RESUMO
The polymorphic APOE gene is the greatest genetic determinant of sporadic Alzheimer's disease risk: the APOE4 allele increases risk, while the APOE2 allele is neuroprotective compared with the risk-neutral APOE3 allele. The neuronal endosomal system is inherently vulnerable during aging, and APOE4 exacerbates this vulnerability by driving an enlargement of early endosomes and reducing exosome release in the brain of humans and mice. We hypothesized that the protective effects of APOE2 are, in part, mediated through the endosomal pathway. Messenger RNA analyses showed that APOE2 leads to an enrichment of endosomal pathways in the brain when compared with both APOE3 and APOE4. Moreover, we show age-dependent alterations in the recruitment of key endosomal regulatory proteins to vesicle compartments when comparing APOE2 to APOE3. In contrast to the early endosome enlargement previously shown in Alzheimer's disease and APOE4 models, we detected similar morphology and abundance of early endosomes and retromer-associated vesicles within cortical neurons of aged APOE2 targeted-replacement mice compared with APOE3. Additionally, we observed increased brain extracellular levels of endosome-derived exosomes in APOE2 compared with APOE3 mice during aging, consistent with enhanced endosomal cargo clearance by exosomes to the extracellular space. Our findings thus demonstrate that APOE2 enhances an endosomal clearance pathway, which has been shown to be impaired by APOE4 and which may be protective due to APOE2 expression during brain aging.
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Envelhecimento , Apolipoproteína E2 , Encéfalo , Endossomos , Exossomos , Animais , Humanos , Camundongos , Envelhecimento/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/genética , Apolipoproteína E2/metabolismo , Apolipoproteína E2/genética , Apolipoproteína E3/metabolismo , Apolipoproteína E3/genética , Apolipoproteína E4/metabolismo , Apolipoproteína E4/genética , Encéfalo/metabolismo , Endossomos/metabolismo , Exossomos/metabolismo , Camundongos Endogâmicos C57BL , Neurônios/metabolismoRESUMO
Early-life stress has been linked to multiple neurodevelopmental and neuropsychiatric deficits. Our previous studies have linked maternal presence/absence from the nest in developing rat pups to changes in prefrontal cortex (PFC) activity. Furthermore, we have shown that these changes are modulated by serotonergic signaling. Here we test whether changes in PFC activity during early life affect the developing cortex leading to behavioral alterations in the adult. We show that inhibiting the PFC of mouse pups leads to cognitive deficits in the adult comparable to those seen following maternal separation. Moreover, we show that activating the PFC during maternal separation can prevent these behavioral deficits. To test how maternal separation affects the transcriptional profile of the PFC we performed single-nucleus RNA-sequencing. Maternal separation led to differential gene expression almost exclusively in inhibitory neurons. Among others, we found changes in GABAergic and serotonergic pathways in these interneurons. Interestingly, both maternal separation and early-life PFC inhibition led to changes in physiological responses in prefrontal activity to GABAergic and serotonergic antagonists that were similar to the responses of more immature brains. Prefrontal activation during maternal separation prevented these changes. These data point to a crucial role of PFC activity during early life in behavioral expression in adulthood.
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We elucidated the molecular fingerprint of vulnerable excitatory neurons within select cortical lamina of individuals with Down syndrome (DS) for mechanistic understanding and therapeutic potential that also informs Alzheimer's disease (AD) pathophysiology. Frontal cortex (BA9) layer III (L3) and layer V (L5) pyramidal neurons were microisolated from postmortem human DS and age- and sex-matched controls (CTR) to interrogate differentially expressed genes (DEGs) and key biological pathways relevant to neurodegenerative programs. We identified > 2300 DEGs exhibiting convergent dysregulation of gene expression in both L3 and L5 pyramidal neurons in individuals with DS versus CTR subjects. DEGs included over 100 triplicated human chromosome 21 genes in L3 and L5 neurons, demonstrating a trisomic neuronal karyotype in both laminae. In addition, thousands of other DEGs were identified, indicating gene dysregulation is not limited to trisomic genes in the aged DS brain, which we postulate is relevant to AD pathobiology. Convergent L3 and L5 DEGs highlighted pertinent biological pathways and identified key pathway-associated targets likely underlying corticocortical neurodegeneration and related cognitive decline in individuals with DS. Select key DEGs were interrogated as potential hub genes driving dysregulation, namely the triplicated DEGs amyloid precursor protein (APP) and superoxide dismutase 1 (SOD1), along with key signaling DEGs including mitogen activated protein kinase 1 and 3 (MAPK1, MAPK3) and calcium calmodulin dependent protein kinase II alpha (CAMK2A), among others. Hub DEGs determined from multiple pathway analyses identified potential therapeutic candidates for amelioration of cortical neuron dysfunction and cognitive decline in DS with translational relevance to AD.
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Síndrome de Down , Lobo Frontal , Células Piramidais , Síndrome de Down/patologia , Síndrome de Down/genética , Síndrome de Down/metabolismo , Humanos , Células Piramidais/patologia , Células Piramidais/metabolismo , Masculino , Feminino , Lobo Frontal/patologia , Lobo Frontal/metabolismo , Pessoa de Meia-Idade , Idoso , Fenótipo , Adulto , Idoso de 80 Anos ou maisRESUMO
Basal forebrain cholinergic neuron (BFCN) degeneration is a hallmark of Down syndrome (DS) and Alzheimer's disease (AD). Current therapeutics in these disorders have been unsuccessful in slowing disease progression, likely due to poorly understood complex pathological interactions and dysregulated pathways. The Ts65Dn trisomic mouse model recapitulates both cognitive and morphological deficits of DS and AD, including BFCN degeneration and has shown lifelong behavioral changes due to maternal choline supplementation (MCS). To test the impact of MCS on trisomic BFCNs, we performed laser capture microdissection to individually isolate choline acetyltransferase-immunopositive neurons in Ts65Dn and disomic littermates, in conjunction with MCS at the onset of BFCN degeneration. We utilized single population RNA sequencing (RNA-seq) to interrogate transcriptomic changes within medial septal nucleus (MSN) BFCNs. Leveraging multiple bioinformatic analysis programs on differentially expressed genes (DEGs) by genotype and diet, we identified key canonical pathways and altered physiological functions within Ts65Dn MSN BFCNs, which were attenuated by MCS in trisomic offspring, including the cholinergic, glutamatergic and GABAergic pathways. We linked differential gene expression bioinformatically to multiple neurological functions, including motor dysfunction/movement disorder, early onset neurological disease, ataxia and cognitive impairment via Ingenuity Pathway Analysis. DEGs within these identified pathways may underlie aberrant behavior in the DS mice, with MCS attenuating the underlying gene expression changes. We propose MCS ameliorates aberrant BFCN gene expression within the septohippocampal circuit of trisomic mice through normalization of principally the cholinergic, glutamatergic, and GABAergic signaling pathways, resulting in attenuation of underlying neurological disease functions.
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Doença de Alzheimer , Prosencéfalo Basal , Síndrome de Down , Camundongos , Animais , Síndrome de Down/genética , Síndrome de Down/metabolismo , Camundongos Transgênicos , Prosencéfalo Basal/metabolismo , Prosencéfalo Basal/patologia , Neurônios Colinérgicos/metabolismo , Neurônios Colinérgicos/patologia , Doença de Alzheimer/metabolismo , Modelos Animais de Doenças , Colina/metabolismo , Suplementos NutricionaisRESUMO
Down syndrome (DS) is a genetic disorder caused by triplication of human chromosome 21. In addition to intellectual disability, DS is defined by a premature aging phenotype and Alzheimer's disease (AD) neuropathology, including septohippocampal circuit vulnerability and degeneration of basal forebrain cholinergic neurons (BFCNs). The Ts65Dn mouse model recapitulates key aspects of DS/AD pathology, namely age-associated atrophy of BFCNs and cognitive decline in septohippocampal-dependent behavioral tasks. We investigated whether maternal choline supplementation (MCS), a well-tolerated treatment modality, protects vulnerable BFCNs from age- and genotype-associated degeneration in trisomic offspring. We also examined the effect of trisomy, and MCS, on GABAergic basal forebrain parvalbumin neurons (BFPNs), an unexplored neuronal population in this DS model. Unbiased stereological analyses of choline acetyltransferase (ChAT)-immunoreactive BFCNs and parvalbumin-immunoreactive BFPNs were conducted using confocal z-stacks of the medial septal nucleus and the vertical limb of the diagonal band (MSN/VDB) in Ts65Dn mice and disomic (2N) littermates at 3-4 and 10-12 months of age. MCS trisomic offspring displayed significant increases in ChAT-immunoreactive neuron number and density compared to unsupplemented counterparts, as well as increases in the area of the MSN/VDB occupied by ChAT-immunoreactive neuropil. MCS also rescued BFPN number and density in Ts65Dn offspring, a novel rescue of a non-cholinergic cell population. Furthermore, MCS prevented age-associated loss of BFCNs and MSN/VDB regional area in 2N offspring, indicating genotype-independent neuroprotective benefits. These findings demonstrate MCS provides neuroprotection of vulnerable BFCNs and non-cholinergic septohippocampal BFPNs, indicating this modality has translational value as an early life therapy for DS, as well as extending benefits to the aging population at large.
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Doença de Alzheimer , Prosencéfalo Basal , Síndrome de Down , Humanos , Animais , Camundongos , Idoso , Parvalbuminas , Neurônios GABAérgicos , Colina O-Acetiltransferase , Modelos Animais de Doenças , Degeneração Neural , Suplementos Nutricionais , ColinaRESUMO
Choline is critical for normative function of 3 major pathways in the brain, including acetylcholine biosynthesis, being a key mediator of epigenetic regulation, and serving as the primary substrate for the phosphatidylethanolamine N-methyltransferase pathway. Sufficient intake of dietary choline is critical for proper brain function and neurodevelopment. This is especially important for brain development during the perinatal period. Current dietary recommendations for choline intake were undertaken without critical evaluation of maternal choline levels. As such, recommended levels may be insufficient for both mother and fetus. Herein, we examined the impact of perinatal maternal choline supplementation (MCS) in a mouse model of Down syndrome and Alzheimer's disease, the Ts65Dn mouse relative to normal disomic littermates, to examine the effects on gene expression within adult offspring at â¼6 and 11 mo of age. We found MCS produces significant changes in offspring gene expression levels that supersede age-related and genotypic gene expression changes. Alterations due to MCS impact every gene ontology category queried, including GABAergic neurotransmission, the endosomal-lysosomal pathway and autophagy, and neurotrophins, highlighting the importance of proper choline intake during the perinatal period, especially when the fetus is known to have a neurodevelopmental disorder such as trisomy.-Alldred, M. J., Chao, H. M., Lee, S. H., Beilin, J., Powers, B. E., Petkova, E., Strupp, B. J., Ginsberg, S. D. Long-term effects of maternal choline supplementation on CA1 pyramidal neuron gene expression in the Ts65Dn mouse model of Down syndrome and Alzheimer's disease.
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Doença de Alzheimer/metabolismo , Região CA1 Hipocampal/citologia , Colina/administração & dosagem , Colina/farmacologia , Síndrome de Down/metabolismo , Fenômenos Fisiológicos da Nutrição Materna , Animais , Suplementos Nutricionais , Modelos Animais de Doenças , Epigênese Genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , GravidezRESUMO
In addition to being the greatest genetic risk factor for Alzheimer's disease, expression of the É4 allele of apolipoprotein E can lead to cognitive decline during ageing that is independent of Alzheimer's amyloid-ß and tau pathology. In human post-mortem tissue and mouse models humanized for apolipoprotein E, we examined the impact of apolipoprotein E4 expression on brain exosomes, vesicles that are produced within and secreted from late-endocytic multivesicular bodies. Compared to humans or mice homozygous for the risk-neutral É3 allele we show that the É4 allele, whether homozygous or heterozygous with an É3 allele, drives lower exosome levels in the brain extracellular space. In mice, we show that the apolipoprotein E4-driven change in brain exosome levels is age-dependent: while not present at age 6 months, it is detectable at 12 months of age. Expression levels of the exosome pathway regulators tumor susceptibility gene 101 (TSG101) and Ras-related protein Rab35 (RAB35) were found to be reduced in the brain at the protein and mRNA levels, arguing that apolipoprotein E4 genotype leads to a downregulation of exosome biosynthesis and release. Compromised exosome production is likely to have adverse effects, including diminishing a cell's ability to eliminate materials from the endosomal-lysosomal system. This reduction in brain exosome levels in 12-month-old apolipoprotein E4 mice occurs earlier than our previously reported brain endosomal pathway changes, arguing that an apolipoprotein E4-driven failure in exosome production plays a primary role in endosomal and lysosomal deficits that occur in apolipoprotein E4 mouse and human brains. Disruption of these interdependent endosomal-exosomal-lysosomal systems in apolipoprotein E4-expressing individuals may contribute to amyloidogenic amyloid-ß precursor protein processing, compromise trophic signalling and synaptic function, and interfere with a neuron's ability to degrade material, all of which are events that lead to neuronal vulnerability and higher risk of Alzheimer's disease development. Together, these data suggest that exosome pathway dysfunction is a previously unappreciated component of the brain pathologies that occur as a result of apolipoprotein E4 expression.
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Apolipoproteína E4/biossíntese , Encéfalo/metabolismo , Exossomos/metabolismo , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/metabolismo , Alelos , Animais , Apolipoproteína E3/genética , Apolipoproteína E4/genética , Proteínas de Ligação a DNA/biossíntese , Regulação para Baixo , Complexos Endossomais de Distribuição Requeridos para Transporte/biossíntese , Exossomos/ultraestrutura , Espaço Extracelular/metabolismo , Feminino , Genótipo , Humanos , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Fatores de Transcrição/biossíntese , Proteínas rab de Ligação ao GTP/biossínteseRESUMO
INTRODUCTION: Downregulation of brain-derived neurotrophic factor (BDNF) and its cognate neurotrophin receptor, TrkB, were observed during the progression of dementia, but whether the Alzheimer's disease (AD) pathological lesions diffuse plaques, (DPs), neuritic plaques (NPs), and neurofibrillary tangles (NFTs) are related to this alteration remains to be clarified. METHODS: Negative binomial (NB) regressions were performed using gene expression data accrued from a single population of CA1 pyramidal neurons and regional hippocampal dissections obtained from participants in the Rush Religious Orders Study (RROS). RESULTS: Downregulation of Bdnf is independently associated with increased entorhinal cortex NPs. Downregulation of TrkB is independently associated with increased entorhinal cortex NFTs and CA1 NPs during the progression of AD. DISCUSSION: Results indicate that BDNF and TrkB dysregulation contribute to AD neuropathology, most notably hippocampal NPs and NFTs. These data suggest attenuating BDNF/TrkB signaling deficits either at the level of BDNF, TrkB, or downstream of TrkB signaling may abrogate NPs and/or NFTs.
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Fator Neurotrófico Derivado do Encéfalo/biossíntese , Hipocampo/metabolismo , Glicoproteínas de Membrana/biossíntese , Emaranhados Neurofibrilares/metabolismo , Placa Amiloide/metabolismo , Receptor trkB/biossíntese , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Fator Neurotrófico Derivado do Encéfalo/genética , Feminino , Expressão Gênica , Hipocampo/patologia , Humanos , Masculino , Glicoproteínas de Membrana/genética , Emaranhados Neurofibrilares/genética , Emaranhados Neurofibrilares/patologia , Placa Amiloide/genética , Placa Amiloide/patologia , Valor Preditivo dos Testes , Receptor trkB/genéticaRESUMO
Hippocampal CA1 pyramidal neurons, a major component of the medial temporal lobe memory circuit, are selectively vulnerable during the progression of Alzheimer's disease (AD). The cellular mechanism(s) underlying degeneration of these neurons and the relationship to cognitive performance remains largely undefined. Here, we profiled neurotrophin and neurotrophin receptor gene expression within microdissected CA1 neurons along with regional hippocampal dissections from subjects who died with a clinical diagnosis of no cognitive impairment (NCI), mild cognitive impairment (MCI), or AD using laser capture microdissection (LCM), custom-designed microarray analysis, and qPCR of CA1 subregional dissections. Gene expression levels were correlated with cognitive test scores and AD neuropathology criteria. We found a significant downregulation of several neurotrophin genes (e.g., Gdnf, Ngfb, and Ntf4) in CA1 pyramidal neurons in MCI compared to NCI and AD subjects. In addition, the neurotrophin receptor transcripts TrkB and TrkC were decreased in MCI and AD compared to NCI. Regional hippocampal dissections also revealed select neurotrophic gene dysfunction providing evidence for vulnerability within the hippocampus proper during the progression of dementia. Downregulation of several neurotrophins of the NGF family and cognate neurotrophin receptor (TrkA, TrkB, and TrkC) genes correlated with antemortem cognitive measures including the Mini-Mental State Exam (MMSE), a composite global cognitive score (GCS), and Episodic, Semantic, and Working Memory, Perceptual Speed, and Visuospatial domains. Significant correlations were found between select neurotrophic expression downregulation and neuritic plaques (NPs) and neurofibrillary tangles (NFTs), but not diffuse plaques (DPs). These data suggest that dysfunction of neurotrophin signaling complexes have profound negative sequelae within vulnerable hippocampal cell types, which play a role in mnemonic and executive dysfunction during the progression of AD.
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Doença de Alzheimer/patologia , Disfunção Cognitiva/patologia , Hipocampo/patologia , Fatores de Crescimento Neural/metabolismo , Células Piramidais/patologia , Receptores de Fator de Crescimento Neural/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/metabolismo , Região CA1 Hipocampal/metabolismo , Região CA1 Hipocampal/patologia , Disfunção Cognitiva/metabolismo , Progressão da Doença , Feminino , Hipocampo/metabolismo , Humanos , Masculino , Células Piramidais/metabolismoRESUMO
OBJECTIVE: CD16+ /CD163+ macrophages (MΦs) and microglia accumulate in the brains of patients with human immunodeficiency virus (HIV) encephalitis (HIVE), a neuropathological correlate of the most severe form of HIV-associated neurocognitive disorders, HIV-associated dementia. Recently, we found that some parenchymal microglia in brain of HIV+ subjects without encephalitis (HIV/noE) but with varying degrees of neurocognitive impairment express CD16 and CD163, even in the absence of detectable virus production. To further our understanding of microglial activation in HIV, we investigated expression of specific genes by profiling parenchymal microglia from archival brain tissue of patients with HIVE and HIV/noE, and HIV- controls. METHODS: Single-population microarray analyses were performed on â¼2,500 laser capture microdissected CD163+ , CD16+ , or CD68+ MΦs/microglia per case, using terminal continuation RNA amplification and a custom-designed array platform. RESULTS: Several classes of microglial transcripts in HIVE and HIV/noE were altered, relative to HIV- subjects, including factors related to cell stress, immune activation, and apoptosis. Additionally, several neurotrophic factors were reduced in HIV infection, suggesting an additional mechanism of neuropathogenesis. The majority of transcripts altered in HIVE displayed intermediate changes in HIV/noE. INTERPRETATION: Our results support the notion that microglia contribute to the maintenance of brain homeostasis and their potential loss of function in the context of chronic inflammation contributes to neuropathogenesis. Furthermore, they indicate the utility of profiling MΦs/microglia to increase our understanding of microglia function, as well as to ascertain alterations in specific pathways, genes, and potentially, encoded proteins that may be amenable to targeted treatment modalities in diseases affecting the brain. Ann Neurol 2018;83:406-417.
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Complexo AIDS Demência/imunologia , Disfunção Cognitiva/imunologia , Infecções por HIV/imunologia , Microglia/imunologia , Complexo AIDS Demência/complicações , Complexo AIDS Demência/patologia , Adulto , Feminino , Perfilação da Expressão Gênica , Infecções por HIV/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Although, by age 40, individuals with Down syndrome (DS) develop amyloid-ß (Aß) plaques and tau-containing neurofibrillary tangles (NFTs) linked to cognitive impairment in Alzheimer's disease (AD), not all people with DS develop dementia. Whether Aß plaques and NFTs are associated with individuals with DS with (DSD +) and without dementia (DSD -) is under-investigated. Here, we applied quantitative immunocytochemistry and fluorescent procedures to characterize NFT pathology using antibodies specific for tau phosphorylation (pS422, AT8), truncation (TauC3, MN423), and conformational (Alz50, MC1) epitopes, as well as Aß and its precursor protein (APP) to frontal cortex (FC) and striatal tissue from DSD + to DSD - cases. Expression profiling of single pS422 labeled FC layer V and VI neurons was also determined using laser capture microdissection and custom-designed microarray analysis. Analysis revealed that cortical and striatal Aß plaque burdens were similar in DSD + and DSD - cases. In both groups, most FC plaques were neuritic, while striatal plaques were diffuse. By contrast, FC AT8-positive NFTs and neuropil thread densities were significantly greater in DSD + compared to DSD -, while striatal NFT densities were similar between groups. FC pS422-positive and TauC3 NFT densities were significantly greater than Alz50-labeled NFTs in DSD + , but not DSD - cases. Putaminal, but not caudate pS422-positive NFT density, was significantly greater than TauC3-positive NFTs. In the FC, AT8 + pS422 + Alz50, TauC3 + pS422 + Alz50, pS422 + Alz50, and TauC3 + pS422 positive NFTs were more frequent in DSD + compared to DSD- cases. Single gene-array profiling of FC pS422 positive neurons revealed downregulation of 63 of a total of 864 transcripts related to Aß/tau biology, glutamatergic, cholinergic, and monoaminergic metabolism, intracellular signaling, cell homeostasis, and cell death in DSD + compared DSD - cases. These observations suggest that abnormal tau aggregation plays a critical role in the development of dementia in DS.
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Encéfalo/patologia , Demência/etiologia , Síndrome de Down/complicações , Emaranhados Neurofibrilares/patologia , Placa Amiloide/patologia , Adulto , Demência/patologia , Síndrome de Down/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Although there are changes in gene expression and alterations in neuronal density and afferent inputs in the forebrain of trisomic mouse models of Down syndrome (DS) and Alzheimer's disease (AD), there is a lack of systematic assessments of gene expression and encoded proteins within individual vulnerable cell populations, precluding translational investigations at the molecular and cellular level. Further, no effective treatment exists to combat intellectual disability and basal forebrain cholinergic neurodegeneration seen in DS. To further our understanding of gene expression changes before and following cholinergic degeneration in a well-established mouse model of DS/AD, the Ts65Dn mouse, we assessed RNA expression levels from CA1 pyramidal neurons at two adult ages (â¼6 months of age and â¼11 months of age) in both Ts65Dn and their normal disomic (2N) littermates. We further examined a therapeutic intervention, maternal choline supplementation (MCS), which has been previously shown to lessen dysfunction in spatial cognition and attention, and have protective effects on the survival of basal forebrain cholinergic neurons in the Ts65Dn mouse model. Results indicate that MCS normalized expression of several genes in key gene ontology categories, including synaptic plasticity, calcium signaling, and AD-associated neurodegeneration related to amyloid-beta peptide (Aß) clearance. Specifically, normalized expression levels were found for endothelin converting enzyme-2 (Ece2), insulin degrading enzyme (Ide), Dyrk1a, and calcium/calmodulin-dependent protein kinase II (Camk2a), among other relevant genes. Single population expression profiling of vulnerable CA1 pyramidal neurons indicates that MCS is a viable therapeutic for long-term reprogramming of key transcripts involved in neuronal signaling that are dysregulated in the trisomic mouse brain which have translational potential for DS and AD.
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Doença de Alzheimer/metabolismo , Região CA1 Hipocampal/metabolismo , Colina/administração & dosagem , Síndrome de Down/metabolismo , Fármacos Neuroprotetores/administração & dosagem , Células Piramidais/metabolismo , Envelhecimento/metabolismo , Doença de Alzheimer/prevenção & controle , Animais , Região CA1 Hipocampal/crescimento & desenvolvimento , Suplementos Nutricionais , Modelos Animais de Doenças , Síndrome de Down/prevenção & controle , Feminino , Expressão Gênica , Masculino , Fenômenos Fisiológicos da Nutrição Materna , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Trisomy 21, or Down's syndrome (DS), is the most common genetic cause of intellectual disability. Altered neurotransmission in the brains of DS patients leads to hippocampus-dependent learning and memory deficiency. Although genetic mouse models have provided important insights into the genes and mechanisms responsible for DS-specific changes, the molecular mechanisms leading to memory deficits are not clear. We investigated whether the segmental trisomy model of DS, Ts[Rb(12.1716)]2Cje (Ts2), exhibits hippocampal glutamatergic transmission abnormalities and whether these alterations cause behavioral deficits. Behavioral assays demonstrated that Ts2 mice display a deficit in nest building behavior, a measure of hippocampus-dependent nonlearned behavior, as well as dysfunctional hippocampus-dependent spatial memory tested in the object-placement and the Y-maze spontaneous alternation tasks. Magnetic resonance spectra measured in the hippocampi revealed a significantly lower glutamate concentration in Ts2 as compared with normal disomic (2N) littermates. The glutamate deficit accompanied hippocampal NMDA receptor1 (NMDA-R1) mRNA and protein expression level downregulation in Ts2 compared with 2N mice. In concert with these alterations, paired-pulse analyses suggested enhanced synaptic inhibition and/or lack of facilitation in the dentate gyrus of Ts2 compared with 2N mice. Ts2 mice also exhibited disrupted synaptic plasticity in slice recordings of the hippocampal CA1 region. Collectively, these findings imply that deficits in glutamate and NMDA-R1 may be responsible for impairments in synaptic plasticity in the hippocampus associated with behavioral dysfunctions in Ts2 mice. Thus, these findings suggest that glutamatergic deficits have a significant role in causing intellectual disabilities in DS.
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Síndrome de Down/metabolismo , Ácido Glutâmico/metabolismo , Potenciação de Longa Duração , Aprendizagem em Labirinto , Memória , Comportamento de Nidação , Animais , Região CA1 Hipocampal/metabolismo , Região CA1 Hipocampal/fisiopatologia , Giro Denteado/metabolismo , Giro Denteado/fisiopatologia , Modelos Animais de Doenças , Síndrome de Down/fisiopatologia , Feminino , Masculino , Camundongos , Neurônios/metabolismo , Neurônios/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismo , Sinapses/fisiologiaRESUMO
Maternal choline supplementation (MCS) induces lifelong cognitive benefits in the Ts65Dn mouse, a trisomic mouse model of Down syndrome and Alzheimer's disease. To gain insight into the mechanisms underlying these beneficial effects, we conducted a study to test the hypothesis that MCS alters choline metabolism in adult Ts65Dn offspring. Deuterium-labeled methyl-d9-choline was administered to adult Ts65Dn and disomic (2N) female littermates born to choline-unsupplemented or choline-supplemented Ts65Dn dams. Enrichment of d9-choline metabolites (derived from intact choline) and d3 + d6-choline metabolites [produced when choline-derived methyl groups are used by phosphatidylethanolamine N-methyltransferase (PEMT)] was measured in harvested tissues. Adult offspring (both Ts65Dn and 2N) of choline-supplemented (vs. choline-unsupplemented) dams exhibited 60% greater (P≤0.007) activity of hepatic PEMT, which functions in de novo choline synthesis and produces phosphatidylcholine (PC) enriched in docosahexaenoic acid. Higher (P<0.001) enrichment of PEMT-derived d3 and d6 metabolites was detected in liver, plasma, and brain in both genotypes but to a greater extent in the Ts65Dn adult offspring. MCS also yielded higher (P<0.05) d9 metabolite enrichments in liver, plasma, and brain. These data demonstrate that MCS exerts lasting effects on offspring choline metabolism, including up-regulation of the hepatic PEMT pathway and enhanced provision of choline and PEMT-PC to the brain.
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Colina/farmacocinética , Síndrome de Down/metabolismo , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Efeitos Tardios da Exposição Pré-Natal , Animais , Colina/administração & dosagem , Colina/farmacologia , Colina/uso terapêutico , Suplementos Nutricionais , Ácidos Docosa-Hexaenoicos/metabolismo , Síndrome de Down/tratamento farmacológico , Feminino , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilcolinas/metabolismo , Gravidez , Distribuição TecidualRESUMO
Background: Individuals with Down syndrome (DS) have intellectual disability and develop Alzheimer's disease (AD) pathology during midlife, particularly in the hippocampal component of the medial temporal lobe memory circuit. However, molecular and cellular mechanisms underlying selective vulnerability of hippocampal CA1 neurons remains a major knowledge gap during DS/AD onset. This is compounded by evidence showing spatial (e.g., deep versus superficial) localization of pyramidal neurons (PNs) has profound effects on activity and innervation within the CA1 region. Objective: We investigated whether there is a spatial profiling difference in CA1 PNs in an aged female DS/AD mouse model. We posit dysfunction may be dependent on spatial localization and innervation patterns within discrete CA1 subfields. Methods: Laser capture microdissection was performed on trisomic CA1 PNs in an established mouse model of DS/AD compared to disomic controls, isolating the entire CA1 pyramidal neuron layer and sublayer microisolations of deep and superficial PNs from the distal CA1 (CA1a) region. Results: RNA sequencing and bioinformatic inquiry revealed dysregulation of CA1 PNs based on spatial location and innervation patterns. The entire CA1 region displayed the most differentially expressed genes (DEGs) in trisomic mice reflecting innate DS vulnerability, while trisomic CA1a deep PNs exhibited fewer but more physiologically relevant DEGs, as evidenced by bioinformatic inquiry. Conclusions: CA1a deep neurons displayed numerous DEGs linked to cognitive functions whereas CA1a superficial neurons, with approximately equal numbers of DEGs, were not linked to pathways of dysregulation, suggesting the spatial location of vulnerable CA1 PNs plays an important role in circuit dissolution.
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Neurodegenerative disorders, including Alzheimer's disease (AD) and Parkinson's disease (PD), represent debilitating conditions with complex, poorly understood pathologies. Epichaperomes, pathologic protein assemblies nucleated on key chaperones, have emerged as critical players in the molecular dysfunction underlying these disorders. In this study, we introduce the synthesis and characterization of clickable epichaperome probes, PU-TCO, positive control, and PU-NTCO, negative control. Through comprehensive in vitro assays and cell-based investigations, we establish the specificity of the PU-TCO probe for epichaperomes. Furthermore, we demonstrate the efficacy of PU-TCO in detecting epichaperomes in brain tissue with a cellular resolution, underscoring its potential as a valuable tool for dissecting single-cell responses in neurodegenerative diseases. This clickable probe is therefore poised to address a critical need in the field, offering unprecedented precision and versatility in studying epichaperomes and opening avenues for novel insights into their role in disease pathology.
RESUMO
Neurons rely on their metabolic coupling with astrocytes to combat oxidative stress. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) appears important for astrocyte-dependent neuroprotection from oxidative insults. Indeed, Nrf2 activators are effective in stroke, Parkinson disease, and Huntington disease models. However, key endogenous signals that initiate adaptive neuroprotective cascades in astrocytes, including activation of Nrf2-mediated gene expression, remain unclear. Hydrogen peroxide (H(2)O(2)) plays an important role in cell signaling and is an attractive candidate mediator of adaptive responses in astrocytes. Here we determine (i) the significance of H(2)O(2) in promoting astrocyte-dependent neuroprotection from oxidative stress, and (ii) the relevance of H(2)O(2) in inducing astrocytic Nrf2 activation. To control the duration and level of cytoplasmic H(2)O(2) production in astrocytes cocultured with neurons, we heterologously expressed the H(2)O(2)-producing enzyme Rhodotorula gracilis D-amino acid oxidase (rgDAAO) selectively in astrocytes. Exposure of rgDAAO-astrocytes to D-alanine lead to the concentration-dependent generation of H(2)O(2). Seven hours of low-level H(2)O(2) production (â¼3.7 nmol·min·mg protein) in astrocytes protected neurons from oxidative stress, but higher levels (â¼130 nmol·min·mg protein) were neurotoxic. Neuroprotection occurred without direct neuronal exposure to astrocyte-derived H(2)O(2), suggesting a mechanism specific to astrocytic intracellular signaling. Nrf2 activation mimicked the effect of astrocytic H(2)O(2) yet H(2)O(2)-induced protection was independent of Nrf2. Astrocytic protein tyrosine phosphatase inhibition also protected neurons from oxidative death, representing a plausible mechanism for H(2)O(2)-induced neuroprotection. These findings demonstrate the utility of rgDAAO for spatially and temporally controlling intracellular H(2)O(2) concentrations to uncover unique astrocyte-dependent neuroprotective mechanisms.
Assuntos
Astrócitos/metabolismo , Peróxido de Hidrogênio/metabolismo , Neurônios/metabolismo , Fármacos Neuroprotetores/metabolismo , Oxidantes/metabolismo , Estresse Oxidativo/fisiologia , Animais , Astrócitos/citologia , Células Cultivadas , Técnicas de Cocultura , D-Aminoácido Oxidase/metabolismo , Glutationa/metabolismo , Análise em Microsséries , Fator 2 Relacionado a NF-E2/metabolismo , Neurônios/citologia , Ratos , Rhodotorula/enzimologiaRESUMO
Single-cell and single-population RNA sequencing (RNA-seq) is a rapidly evolving new field of intense investigation. Recent studies indicate unique transcriptomic profiles are derived based on the spatial localization of neurons within circuits and regions. Individual neuronal subtypes can have vastly different transcriptomic fingerprints, well beyond the basic excitatory neuron and inhibitory neuron designations. To study single-population gene expression profiles of spatially characterized neurons, we have developed a methodology combining laser capture microdissection (LCM), RNA purification of single populations of neurons, and subsequent library preparation for downstream applications, including RNA-seq. LCM provides the benefit of isolating single neurons characterized by morphology or via transmitter-identified and/or receptor immunoreactivity and enables spatial localization within the sample. We utilize unfixed human postmortem and mouse brain tissue that is frozen to preserve RNA quality in order to isolate the desired neurons of interest. Microisolated neurons are then pooled for RNA purification utilizing as few as 250 individual neurons from a tissue section, precluding extraneous nonspecific tissue contaminants. Library preparation is performed from picogram RNA quantities extracted from LCM-captured neurons. Single-population RNA-seq analysis demonstrates that microisolated neurons from both postmortem human and mouse brain tissues are viable for transcriptomic profiling, including differential gene expression assessment and bioinformatic pathway inquiry.
RESUMO
Linear regression is one of the most used statistical techniques in neuroscience, including the study of the neuropathology of Alzheimer's disease (AD) dementia. However, the practical utility of this approach is often limited because dependent variables are often highly skewed and fail to meet the assumption of normality. Applying linear regression analyses to highly skewed datasets can generate imprecise results, which lead to erroneous estimates derived from statistical models. Furthermore, the presence of outliers can introduce unwanted bias, which affect estimates derived from linear regression models. Although a variety of data transformations can be utilized to mitigate these problems, these approaches are also associated with various caveats. By contrast, a robust regression approach does not impose distributional assumptions on data allowing for results to be interpreted in a similar manner to that derived using a linear regression analysis. Here, we demonstrate the utility of applying robust regression to the analysis of data derived from studies of human brain neurodegeneration where the error distribution of a dependent variable does not meet the assumption of normality. We show that the application of a robust regression approach to two independent published human clinical neuropathologic data sets provides reliable estimates of associations. We also demonstrate that results from a linear regression analysis can be biased if the dependent variable is significantly skewed, further indicating robust regression as a suitable alternate approach.
RESUMO
To evaluate molecular signatures of an individual cell type in comparison to the associated region relevant towards understanding the pathogenesis of Alzheimer's disease (AD), CA1 pyramidal neurons and the surrounding hippocampal formation were microaspirated via laser capture microdissection (LCM) from neuropathologically confirmed AD and age-matched control (CTR) subjects as well as from wild type mouse brain using single population RNA amplification methodology coupled with custom-designed microarray analysis with real-time quantitative polymerase-chain reaction (qPCR) validation. CA1 pyramidal neurons predominantly displayed downregulation of classes of transcripts related to synaptic transmission in AD versus CTR. Regional hippocampal dissections displayed downregulation of several overlapping genes found in the CA1 neuronal population related to neuronal expression, as well as upregulation of select transcripts indicative of admixed cell types including glial-associated markers and immediate-early and cell death genes. Gene level distributions observed in CA1 neurons and regional hippocampal dissections in wild type mice paralleled expression mosaics seen in postmortem human tissue. Microarray analysis was validated in qPCR studies using human postmortem brain tissue and CA1 sector and regional hippocampal dissections obtained from a mouse model of AD/Down syndrome (Ts65Dn mice) and normal disomic (2N) littermates. Classes of transcripts that have a greater percentage of the overall hybridization signal intensity within single neurons tended to be genes related to neuronal communication. The converse was also found, as classes of transcripts such as glial-associated markers were under represented in CA1 pyramidal neuron expression profiles relative to regional hippocampal dissections. These observations highlight a dilution effect that is likely to occur in conventional regional microarray and qPCR studies. Thus, single population studies of specific neurons and intrinsic circuits will likely yield informative gene expression profile data that may be subthreshold and/or underrepresented in regional studies with an admixture of cell types.