Roberto Piacentini

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.

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.

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.

In vivo protective effect of ferulic acid against noise-induced hearing loss in the guinea-pig

Ferulic acid (FA) is a phenolic compound whose neuroprotective activity was extensively studied in vitro. In this study, we provided functional in vivo evidence that FA limits noise-induced hearing loss. Guinea-pigs exposed to acoustic trauma for 1 h exhibited a significant impairment in auditory function; this injury was evident as early as 1 day from noise exposure and persisted over 21 days. Ferulic acid (150 mg/kg i.p. for 4 days) counteracted noise-induced hearing loss at days 1, 3, 7 and 21 from noise exposure. The improvement of auditory function by FA was paralleled by a significant reduction in oxidative stress, apoptosis and increase in hair cell viability in the organ of Corti. Interestingly in the guinea-pig cochleae, the neuroprotective effect of FA was functionally related not only to its scavenging ability in the peri-traumatic period but also to the up-regulation of the cytoprotective enzyme heme oxygenase-1 (HO-1); in fact, FA-induced improvement of auditory function was counteracted by the HO inhibitor zinc-protoporphyrin-IX and paralleled the time-course of HO-1 induction over 3-7 days. These results confirm the antioxidant properties of FA as free-radical scavenger and suggest a role of HO-1 as an additional mediator against noise-induced ototoxicity.

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.

Water-soluble Coenzyme Q10 formulation (Q-ter) promotes outer hair cell survival in a guinea pig model of noise induced hearing loss (NIHL).

The mitochondrial respiratory chain is a powerful source of reactive oxygen species (ROS) also in noise induced hearing loss (NIHL) and anti-oxidants and free-radicals scavengers have been shown to attenuate the damage. Coenzyme Q(10) (CoQ(10)) or ubiquinone has a bioenergetic role as a component of the mithocondrial respiratory chain, it inhibits mitochondrial lipid peroxidation, inducing ATP production and it is involved in ROS removal and prevention of oxidative stress-induced apoptosis. However the therapeutic application of CoQ(10) is limited by the lack of solubility and poor bio- availability, therefore it is a challenge to improve its water solubility in order to ameliorate the efficacy in tissues and fluids. This study was conducted in a model of acoustic trauma in the guinea pig where the effectiveness of CoQ(10) was compared with a soluble formulation of CoQ(10) (multicomposite CoQ(10) Terclatrate, Q-ter) given intraperitoneally 1 h before and once daily for 3 days after pure tone noise exposure (6 kHz for 1 h at 120 dB SPL). Functional and morphological studies were carried out by measuring auditory brainstem responses, scanning electron microscopy for hair cell loss count, active caspase 3 staining and terminal deoxynucleotidyl transferase-mediated dUTP labelling assay in order to identify initial signs of apoptosis. Treatments decreased active caspase 3 expression and the number of apoptotic cells, but animals injected with Q-ter showed a greater degree of activity in preventing apoptosis and thus in improving hearing. These data confirm that solubility of Coenzyme Q(10) improves the ability of CoQ(10) in preventing oxidative injuries that result from mitochondrial dysfunction.

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.

APP Processing Induced by Herpes Simplex Virus Type 1 (HSV-1) Yields Several APP Fragments in Human and Rat Neuronal Cells

Lifelong latent infections of the trigeminal ganglion by the neurotropic herpes simplex virus type 1 (HSV-1) are characterized by periodic reactivation. During these episodes, newly produced virions may also reach the central nervous system (CNS), causing productive but generally asymptomatic infections. Epidemiological and experimental findings suggest that HSV-1 might contribute to the pathogenesis of Alzheimers disease (AD). This multifactorial neurodegenerative disorder is related to an overproduction of amyloid beta (Aβ) and other neurotoxic peptides, which occurs during amyloidogenic endoproteolytic processing of the transmembrane amyloid precursor protein (APP). The aim of our study was to identify the effects of productive HSV-1 infection on APP processing in neuronal cells. We found that infection of SH-SY5Y human neuroblastoma cells and rat cortical neurons is followed by multiple cleavages of APP, which result in the intra- and/or extra-cellular accumulation of various neurotoxic species. These include: i) APP fragments (APP-Fs) of 35 and 45 kDa (APP-F35 and APP-F45) that comprise portions of Aβ; ii) N-terminal APP-Fs that are secreted; iii) intracellular C-terminal APP-Fs; and iv) Aβ1-40 and Aβ1-42. Western blot analysis of infected-cell lysates treated with formic acid suggests that APP-F35 may be an Aβ oligomer. The multiple cleavages of APP that occur in infected cells are produced in part by known components of the amyloidogenic APP processing pathway, i.e., host-cell β-secretase, γ-secretase, and caspase-3-like enzymes. These findings demonstrate that HSV-1 infection of neuronal cells can generate multiple APP fragments with well-documented neurotoxic potentials. It is tempting to speculate that intra- and extracellular accumulation of these species in the CNS resulting from repeated HSV-1 reactivation could, in the presence of other risk factors, play a co-factorial role in the development of AD.

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.

Extracellular Tau Oligomers Produce An Immediate Impairment of LTP and Memory

Non-fibrillar soluble oligomeric forms of amyloid-β peptide (oAβ) and tau proteins are likely to play a major role in Alzheimer’s disease (AD). The prevailing hypothesis on the disease etiopathogenesis is that oAβ initiates tau pathology that slowly spreads throughout the medial temporal cortex and neocortices independently of Aβ, eventually leading to memory loss. Here we show that a brief exposure to extracellular recombinant human tau oligomers (oTau), but not monomers, produces an impairment of long-term potentiation (LTP) and memory, independent of the presence of high oAβ levels. The impairment is immediate as it raises as soon as 20 min after exposure to the oligomers. These effects are reproduced either by oTau extracted from AD human specimens, or naturally produced in mice overexpressing human tau. Finally, we found that oTau could also act in combination with oAβ to produce these effects, as sub-toxic doses of the two peptides combined lead to LTP and memory impairment. These findings provide a novel view of the effects of tau and Aβ on memory loss, offering new therapeutic opportunities in the therapy of AD and other neurodegenerative diseases associated with Aβ and tau pathology.