Lucia Leone

A role for neuronal cAMP responsive-element binding (CREB)-1 in brain responses to calorie restriction

Calorie restriction delays brain senescence and prevents neurodegeneration, but critical regulators of these beneficial responses other than the NAD+-dependent histone deacetylase Sirtuin-1 (Sirt-1) are unknown. We report that effects of calorie restriction on neuronal plasticity, memory and social behavior are abolished in mice lacking cAMP responsive-element binding (CREB)-1 in the forebrain. Moreover, CREB deficiency drastically reduces the expression of Sirt-1 and the induction of genes relevant to neuronal metabolism and survival in the cortex and hippocampus of dietary-restricted animals. Biochemical studies reveal a complex interplay between CREB and Sirt-1: CREB directly regulates the transcription of the sirtuin in neuronal cells by binding to Sirt-1 chromatin; Sirt-1, in turn, is recruited by CREB to DNA and promotes CREB-dependent expression of target gene peroxisome proliferator-activated receptor-γ coactivator-1α and neuronal NO Synthase. Accordingly, expression of these CREB targets is markedly reduced in the brain of Sirt KO mice that are, like CREB-deficient mice, poorly responsive to calorie restriction. Thus, the above circuitry, modulated by nutrient availability, links energy metabolism with neurotrophin signaling, participates in brain adaptation to nutrient restriction, and is potentially relevant to accelerated brain aging by overnutrition and diabetes.

Exposure to extremely low-frequency (50 Hz) electromagnetic fields enhances adult hippocampal neurogenesis in C57BL/6 mice

Throughout life, new neurons are continuously generated in the hippocampus, which is therefore a major site of structural plasticity in the adult brain. We recently demonstrated that extremely low-frequency electromagnetic fields (ELFEFs) promote the neuronal differentiation of neural stem cells in vitro by up-regulating Ca(v)1-channel activity. The aim of the present study was to determine whether 50-Hz/1 mT ELFEF stimulation also affects adult hippocampal neurogenesis in vivo, and if so, to identify the molecular mechanisms underlying this action and its functional impact on synaptic plasticity. ELFEF exposure (1 to 7 h/day for 7 days) significantly enhanced neurogenesis in the dentate gyrus (DG) of adult mice, as documented by increased numbers of cells double-labeled for 5-bromo-deoxyuridine (BrdU) and doublecortin. Quantitative RT-PCR analysis of hippocampal extracts revealed significant ELFEF exposure-induced increases in the transcription of pro-neuronal genes (Mash1, NeuroD2, Hes1) and genes encoding Ca(v)1.2 channel α(1C) subunits. Increased expression of NeuroD1, NeuroD2 and Ca(v)1 channels was also documented by Western blot analysis. Immunofluorescence experiments showed that, 30 days after ELFEF stimulation, roughly half of the newly generated immature neurons had survived and become mature dentate granule cells (as shown by their immunoreactivity for both BrdU and NeuN) and were integrated into the granule cell layer of the DG. Electrophysiological experiments demonstrated that the new mature neurons influenced hippocampal synaptic plasticity, as reflected by increased long-term potentiation. Our findings show that ELFEF exposure can be an effective tool for increasing in vivo neurogenesis, and they could lead to the development of novel therapeutic approaches in regenerative medicine.

P22.22 Exposure to extremely low-frequency (50 Hz) electromagnetic fields enhances adult hippocampal neurogenesis in C57BL/6 mice

Introduction: Paired associative stimulation (PAS) at an interstimulus interval (ISI) of 25 ms produces long term potentiation (LTP)-like effect, but each pair occurs at intervals for producing short afferent inhibition (SAI). This implies that inhibitory mechanisms may play a role in producing LTP-like effects of PAS. Objectives: We assessed the inhibitory synaptic pathways by measuring short-interval intracortical inhibition (SICI). Methods: Twenty-two healthy volunteers (9 females, 34 yrs on average) were recruited. Stimulus intensities were adjusted so that at the start of PAS, the test motor evoked potential (MEP) was suppressed to 60 80% control (SAI). SICI was assessed with a threshold tracking technique using a standard 0.2 mV MEP. Inhibition is expressed as the increase in stimulation intensity needed to maintain 0.2 mV MEP constant in the presence of a conditioning stimulus (CS) of 70% resting motor threshold. Thus high values indicate strong inhibition. Results: MEPs increased by an average of 1.55±0.19 (SE) after PAS, but ranged from 0.54 to 3.67. We divided the subjects into three groups; good responders (1 < PAS effect < 2, n = 11), poor responders (PAS effect < 1, n = 7) and outliers (PAS effect 2, n = 4). Before PAS, good responders had strong SICI at ISI 1.8 to 5 ms compared to poor responders. SICI at ISI 3 ms was 27.3±4.5% in good responders and 4.9±4.7% in poor responders (p = 0.004). SICI was significantly correlated with PAS effect (r = 0.61, p = 0.007). SICI in the outliers (who were musicians) fell out of the 95% confidence interval in this correlation. Conclusions: The relationship between the initial level of SICI and the response to PAS is compatible with the following. The PAS effect relies on increased excitability of late indirect (I)-wave generating mechanisms. SICI has its primary effect on late I-waves. Thus individuals with good SICI may have prominent late I-waves that are readily inhibited by CS; however the same I-waves may be beneficial for PAS.

Anodal transcranial direct current stimulation boosts synaptic plasticity and memory in mice via epigenetic regulation of Bdnf expression

The effects of transcranial direct current stimulation (tDCS) on brain functions and the underlying molecular mechanisms are yet largely unknown. Here we report that mice subjected to 20-min anodal tDCS exhibited one-week lasting increases in hippocampal LTP, learning and memory. These effects were associated with enhanced: i) acetylation of brain-derived neurotrophic factor (Bdnf) promoter I; ii) expression of Bdnf exons I and IX; iii) Bdnf protein levels. The hippocampi of stimulated mice also exhibited enhanced CREB phosphorylation, pCREB binding to Bdnf promoter I and recruitment of CBP on the same regulatory sequence. Inhibition of acetylation and blockade of TrkB receptors hindered tDCS effects at molecular, electrophysiological and behavioral levels. Collectively, our findings suggest that anodal tDCS increases hippocampal LTP and memory via chromatin remodeling of Bdnf regulatory sequences leading to increased expression of this gene, and support the therapeutic potential of tDCS for brain diseases associated with impaired neuroplasticity.

Reduced D-serine levels in the nucleus accumbens of cocaine-treated rats hinder the induction of NMDA receptor-dependent synaptic plasticity

Cocaine seeking behaviour and relapse have been linked to impaired potentiation and depression at excitatory synapses in the nucleus accumbens, but the mechanism underlying this process is poorly understood. We show that, in the rat nucleus accumbens core, D-serine is the endogenous coagonist of N-methyl-D-aspartate receptors, and its presence is essential for N-methyl-D-aspartate receptor-dependent potentiation and depression of synaptic transmission. Nucleus accumbens core slices obtained from cocaine-treated rats after 1 day of abstinence presented significantly reduced D-serine concentrations, increased expression of the D-serine degrading enzyme, D-amino acid oxidase, and downregulated expression of serine racemase, the enzyme responsible for D-serine synthesis. The D-serine deficit was associated with impairment of potentiation and depression of glutamatergic synaptic transmission, which was restored by slice perfusion with exogenous D-serine. Furthermore, in vivo administration of D-serine directly into the nucleus accumbens core blocked behavioural sensitization to cocaine. These results provide evidence for a critical role of D-serine signalling in synaptic plasticity relevant to cocaine addiction.

Effects of different amyloid β-protein analogues on synaptic function

Perisynaptic accumulations of amyloid β-protein (Aβ) play a critical role in the synaptic dysfunction underlying the cognitive impairment observed in Alzheimers disease. The methionine residue at position 35 (Met35) in Aβ is highly subject to oxidation in Alzheimers disease brains. In hippocampal brain slices we found that long-term potentiation at CA3-CA1 synapses was significantly inhibited by wild type Aβ42 in which Met35 is reduced, but not by Aβ42 harboring Met35 sulfoxide. Similar differences were observed when basal synaptic transmission was investigated in autaptic hippocampal neurons. The significant decreases in excitatory postsynaptic current amplitude, vesicle release probability and miniature excitatory postsynaptic current frequency caused by 20-minute exposure to wild type Aβ42 were not observed after exposure to Aβ42 harboring Met35 sulfoxide. With longer (24-hour) Aβ treatments, this early impairment of the presynaptic terminal function extended to involve the postsynaptic side as well. The Met35 oxidation also affected Aβ42 negative impact on dendritic spine density and expression of pre- and postsynaptic proteins (synaptophysin and postsynaptic density protein-95). Our findings suggest that oxidation of Met35 is critical for molecular, structural, and functional determinants of Aβ42 synaptotoxicity.

Epigenetic Modulation of Adult Hippocampal Neurogenesis by Extremely Low-Frequency Electromagnetic Fields

Throughout life, adult neurogenesis generates new neurons in the dentate gyrus of hippocampus that have a critical role in memory formation. Strategies able to stimulate this endogenous process have raised considerable interest because of their potential use to treat neurological disorders entailing cognitive impairment. We previously reported that mice exposed to extremely low-frequency electromagnetic fields (ELFEFs) showed increased hippocampal neurogenesis. Here, we demonstrate that the ELFEF-dependent enhancement of hippocampal neurogenesis improves spatial learning and memory. To gain insights on the molecular mechanisms underlying ELFEFs’ effects, we extended our studies to an in vitro model of neural stem cells (NSCs) isolated from the hippocampi of newborn mice. We found that ELFEFs enhanced proliferation and neuronal differentiation of hippocampal NSCs by regulation of epigenetic mechanisms leading to pro-neuronal gene expression. Upon ELFEF stimulation of NSCs, we observed a significant enhancement of expression of the pro-proliferative gene hairy enhancer of split 1 and the neuronal determination genes NeuroD1 and Neurogenin1. These events were preceded by increased acetylation of H3K9 and binding of the phosphorylated transcription factor cAMP response element-binding protein (CREB) on the regulatory sequence of these genes. Such ELFEF-dependent epigenetic modifications were prevented by the Cav1-channel blocker nifedipine, and were associated with increased occupancy of CREB-binding protein (CBP) to the same loci within the analyzed promoters. Our results unravel the molecular mechanisms underlying the ELFEFs’ ability to improve endogenous neurogenesis, pointing to histone acetylation–related chromatin remodeling as a critical determinant. These findings could pave the way to the development of novel therapeutic approaches in regenerative medicine.

Extremely low-frequency electromagnetic fields enhance the survival of newborn neurons in the mouse hippocampus

In recent years, much effort has been devoted to identifying stimuli capable of enhancing adult neurogenesis, a process that generates new neurons throughout life, and that appears to be dysfunctional in the senescent brain and in several neuropsychiatric and neurodegenerative diseases. We previously reported that in vivo exposure to extremely low‐frequency electromagnetic fields (ELFEFs) promotes the proliferation and neuronal differentiation of hippocampal neural stem cells (NSCs) that functionally integrate in the dentate gyrus. Here, we extended our studies to specifically assess the influence of ELFEFs on hippocampal newborn cell survival, which is a very critical issue in adult neurogenesis regulation. Mice were injected with 5‐bromo‐2′‐deoxyuridine (BrdU) to label newborn cells, and were exposed to ELFEFs 9 days later, when the most dramatic decrease in the number of newly generated neurons occurs. The results showed that ELFEF exposure (3.5 h/day for 6 days) enhanced newborn neuron survival as documented by double staining for BrdU and doublecortin, to identify immature neurons, or NeuN labeling of mature neurons. The effects of ELFEFs were associated with enhanced spatial learning and memory. In an in vitro model of hippocampal NSCs, ELFEFs exerted their pro‐survival action by rescuing differentiating neurons from apoptotic cell death. Western immunoblot assay revealed reduced expression of the pro‐apoptotic protein Bax, and increased levels of the anti‐apoptotic protein Bcl‐2, in the hippocampi of ELFEF‐exposed mice as well as in ELFEF‐exposed NSC cultures, as compared with their sham‐exposed counterparts. Our results may have clinical implications for the treatment of impaired neurogenesis associated with brain aging and neurodegenerative diseases.

Role of methionine 35 in the intracellular Ca2+ homeostasis dysregulation and Ca2+-dependent apoptosis induced by amyloid β-peptide in human neuroblastoma IMR32 cells

Amyloid β‐peptide (Aβ) plays a fundamental role in the pathogenesis of Alzheimer’s disease. We recently reported that the redox state of the methionine residue in position 35 of amyloid β‐peptide (Aβ) 1–42 (Met35) strongly affects the peptide’s ability to trigger apoptosis and is thus a major determinant of its neurotoxicity. Dysregulation of intracellular Ca2+ homeostasis resulting in the activation of pro‐apoptotic pathways has been proposed as a mechanism underlying Aβ toxicity. Therefore, we investigated correlations between the Met35 redox state, Aβ toxicity, and altered intracellular Ca2+ signaling in human neuroblastoma IMR32 cells. Cells incubated for 6–24 h with 10 μM Aβ1–42 exhibited significantly increased KCl‐induced Ca2+ transient amplitudes and resting free Ca2+ concentrations. Nifedipine‐sensitive Ca2+ current densities and Cav1 channel expression were markedly enhanced by Aβ1–42. None of these effects were observed when cells were exposed to Aβ containing oxidized Met35 (Aβ1–42Met35‐Ox). Cell pre‐treatment with the intracellular Ca2+ chelator 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N’,N’‐tetraacetic acid acetoxymethyl ester (1 μM) or the Cav1 channel blocker nifedipine (5 μM) significantly attenuated Aβ1–42‐induced apoptosis but had no effect on Aβ1–42Met35‐Ox toxicity. Collectively, these data suggest that reduced Met35 plays a critical role in Aβ1–42 toxicity by rendering the peptide capable of disrupting intracellular Ca2+ homeostasis and thereby provoking apoptotic cell death.

A CREB-Sirt1-Hes1 Circuitry Mediates Neural Stem Cell Response to Glucose Availability

Adult neurogenesis plays increasingly recognized roles in brain homeostasis and repair and is profoundly affected by energy balance and nutrients. We found that the expression of Hes-1 (hairy and enhancer of split 1) is modulated in neural stem and progenitor cells (NSCs) by extracellular glucose through the coordinated action of CREB (cyclic AMP responsive element binding protein) and Sirt-1 (Sirtuin 1), two cellular nutrient sensors. Excess glucose reduced CREB-activated Hes-1 expression and results in impaired cell proliferation. CREB-deficient NSCs expanded poorly in vitro and did not respond to glucose availability. Elevated glucose also promoted Sirt-1-dependent repression of the Hes-1 promoter. Conversely, in low glucose, CREB replaced Sirt-1 on the chromatin associated with the Hes-1 promoter enhancing Hes-1 expression and cell proliferation. Thus, the glucose-regulated antagonism between CREB and Sirt-1 for Hes-1 transcription participates in the metabolic regulation of neurogenesis.