Claudio Grassi

Extremely low‐frequency electromagnetic fields promote in vitro neurogenesis via upregulation of Cav1‐channel activity

We previously reported that exposure to extremely low‐frequency electromagnetic fields (ELFEFs) increases the expression and function of voltage‐gated Ca2+ channels and that Ca2+ influx through Cav1 channels plays a key role in promoting the neuronal differentiation of neural stem/progenitor cells (NSCs). The present study was conducted to determine whether ELFEFs influence the neuronal differentiation of NSCs isolated from the brain cortices of newborn mice by modulating Cav1‐channel function. In cultures of differentiating NSCs exposed to ELFEFs (1 mT, 50 Hz), the percentage of cells displaying immunoreactivity for neuronal markers (β‐III‐tubulin, MAP2) and for Cav1.2 and Cav1.3 channels was markedly increased. NSC‐differentiated neurons in ELFEF‐exposed cultures also exhibited significant increases in spontaneous firing, in the percentage of cells exhibiting Ca2+ transients in response to KCl stimulation, in the amplitude of these transients and of Ca2+ currents generated by the activation of Cav1 channels. When the Cav1‐channel blocker nifedipine (5 µM) was added to the culture medium, the neuronal yield of NSC differentiation dropped significantly, and ELFEF exposure no longer produced significant increases in β‐III‐tubulin‐ and MAP2‐immunoreactivity rates. In contrast, the effects of ELFEFs were preserved when NSCs were cultured in the presence of either glutamate receptor antagonists or Cav2.1‐ and Cav2.2‐channel blockers. ELFEF stimulation during the first 24 h of differentiation caused Cav1‐dependent increases in the number of cells displaying CREB phosphorylation. Our data suggest that ELFEF exposure promotes neuronal differentiation of NSCs by upregulating Cav1‐channel expression and function. J. Cell. Physiol. 215: 129–139, 2008.

Microbes and Alzheimer's Disease

We are researchers and clinicians working on Alzheimer’s disease (AD) or related topics, and we write to express our concern that one particular aspect of the disease has been neglected, even thoug ...

Role of L-type Ca2+ channels in neural stem/progenitor cell differentiation.

Ca2+ influx through voltage‐gated Ca2+ channels, especially the L‐type (Cav1), activates downstream signaling to the nucleus that affects gene expression and, consequently, cell fate. We hypothesized that these Ca2+ signals may also influence the neuronal differentiation of neural stem/progenitor cells (NSCs) derived from the brain cortex of postnatal mice. We first studied Ca2+ transients induced by membrane depolarization in Fluo 4‐AM‐loaded NSCs using confocal microscopy. Undifferentiated cells (nestin+) exhibited no detectable Ca2+ signals whereas, during 12 days of fetal bovine serum‐induced differentiation, neurons (β‐III‐tubulin+/MAP2+) displayed time‐dependent increases in intracellular Ca2+ transients, with ΔF/F ratios ranging from 0.4 on day 3 to 3.3 on day 12. Patch‐clamp experiments revealed similar correlation between NSC differentiation and macroscopic Ba2+ current density. These currents were markedly reduced (−77%) by Cav1 channel blockade with 5 µm nifedipine. To determine the influence of Cav1‐mediated Ca2+ influx on NSC differentiation, cells were cultured in differentiative medium with either nifedipine (5 µm) or the L‐channel activator Bay K 8644 (10 µm). The latter treatment significantly increased the percentage of β‐III‐tubulin+/MAP2+ cells whereas nifedipine produced opposite effects. Pretreatment with nifedipine also inhibited the functional maturation of neurons, which responded to membrane depolarization with weak Ca2+ signals. Conversely, Bay K 8644 pretreatment significantly enhanced the percentage of responsive cells and the amplitudes of Ca2+ transients. These data suggest that NSC differentiation is strongly correlated with the expression of voltage‐gated Ca2+ channels, especially the Cav1, and that Ca2+ influx through these channels plays a key role in promoting neuronal differentiation.

Modulation of LTP at rat hippocampal CA3-CA1 synapses by direct current stimulation

Transcranial direct current stimulation (tDCS) can produce a lasting polarity-specific modulation of cortical excitability in the brain, and it is increasingly used in experimental and clinical settings. Recent studies suggest that the after-effects of tDCS are related to molecular mechanisms of activity-dependent synaptic plasticity. Here we investigated the effect of DCS on the induction of one of the most studied N-methyl-d-aspartate receptor-dependent forms of long-term potentiation (LTP) of synaptic activity at CA3-CA1 synapses in the hippocampus. We show that DCS applied to rat brain slices determines a modulation of LTP that is increased by anodal and reduced by cathodal DCS. Immediate early genes, such as c-fos and zif268 (egr1/NGFI-A/krox24), are rapidly induced following neuronal activation, and a specific role of zif268 in the induction and maintenance of LTP has been demonstrated. We found that both anodal and cathodal DCS produce a marked subregion-specific increase in the expression of zif268 protein in the cornus ammonis (CA) region, whereas the same protocols of stimulation produce a less pronounced increase in c-fos protein expression in the CA and in dentate gyrus regions of the hippocampus. Brain-derived neurotrophic factor expression was also investigated, and it was found to be reduced in cathodal-stimulated slices. The present data demonstrate that it is possible to modulate LTP by using DCS and provide the rationale for the use of DCS in neurological diseases to promote the adaptive and suppress the maladaptive forms of brain plasticity.

Infectious agents and neurodegeneration.

A growing body of epidemiologic and experimental data point to chronic bacterial and viral infections as possible risk factors for neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis. Infections of the central nervous system, especially those characterized by a chronic progressive course, may produce multiple damage in infected and neighbouring cells. The activation of inflammatory processes and host immune responses cause chronic damage resulting in alterations of neuronal function and viability, but different pathogens can also directly trigger neurotoxic pathways. Indeed, viral and microbial agents have been reported to produce molecular hallmarks of neurodegeneration, such as the production and deposit of misfolded protein aggregates, oxidative stress, deficient autophagic processes, synaptopathies and neuronal death. These effects may act in synergy with other recognized risk factors, such as aging, concomitant metabolic diseases and the host’s specific genetic signature. This review will focus on the contribution given to neurodegeneration by herpes simplex type-1, human immunodeficiency and influenza viruses, and by Chlamydia pneumoniae.

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.

Electrophysiological and molecular evidence of L-(Cav1), N- (Cav2.2), and R- (Cav2.3) type Ca2+ channels in rat cortical astrocytes

Changes in intracellular Ca2+ levels are an important signal underlying neuron‐glia cross‐talk, but little is known about the possible role of voltage‐gated Ca2+ channels (VGCCs) in controlling glial cell Ca2+ influx. We investigated the pharmacological and biophysical features of VGCCs in cultured rat cortical astrocytes. In whole‐cell patch‐clamp experiments, L‐channel blockade (5 μM nifedipine) reduced Ba2+ current amplitude by 28% of controls, and further decrease (32%) was produced by N‐channel blockade (3 μM ω‐conotoxin‐GVIA). No significant additional changes were observed after P/Q channel blockade (3 μM ω‐conotoxin‐MVIIC). Residual current (36% of controls) amounted to roughly the same percentage (34%) that was abolished by R‐channel blockade (100 nM SNX‐482). Electrophysiological evidence of L‐, N‐, and R‐channels was associated with RT‐PCR detection of mRNA transcripts for VGCC subunits α1C (L‐type), α1B (N‐type), and α1E (R‐type). In cell‐attached recordings, single‐channel properties (L‐currents: amplitude, −1.21 ± 0.02 pA at 10 mV; slope conductance, 22.0 ± 1.1 pS; mean open time, 5.95 ± 0.24 ms; N‐currents: amplitude, −1.09 ± 0.02 pA at 10 mV; slope conductance, 18.0 ± 1.1 pS; mean open time, 1.14 ± 0.02 ms; R‐currents: amplitude, −0.81 ± 0.01 pA at 20 mV; slope conductance, 10.5 ± 0.3 pS; mean open time, 0.88 ± 0.02 ms) resembled those of corresponding VGCCs in neurons. These novel findings indicate that VGCC expression by cortical astrocytes may be more varied than previously thought, suggesting that these channels may indeed play substantial roles in the regulation of astrocyte Ca2+ influx, which influences neuron‐glia cross‐talk and numerous other calcium‐mediated glial‐cell functions.

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.

HSV-1 promotes Ca2+-mediated APP phosphorylation and Aβ accumulation in rat cortical neurons

Epidemiological and experimental findings suggest that chronic infection with Herpes simplex virus type 1 (HSV-1) may be a risk factor for Alzheimers disease (AD), but the molecular mechanisms underlying this association have not been fully identified. We investigated the effects of HSV-1 on excitability and intracellular calcium signaling in rat cortical neurons and the impact of these effects on amyloid precursor protein (APP) processing and the production of amyloid-β peptide (Aβ). Membrane depolarization triggering firing rate increases was observed shortly after neurons were challenged with HSV-1 and was still evident 12 hours postinfection. These effects depended on persistent sodium current activation and potassium current inhibition. The virally induced hyperexcitability triggered intracellular Ca(2+) signals that significantly increased intraneuronal Ca(2+) levels. It also enhanced activity- and Ca(2+)-dependent APP phosphorylation and intracellular accumulation of Aβ42. These findings indicate that HSV-1 causes functional changes in cortical neurons that promote APP processing and Aβ production, and they are compatible with the co-factorial role for HSV-1 in the pathogenesis of AD suggested by previous findings.

Nitric oxide inhibits neuroendocrine CaV1 L-channel gating via cGMP-dependent protein kinase in cell-attached patches of bovine chromaffin cells

Nitric oxide (NO) regulates the release of catecholamines from the adrenal medulla but the molecular targets of its action are not yet well identified. Here we show that the NO donor sodium nitroprusside (SNP, 200 μM) causes a marked depression of the single CaV1 L‐channel activity in cell‐attached patches of bovine chromaffin cells. SNP action was complete within 3‐5 min of cell superfusion. In multichannel patches the open probability (NPo) decreased by ∼60 % between 0 and +20 mV. Averaged currents over a number of traces were proportionally reduced and showed no drastic changes to their time course. In single‐channel patches the open probability (Po) at +10 mV decreased by the same amount as that of multichannel patches (∼61 %). Such a reduction was mainly associated with an increased probability of null sweeps and a prolongation of mean shut times, while first latency, mean open time and single‐channel conductance were not significantly affected. Addition of the NO scavenger carboxy‐PTIO or cell treatment with the guanylate cyclase inhibitor ODQ prevented the SNP‐induced inhibition. 8‐Bromo‐cyclicGMP (8‐Br‐cGMP; 400 μM) mimicked the action of the NO donor and the protein kinase G blocker KT‐5823 prevented this effect. The depressive action of SNP was preserved after blocking the cAMP‐dependent up‐regulatory pathway with the protein kinase A inhibitor H89. Similarly, the inhibitory action of 8‐Br‐cGMP proceeded regardless of the elevation of cAMP levels, suggesting that cGMP/PKG and cAMP/PKA act independently on L‐channel gating. The inhibitory action of 8‐Br‐cGMP was also independent of the G protein‐induced inhibition of L‐channels mediated by purinergic and opiodergic autoreceptors. Since Ca2+ channels contribute critically to both the local production of NO and catecholamine release, the NO/PKG‐mediated inhibition of neuroendocrine L‐channels described here may represent an important autocrine signalling mechanism for controlling the rate of neurotransmitter release from adrenal glands.