Epigenetic mechanisms involved in neuronal Bdnf gene expression in adult and aged mouse in response to cognitive stimulation

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 12-02-2016Esta tesis tiene embargado el acceso al texto completo hasta el 12-08-2017Transcription of immediate early memory genes is an essential process in brain function, regulating, among other processes, synaptic plasticity. It is well established the necessity of gene transcription in order to maintain the late phases of the long-term potentiation (LTP) and the long-term depression (LTD). Several works performed in animals subjected to different memory paradigms have shown that there are epigenetic mechanisms involved in memory genes regulation. However, little is known about the contribution of these epigenetics mechanisms in response to a single stimulus, in the adult and in the old brain. The aim of this thesis was to characterise such mechanisms in response to LTD, in order to better understand the regulation of Bdnf gene expression, and its possible relation with the aged associated learning and memory deficits. In this thesis we present that LTD stimulation triggered by low NMDA dose in young adult animals, induces the transcription of Bdnf gene from promoters I, II, IV and VI by H3K27Me3 demethylation and H3K27Me3 phosphorylation at Serine 28, leading to displacement of EZH2, the catalytic subunit of Polycomb Repressor Complex 2. LTD not only does induce EZH2 repressor detachment, but also the dissociation of another transcriptionally repressive enzyme such as histone deacetylase 4 (HDAC4). We also show that LTD enhances acetylation of histone H3K27 via pCREB/CBP. Differently from the described situation, typical of the mature brain, we present data showing that the normal singling transduction of the young upon LTD is impaired in the aged hippocampus, leading to a different basal chromatin state at Bdnf promoters in the old. The consequence of this impairment is the loss of Bdnf induction in the old when exposed to LTD, as a result of impaired HDAC4 dissociation, CBP recruitment and Histone H3K27 acetylation at Bdnf promoters. We also have observed that the loss of cholesterol at the neuronal plasma membrane, a physiological feature of the old, plays a role in these epigenetic deficits. In fact, cholesterol addition to old hippocampal slices rescued Bdnf epigenetic regulation and expression in response to LTD. Furthermore, cholesterol reduction in young adult hippocampal slices led to similar deficits to the ones found in the old animals. In further support of the cholesterol loss-epigenetic dysregulation in the old, oral administration of Voriconazole, an inhibitor of the enzyme responsible for cerebral cholesterol loss (Cyp46A1), rescued hippocampal cholesterol loss and enhanced cognitive abilities in the old animals, improving Bdnf epigenetic regulation and expression in response to LTD. These results unveil one of the mechanisms involved in the cognitive decline of the old and propose Cyp46A1 as valuable therapeutic possibility

Wnt Signaling Deregulation in the Aging and Alzheimer’s Brain

Growing evidence suggests that synaptic signaling is compromised in the aging brain and in Alzheimer’s disease (AD), contributing to synaptic decline. Wnt signaling is a prominent pathway at the synapse and is required for synaptic plasticity and maintenance in the adult brain. In this review, we summarize the current knowledge on deregulation of Wnt signaling in the context of aging and AD. Emerging studies suggest that enhancing Wnt signaling could boost synaptic function during aging, and ameliorate synaptic pathology in AD. Although further research is needed to determine the precise contribution of deficient Wnt signaling to AD pathogenesis, targeting Wnt signaling components may provide novel therapeutic avenues for synapse protection or restoration in the brain

A genetic variant of the Wnt receptor LRP6 accelerates synapse degeneration during aging and in Alzheimer's disease

Synapse loss strongly correlates with cognitive decline in Alzheimer's disease (AD), but the underlying mechanisms are poorly understood. Deficient Wnt signaling contributes to synapse dysfunction and loss in AD. Consistently, a variant of the LRP6 receptor, (LRP6-Val), with reduced Wnt signaling, is linked to late-onset AD. However, the impact of LRP6-Val on the healthy and AD brain has not been examined. Knock-in mice, generated by gene editing, carrying this Lrp6 variant develop normally. However, neurons from Lrp6-val mice do not respond to Wnt7a, a ligand that promotes synaptic assembly through the Frizzled-5 receptor. Wnt7a stimulates the formation of the low-density lipoprotein receptor-related protein 6 (LRP6)-Frizzled-5 complex but not if LRP6-Val is present. Lrp6-val mice exhibit structural and functional synaptic defects that become pronounced with age. Lrp6-val mice present exacerbated synapse loss around plaques when crossed to the NL-G-F AD model. Our findings uncover a previously unidentified role for Lrp6-val in synapse vulnerability during aging and AD

Single-Cell Quantification of mRNA Expression in The Human Brain

RNA analysis at the cellular resolution in the human brain is challenging. Here, we describe an optimised approach for detecting single RNA transcripts in a cell-type specific manner in frozen human brain tissue using multiplexed fluorescent RNAscope probes. We developed a new robust analytical approach for RNAscope quantification. Our method shows that low RNA integrity does not significantly affect RNAscope signal, recapitulates bulk RNA analysis and provides spatial context to transcriptomic analysis of human post-mortem brain at single-cell resolution. In summary, our optimised method allows the usage of frozen human samples from brain banks to perform quantitative RNAscope analysis

LTP-triggered cholesterol redistribution activates Cdc42 and drives AMPA receptor synaptic delivery

Neurotransmitter receptor trafficking during synaptic plasticity requires the concerted action of multiple signaling pathways and the protein transport machinery. However, little is known about the contribution of lipid metabolism during these processes. In this paper, we addressed the question of the role of cholesterol in synaptic changes during long-term potentiation (LTP). We found that N-methyl-d-aspartate-type glutamate receptor (NMDAR) activation during LTP induction leads to a rapid and sustained loss or redistribution of intracellular cholesterol in the neuron. A reduction in cholesterol, in turn, leads to the activation of Cdc42 and the mobilization of GluA1-containing α-amino-3-hydroxy-5- methyl-4-isoxazolepropionic acid-type glutamate receptors (AMPARs) from Rab11-recycling endosomes into the synaptic membrane, leading to synaptic potentiation. This process is accompanied by an increase of NMDAR function and an enhancement of LTP. These results imply that cholesterol acts as a sensor of NMDAR activation and as a trigger of downstream signaling to engage small GTPase (guanosine triphosphatase) activation and AMPAR synaptic delivery during LTP.Peer Reviewe

Activation of PKR Causes Amyloid ß-Peptide Accumulation via De-Repression of BACE1 Expression

BACE1 is a key enzyme involved in the production of amyloid ß-peptide (Aß) in Alzheimer's disease (AD) brains. Normally, its expression is constitutively inhibited due to the presence of the 5′untranslated region (5′UTR) in the BACE1 promoter. BACE1 expression is activated by phosphorylation of the eukaryotic initiation factor (eIF)2-alpha, which reverses the inhibitory effect exerted by BACE1 5′UTR. There are four kinases associated with different types of stress that could phosphorylate eIF2-alpha. Here we focus on the double-stranded (ds) RNA-activated protein kinase (PKR). PKR is activated during viral infection, including that of herpes simplex virus type 1 (HSV1), a virus suggested to be implicated in the development of AD, acting when present in brains of carriers of the type 4 allele of the apolipoprotein E gene. HSV1 is a dsDNA virus but it has genes on both strands of the genome, and from these genes complementary RNA molecules are transcribed. These could activate BACE1 expression by the PKR pathway. Here we demonstrate in HSV1-infected neuroblastoma cells, and in peripheral nervous tissue from HSV1-infected mice, that HSV1 activates PKR. Cloning BACE1 5′UTR upstream of a luciferase (luc) gene confirmed its inhibitory effect, which can be prevented by salubrinal, an inhibitor of the eIF2-alpha phosphatase PP1c. Treatment with the dsRNA analog poly (I∶C) mimicked the stimulatory effect exerted by salubrinal over BACE1 translation in the 5′UTR-luc construct and increased Aß production in HEK-APPsw cells. Summarizing, our data suggest that PKR activated in brain by HSV1 could play an important role in the development of AD

Neuronal activity controls Bdnf expression via Polycomb de-repression and CREB/CBP/JMJD3 activation in mature neurons

It has been recently described that in embryonic stem cells, the expression of some important developmentally regulated genes is repressed, but poised for fast activation under the appropriate stimuli. In this work we show that Bdnf promoters are repressed by Polycomb Complex 2 in mature hippocampal neurons, and basal expression is guaranteed by the coexistence with activating histone marks. Neuronal stimulation triggered by N-methyl-D-aspartate application induces the transcription of these promoters by H3K27Me3 demethylation and H3K27Me3 phosphorylation at Serine 28 leading to displacement of EZH2, the catalytic subunit of Polycomb Repressor Complex 2. Our data show that the fast transient expression of Bdnf promoters II and VI after neuronal stimulation is dependent on acetylation of histone H3K27 by CREB-p/CBP. Thus, regulatory mechanisms established during development seem to remain after differentiation controlling genes induced by different stimuli, as would be the case of early memory genes in mature neurons.status: publishe

Neuronal activity controls Bdnf expression via Polycomb de-repression and CREB/CBP/JMJD3 activation in mature neurons

It has been recently described that in embryonic stem cells, the expression of some important developmentally regulated genes is repressed, but poised for fast activation under the appropriate stimuli. In this work we show that Bdnf promoters are repressed by Polycomb Complex 2 in mature hippocampal neurons, and basal expression is guaranteed by the coexistence with activating histone marks. Neuronal stimulation triggered by N-methyl-D-aspartate application induces the transcription of these promoters by H3K27Me3 demethylation and H3K27Me3 phosphorylation at Serine 28 leading to displacement of EZH2, the catalytic subunit of Polycomb Repressor Complex 2. Our data show that the fast transient expression of Bdnf promoters II and VI after neuronal stimulation is dependent on acetylation of histone H3K27 by CREB-p/CBP. Thus, regulatory mechanisms established during development seem to remain after differentiation controlling genes induced by different stimuli, as would be the case of early memory genes in mature neurons.Fil: Palomer, Ernest. Universidad Autonoma de Madrid. Centro de Biología Molecular; EspañaFil: Carretero, Javier. Universidad Autonoma de Madrid. Centro de Biología Molecular; EspañaFil: Benvegnù, Stefano. Universidad Autonoma de Madrid. Centro de Biología Molecular; EspañaFil: Dotti, Carlos G.. Universidad Autonoma de Madrid. Centro de Biología Molecular; EspañaFil: Martín, Mauricio Gerardo. Universidad Autonoma de Madrid. Centro de Biología Molecular; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentin

Age-associated cholesterol reduction triggers brain insulin resistance by facilitating ligand-independent receptor activation and pathway desensitization

In the brain, insulin plays an important role in cognitive processes. During aging, these faculties decline, as does insulin signaling. The mechanism behind this last phenomenon is unclear. In recent studies, we reported that the mild and gradual loss of cholesterol in the synaptic fraction of hippocampal neurons during aging leads to a decrease in synaptic plasticity evoked by glutamate receptor activation and also by receptor tyrosine kinase (RTK) signaling. As insulin and insulin growth factor activity are dependent on tyrosine kinase receptors, we investigated whether the constitutive loss of brain cholesterol is also involved in the decay of insulin function with age. Using long-term depression (LTD) induced by application of insulin to hippocampal slices as a read-out, we found that the decline in insulin function during aging could be monitored as a progressive impairment of insulin-LTD. The application of a cholesterol inclusion complex, which donates cholesterol to the membrane and increases membrane cholesterol levels, rescued the insulin signaling deficit and insulin-LTD. In contrast, extraction of cholesterol from hippocampal neurons of adult mice produced the opposite effect. Furthermore, in vivo inhibition of Cyp46A1, an enzyme involved in brain cholesterol loss with age, improved insulin signaling. Fluorescence resonance energy transfer (FRET) experiments pointed to a change in receptor conformation by reduced membrane cholesterol, favoring ligand-independent autophosphorylation. Together, these results indicate that changes in membrane fluidity of brain cells during aging play a key role in the decay of synaptic plasticity and cognition that occurs at this late stage of life.Ministry of Science and Spanish Ministry of Economy and Competitivenes