Intraneuronal Amyloid Beta Accumulation Disrupts Hippocampal CRTC1-Dependent Gene Expression and Cognitive Function in a Rat Model of Alzheimer Disease.
ABSTRACT: In Alzheimer disease (AD), the accumulation of amyloid beta (A?) begins decades before cognitive symptoms and progresses from intraneuronal material to extracellular plaques. To date, however, the precise mechanism by which the early buildup of A? peptides leads to cognitive dysfunction remains unknown. Here, we investigate the impact of the early A? accumulation on temporal and frontal lobe dysfunction. We compared the performance of McGill-R-Thy1-APP transgenic AD rats with wild-type littermate controls on a visual discrimination task using a touchscreen operant platform. Subsequently, we conducted studies to establish the biochemical and molecular basis for the behavioral alterations. It was found that the presence of intraneuronal A? caused a severe associative learning deficit in the AD rats. This coincided with reduced nuclear translocation and genomic occupancy of the CREB co-activator, CRTC1, and decreased production of synaptic plasticity-associated transcripts Arc, c-fos, Egr1, and Bdnf. Thus, blockade of CRTC1-dependent gene expression in the early, preplaque phase of AD-like pathology provides a molecular basis for the cognitive deficits that figure so prominently in early AD.
Project description:Chronic inflammation during Alzheimer's disease (AD) is most often attributed to sustained microglial activation in response to amyloid-? (A?) plaque deposits and cell death. However, cytokine release and microgliosis are consistently observed in AD transgenic animal models devoid of such pathologies, bringing into question the underlying processes that may be at play during the earliest AD-related immune response. We propose that this plaque-independent inflammatory reaction originates from neurons burdened with increasing levels of soluble and oligomeric A?, which are known to be the most toxic amyloid species within the brain. Laser microdissected neurons extracted from preplaque amyloid precursor protein (APP) transgenic rats were found to produce a variety of potent immune factors, both at the transcript and protein levels. Neuron-derived cytokines correlated with the extent of microglial activation and mobilization, even in the absence of extracellular plaques and cell death. Importantly, we identified an inflammatory profile unique to A?-burdened neurons, since neighboring glial cells did not express similar molecules. Moreover, we demonstrate within disease-vulnerable regions of the human brain that a neuron-specific inflammatory response may precede insoluble A? plaque and tau tangle formation. Thus, we reveal the A?-burdened neuron as a primary proinflammatory agent, implicating the intraneuronal accumulation of A? as a significant immunological component in the AD pathogenesis.
Project description:CRTC1 (CREB regulated transcription coactivator 1) gene plays a role in synaptic plasticity, learning and long-term memory formation in the hippocampus. Recently, CRTC1 has been shown to be downregulated in Alzheimer's disease (AD). Nevertheless, the mechanisms underlying CRTC1 dysregulation in AD remain unclear.To understand better the epigenetic mechanisms regulating CRTC1 expression that may be altered in AD, we profiled DNA methylation at CpG site resolution by bisulfite cloning sequencing in two promoter regions (referred to as Prom1 and Prom2) of the CRTC1 gene in human hippocampus from controls and AD cases. Next, we correlated DNA methylation levels with AD-related pathology, i.e., ?-amyloid and phosphorylated-tau (p-tau) burden and also measured CRTC1 mRNA levels by RT-qPCR.Methylation levels were lower in AD cases as compared to controls within both promoter regions (Prom1: 0.95% vs. 5%, p-value?<?0.01 and Prom2: 2.80% vs. 17.80%, p-value?<?0.001). Interestingly, CRTC1 methylation levels inversely correlated with AD-related neuropathological changes, particularly with p-tau deposition (rSpearman?=?-0.903, p?<?0.001). Moreover, a 1.54-fold decrease in CRTC1 mRNA levels was observed in hippocampus of AD cases compared to controls (p?<?0.05) supporting the notion that CRTC1 is downregulated in the AD hippocampus.DNA methylation levels within two distinct promoter regions of the CRTC1 gene were decreased in human hippocampus affected by AD compared with controls and methylation within Prom1 showed a strong inverse correlation with p-tau deposition. Further studies are guaranteed to elucidate the precise role that CRTC1 methylation plays in AD pathophysiology.
Project description:Pathologic aggregation of ?-amyloid (A?) peptide and the axonal microtubule-associated protein tau protein are hallmarks of Alzheimer's disease (AD). Evidence supports that A? peptide accumulation precedes microtubule-related pathology, although the link between A? and tau remains unclear. We previously provided evidence for early co-localization of A?42 peptides and hyperphosphorylated tau within postsynaptic terminals of CA1 dendrites in the hippocampus of AD transgenic mice. Here, we explore the relation between A? peptide accumulation and the dendritic, microtubule-associated protein 2 (MAP2) in the well-characterized amyloid precursor protein Swedish mutant transgenic mouse (Tg2576). We provide evidence that localized intraneuronal accumulation of A?42 peptides is spatially associated with reductions of MAP2 in dendrites and postsynaptic compartments of Tg2576 mice at early ages. Our data support that reduction in MAP2 begins at sites of A?42 monomer and low molecular weight oligomer (M/LMW) peptide accumulation. Cumulative evidence suggests that accumulation of M/LMW A?42 peptides occurs early, before high molecular weight oligomerization and plaque formation. Since synaptic alteration is the best pathologic correlate of cognitive dysfunction in AD, the spatial association of M/LMW A? peptide accumulation with pathology of MAP2 within neuronal processes and synaptic compartments early in the disease process reinforces the importance of intraneuronal A? accumulation in AD pathogenesis.
Project description:In contrast to extracellular plaque and intracellular tangle pathology, the presence and relevance of intraneuronal Abeta in Alzheimer's disease (AD) is still a matter of debate. Human brain tissue offers technical challenges such as post-mortem delay and uneven or prolonged tissue fixation that might affect immunohistochemical staining. In addition, previous studies on intracellular Abeta accumulation in human brain often used antibodies targeting the C-terminus of Abeta and differed strongly in the pretreatments used. To overcome these inconsistencies, we performed extensive parametrical testing using a highly specific N-terminal Abeta antibody detecting the aspartate at position 1, before developing an optimal staining protocol for intraneuronal Abeta detection in paraffin-embedded sections from AD patients. To rule out that this antibody also detects the beta-cleaved APP C-terminal fragment (beta-CTF, C99) bearing the same epitope, paraffin-sections of transgenic mice overexpressing the C99-fragment were stained without any evidence for cross-reactivity in our staining protocol. The staining intensity of intraneuronal Abeta in cortex and hippocampal tissue of 10 controls and 20 sporadic AD cases was then correlated to patient data including sex, Braak stage, plaque load, and apolipoprotein E (ApoE) genotype. In particular, the presence of one or two ApoE4 alleles strongly correlated with an increased accumulation of intraneuronal Abeta peptides. Given that ApoE4 is a major genetic risk factor for AD and is involved in neuronal cholesterol transport, it is tempting to speculate that perturbed intracellular trafficking is involved in the increased intraneuronal Abeta aggregation in AD.
Project description:The form(s) of amyloid-? peptide (A?) associated with the pathology characteristic of Alzheimer's disease (AD) remains unclear. In particular, the neurotoxicity of intraneuronal A? accumulation is an issue of considerable controversy; even the existence of A? deposits within neurons has recently been challenged by Winton and co-workers. These authors purport that it is actually intraneuronal APP that is being detected by antibodies thought to be specific for A?. To further address this issue, an anti-A? antibody was developed (MOAB-2) that specifically detects A?, but not APP. This antibody allows for the further evaluation of the early accumulation of intraneuronal A? in transgenic mice with increased levels of human A? in 5xFAD and 3xTg mice.MOAB-2 (mouse IgG2b) is a pan-specific, high-titer antibody to A? residues 1-4 as demonstrated by biochemical and immunohistochemical analyses (IHC), particularly compared to 6E10 (a commonly used commercial antibody to A? residues 3-8). MOAB-2 did not detect APP or APP-CTFs in cell culture media/lysates (HEK-APPSwe or HEK-APPSwe/BACE1) or in brain homogenates from transgenic mice expressing 5 familial AD (FAD) mutation (5xFAD mice). Using IHC on 5xFAD brain tissue, MOAB-2 immunoreactivity co-localized with C-terminal antibodies specific for A?40 and A?42. MOAB-2 did not co-localize with either N- or C-terminal antibodies to APP. In addition, no MOAB-2-immunoreactivity was observed in the brains of 5xFAD/BACE-/- mice, although significant amounts of APP were detected by N- and C-terminal antibodies to APP, as well as by 6E10. In both 5xFAD and 3xTg mouse brain tissue, MOAB-2 co-localized with cathepsin-D, a marker for acidic organelles, further evidence for intraneuronal A?, distinct from A? associated with the cell membrane. MOAB-2 demonstrated strong intraneuronal and extra-cellular immunoreactivity in 5xFAD and 3xTg mouse brain tissues.Both intraneuronal A? accumulation and extracellular A? deposition was demonstrated in 5xFAD mice and 3xTg mice with MOAB-2, an antibody that will help differentiate intracellular A? from APP. However, further investigation is required to determine whether a molecular mechanism links the presence of intraneuronal A? with neurotoxicity. As well, understanding the relevance of these observations to human AD patients is critical.
Project description:Numerous studies have implicated the abnormal accumulation of intraneuronal amyloid-? (A?) as an important contributor to Alzheimer's disease (AD) pathology, capable of triggering neuroinflammation, tau hyperphosphorylation and cognitive deficits. However, the occurrence and pathological relevance of intracellular A? remain a matter of controversial debate. In this study, we have used a multidimensional approach including high-magnification and super-resolution microscopy, cerebro-spinal fluid (CSF) mass spectrometry analysis and ELISA to investigate the A? pathology and its associated cognitive impairments, in a novel transgenic rat model overexpressing human APP. Our microscopy studies with quantitative co-localization analysis revealed the presence of intraneuronal A? in transgenic rats, with an immunological signal that was clearly distinguished from that of the amyloid precursor protein (APP) and its C-terminal fragments (CTFs). The early intraneuronal pathology was accompanied by a significant elevation of soluble A?42 peptides that paralleled the presence and progression of early cognitive deficits, several months prior to amyloid plaque deposition. A?38, A?39, A?40 and A?42 peptides were detected in the rat CSF by MALDI-MS analysis even at the plaque-free stages; suggesting that a combination of intracellular and soluble extracellular A? may be responsible for impairing cognition at early time points. Taken together, our results demonstrate that the intraneuronal development of AD-like amyloid pathology includes a mixture of molecular species (A?, APP and CTFs) of which a considerable component is A?; and that the early presence of these species within neurons has deleterious effects in the CNS, even before the development of full-blown AD-like pathology.
Project description:Transcription of hepatitis B virus (HBV) from the covalently closed circular DNA (cccDNA) template is essential for its replication. Suppressing the level and transcriptional activity of cccDNA might have anti-HBV effect. Although cellular transcription factors, such as CREB, which mediate HBV transcription, have been well described, transcriptional coactivators that facilitate this process are incompletely understood. In this study we showed that CREB-regulated transcriptional coactivator 1 (CRTC1) is required for HBV transcription and replication. The steady-state levels of CRTC1 protein were elevated in HBV-positive hepatoma cells and liver tissues. Ectopic expression of CRTC1 or its homolog CRTC2 or CRTC3 in hepatoma cells stimulated the activity of the preS2/S promoter of HBV, whereas overexpression of a dominant inactive form of CRTC1 inhibited HBV transcription. CRTC1 interacts with CREB and they are mutually required for the recruitment to the preS2/S promoter on cccDNA and for the activation of HBV transcription. Accumulation of pregenomic RNA (pgRNA) and cccDNA was observed when CRTC1 or its homologs were overexpressed, whereas the levels of pgRNA, cccDNA and secreted HBsAg were diminished when CRTC1 was compromised. In addition, HBV transactivator protein HBx stabilized CRTC1 and promoted its activity on HBV transcription. Our work reveals an essential role of CRTC1 coactivator in facilitating and supporting HBV transcription and replication.
Project description:The adipocyte-derived hormone leptin maintains energy balance by acting on hypothalamic leptin receptors (Leprs) that act on the signal transducer and activator of transcription 3 (Stat3). Although disruption of Lepr-Stat3 signaling promotes obesity in mice, other features of Lepr function, such as fertility, seem normal, pointing to the involvement of additional regulators. Here we show that the cyclic AMP responsive element-binding protein-1 (Creb1)-regulated transcription coactivator-1 (Crtc1) is required for energy balance and reproduction-Crtc1(-/-) mice are hyperphagic, obese and infertile. Hypothalamic Crtc1 was phosphorylated and inactive in leptin-deficient ob/ob mice, while leptin administration increased amounts of dephosphorylated nuclear Crtc1. Dephosphorylated Crtc1 stimulated expression of the Cartpt and Kiss1 genes, which encode hypothalamic neuropeptides that mediate leptin's effects on satiety and fertility. Crtc1 overexpression in hypothalamic cells increased Cartpt and Kiss1 gene expression, whereas Crtc1 depletion decreased it. Indeed, leptin enhanced Crtc1 activity over the Cartpt and Kiss1 promoters in cells overexpressing Lepr, and these effects were disrupted by expression of a dominant-negative Creb1 polypeptide. As leptin administration increased recruitment of hypothalamic Crtc1 to Cartpt and Kiss1 promoters, our results indicate that the Creb1-Crtc1 pathway mediates the central effects of hormones and nutrients on energy balance and fertility.
Project description:Obesity and depression are major public health concerns, and there is increasing evidence that they share etiological mechanisms. CREB-regulated transcription coactivator 1 (CRTC1) participates in neurobiological pathways involved in both mood and energy balance regulation. Crtc1 -/- mice rapidly develop a depressive-like and obese phenotype in early adulthood, and are therefore a relevant animal model to explore possible common mechanisms underlying mood disorders and obesity. Here, the obese phenotype of male and female Crtc1 -/- mice was further characterized by investigating CRTC1's role in the homeostatic and hedonic regulation of food intake, as well as its influence on daily locomotor activity. Crtc1 -/- mice showed a strong gender difference in the homeostatic regulation of energy balance. Mutant males were hyperphagic and rapidly developed obesity on normal chow diet, whereas Crtc1 -/- females exhibited mild late-onset obesity without hyperphagia. Overeating of mutant males was accompanied by alterations in the expression of several orexigenic and anorexigenic hypothalamic genes, thus confirming a key role of CRTC1 in the central regulation of food intake. No alteration in preference and conditioned response for saccharine was observed in Crtc1 -/- mice, suggesting that mutant males' hyperphagia was not due to an altered hedonic regulation of food intake. Intriguingly, mutant males exhibited a hyperphagic behavior only during the resting (diurnal) phase of the light cycle. This abnormal feeding behavior was associated with a higher diurnal locomotor activity indicating that the lack of CRTC1 may affect circadian rhythmicity. Collectively, these findings highlight the male-specific involvement of CRTC1 in the central control of energy balance and circadian locomotor activity.
Project description:The adipocyte-derived hormone leptin maintains energy balance by acting on hypothalamic leptin receptors (Leprs) that trigger the signal transducer and activator of transcription 3 (Stat3). Although disruption of Lepr-Stat3 signaling promotes obesity in mice, other features of Lepr function, such as fertility, seem normal, pointing to the involvement of additional regulators. Here we show that the cyclic AMP responsive element–binding protein-1 (Creb1)-regulated transcription coactivator-1 (Crtc1) is required for energy balance and reproduction—Crtc1-/- mice are hyperphagic, obese and infertile. Hypothalamic Crtc1 was phosphorylated and inactive in leptin-deficient ob/ob mice; leptin administration increased amounts of dephosphorylated nuclear Crtc1. Dephosphorylated Crtc1 stimulated expression of the Cartpt and Kiss1 genes, which encode hypothalamic neuropeptides that mediate leptin’s effects on satiety and fertility. Crtc1 overexpression in hypothalamic cells increased Cartpt and Kiss1 gene expression, whereas Crtc1 depletion decreased it. Indeed, leptin enhanced Crtc1 activity over the Cartpt and Kiss1 promoters in cells overexpressing Lepr and these effects were disrupted by expression of a dominant-negative Creb1 polypeptide. As leptin administration increased recruitment of hypothalamic Crtc1 to Cartpt and Kiss1 promoters, our results indicate that the Creb1-Crtc1 pathway mediates the central effects of hormones and nutrients on energy balance and fertility. Overall design: Mice were fasted overnight for 18h and refed for 6h. Hypothalami were obtained from 3 wild-type and 3 Crtc1 knockout mice. Total RNA was isolated from each sample and equal amounts from each sample were pooled for the microarray.