homólogo 4 de la Proteína Discs Large , Embrión de Mamíferos , Ácido Glutámico/metabolismo , Proteínas Fluorescentes Verdes/biosíntesis , Guanilato-Quinasas , Hipocampo/patología , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Potenciales de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Neuronas/fisiología , Sinapsis/genética , Sinapsis/patología , Sinapsis/fisiología , Transfección/métodosRESUMEN
Individuals with 22q11.2 microdeletions show behavioral and cognitive deficits and are at high risk of developing schizophrenia. We analyzed an engineered mouse strain carrying a chromosomal deficiency spanning a segment syntenic to the human 22q11.2 locus. We uncovered a previously unknown alteration in the biogenesis of microRNAs (miRNAs) and identified a subset of brain miRNAs affected by the microdeletion. We provide evidence that the abnormal miRNA biogenesis emerges because of haploinsufficiency of the Dgcr8 gene, which encodes an RNA-binding moiety of the 'microprocessor' complex and contributes to the behavioral and neuronal deficits associated with the 22q11.2 microdeletion.
Asunto(s)
Conducta Animal/fisiología , Encéfalo/fisiología , Deleción Cromosómica , Cromosomas Humanos Par 22/genética , Modelos Animales de Enfermedad , MicroARNs/biosíntesis , MicroARNs/genética , Animales , Trastornos del Conocimiento/genética , Femenino , Perfilación de la Expresión Génica , Habituación Psicofisiológica/genética , Heterocigoto , Humanos , Discapacidades para el Aprendizaje/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Proteínas/fisiología , Proteínas de Unión al ARN , Trastornos de la Sensación/genética , Columna Vertebral/anatomía & histología , Columna Vertebral/crecimiento & desarrolloRESUMEN
The characterization and anatomical distribution of 5-hydroxytryptamine (5-HT)(7) receptor binding sites in brain tissue has been hampered by the lack of a specific radioligand. In the present autoradiographic study, we took advantage of 5-HT(1A) knockout and 5-HT(1A/1B) double-knockout mice to revisit the pharmacological characterization and anatomical localization of 5-HT(7) binding sites in mouse brain using [(3)H]5-carboxamidotryptamine (5-CT) and [(3)H]8-hydroxy-2-(di-n-propylamino)tetraline (8-OH-DPAT). The distribution pattern of [(3)H]5-CT binding sites (2 nM) in the brain of mice lacking the 5-HT(1A/1B) receptor was scarce and confined to the septum, globus pallidus, thalamus, hypothalamus, amygdala, cortex, and substantia nigra. The low densities of [(3)H]5-CT binding sites detected in septum, thalamus, hypothalamus, amygdala, and cortex were displaced by 10 microM of the selective 5-HT(7) receptor antagonist (R)-3-(2-(2-(4-methylpiperidin-1-yl) ethyl)pyrrolidine-1-sulfonyl) phenol (SB-269970). The SB-269970-insensitive [(3)H]5-CT binding sites detected in globus pallidus and substantia nigra of 5-HT(1A/1B) knockout mice were displaced by N-[3-(2-dimethylamino)ethoxy-4-methoxy-phenyl]-2'-methyl-4'- (5-methyl-1,2,4-oxadiazol-3-yl)-(1,1'-biphenyl)-4-carboxamide hydrochloride (SB-216641) (1 microM), demonstrating the 5-HT(1D) nature of these binding sites. In contrast to the low densities of [(3)H]5-CT binding sites, high-to-moderate densities of [(3)H]8-OH-DPAT binding sites (10 nM) were found throughout the brain of 5-HT(1A) and 5-HT(1A/1B) knockout mice (olfactory system, septum, thalamus, hypothalamus, amygdala, CA3 field of the hippocampus, cortical mantle, and central gray). These [(3)H]8-OH-DPAT binding sites were displaced by 10 microM SB-269970, risperidone, and methiothepin but not by pindolol, N-tert-butyl-3-[4-(2-methoxyphenyl)piperazin-1-yl]-2-phenylpropanamide (WAY- 100135), or citalopram. We conclude that despite its high affinity for the 5-HT(7) receptor in tissue homogenates, [(3)H]5-CT is not a good tracer for measuring 5-HT(7) receptor binding sites autoradiographically. Also, the lower affinity ligand [(3)H]8-OH-DPAT is a much better tracer for autoradiographic studies at the 5-HT(7) receptor binding sites.