Francesco Tamagnini

Intrinsic excitability changes induced by acute treatment of hippocampal CA1 pyramidal neurons with exogenous amyloid β peptide

Accumulation of beta‐amyloid (Aβ) peptides in the human brain is a canonical pathological hallmark of Alzheimers disease (AD). Recent work in Aβ‐overexpressing transgenic mice indicates that increased brain Aβ levels can be associated with aberrant epileptiform activity. In line with this, such mice can also exhibit altered intrinsic excitability (IE) of cortical and hippocampal neurons: these observations may relate to the increased prevalence of seizures in AD patients. In this study, we examined what changes in IE are produced in hippocampal CA1 pyramidal cells after 2–5 h treatment with an oligomeric preparation of synthetic human Aβ 1–42 peptide. Whole cell current clamp recordings were compared between Aβ‐(500 nM) and vehicle‐(DMSO 0.05%) treated hippocampal slices obtained from mice. The soluble Aβ treatment did not produce alterations in sub‐threshold intrinsic properties, including membrane potential, input resistance, and hyperpolarization activated “sag”. Similarly, no changes were noted in the firing profile evoked by 500 ms square current supra‐threshold stimuli. However, Aβ 500 nM treatment resulted in the hyperpolarization of the action potential (AP) threshold. In addition, treatment with Aβ at 500 nM depressed the after‐hyperpolarization that followed both a single AP or 50 Hz trains of a number of APs between 5 and 25. These data suggest that acute exposure to soluble Aβ oligomers affects IE properties of CA1 pyramidal neurons differently from outcomes seen in transgenic models of amyloidopathy. However, in both chronic and acute models, the IE changes are toward hyperexcitability, reinforcing the idea that amyloidopathy and increased incidence in seizures might be causally related in AD patients.

Functionalized α-Helical Peptide Hydrogels for Neural Tissue Engineering

Trauma to the central and peripheral nervous systems often lead to serious morbidity. Current surgical methods for repairing or replacing such damage have limitations. Tissue engineering offers a potential alternative. Here we show that functionalized α-helical-peptide hydrogels can be used to induce attachment, migration, proliferation and differentiation of murine embryonic neural stem cells (NSCs). Specifically, compared with undecorated gels, those functionalized with Arg-Gly-Asp-Ser (RGDS) peptides increase the proliferative activity of NSCs; promote their directional migration; induce differentiation, with increased expression of microtubule-associated protein-2, and a low expression of glial fibrillary acidic protein; and lead to the formation of larger neurospheres. Electrophysiological measurements from NSCs grown in RGDS-decorated gels indicate developmental progress toward mature neuron-like behavior. Our data indicate that these functional peptide hydrogels may go some way toward overcoming the limitations of current approaches to nerve-tissue repair.

Long-term culture of pluripotent stem-cell-derived human neurons on diamond - A substrate for neurodegeneration research and therapy

Brain Computer Interfaces (BCI) currently represent a field of intense research aimed both at understanding neural circuit physiology and at providing functional therapy for traumatic or degenerative neurological conditions. Due to its chemical inertness, biocompatibility and stability, diamond is currently being actively investigated as a potential substrate material for culturing cells and for use as the electrically active component of a neural sensor. Here we provide a protocol for the differentiation of mature, electrically active neurons on microcrystalline synthetic thin-film diamond substrates starting from undifferentiated pluripotent stem cells. Furthermore, we investigate the optimal characteristics of the diamond microstructure for long-term neuronal sustainability. We also analyze the effect of boron as a dopant for such a culture. We found that the diamond crystalline structure has a significant influence on the neuronal culture unlike the boron doping. Specifically, small diamond microcrystals promote higher neurite density formation. We find that boron incorporated into the diamond does not influence the neurite density and has no deleterious effect on cell survival.

Altered intrinsic excitability of hippocampal CA1 pyramidal neurons in aged PDAPP mice.

Amyloidopathy involves the accumulation of insoluble amyloid β (Aβ) species in the brain’s parenchyma and is a key histopathological hallmark of Alzheimer’s disease (AD). Work on transgenic mice that overexpress Aβ suggests that elevated Aβ levels in the brain are associated with aberrant epileptiform activity and increased intrinsic excitability (IE) of CA1 hippocampal neurons. In this study we examined if similar changes could be observed in hippocampal CA1 pyramidal neurons from aged PDAPP mice (20–23 month old, Indiana mutation: V717F on APP gene) compared to their age-matched wild-type littermate controls. Whole-cell current clamp recordings revealed that sub-threshold intrinsic properties, such as input resistance, resting membrane potential and hyperpolarization activated “sag” were unaffected, but capacitance was significantly decreased in the transgenic animals. No differences between genotypes were observed in the overall number of action potentials (AP) elicited by 500 ms supra-threshold current stimuli. PDAPP neurons, however, exhibited higher instantaneous firing frequencies after accommodation in response to high intensity current injections. The AP waveform was narrower and shorter in amplitude in PDAPP mice: these changes, according to our in silico model of a CA1/3 pyramidal neuron, depended on the respective increase and reduction of K+ and Na+ voltage-gated channels maximal conductances. Finally, the after-hyperpolarization, seen after the first AP evoked by a +300 pA current injection and after 50 Hz AP bursts, was more pronounced in PDAPP mice. These data show that Aβ-overexpression in aged mice altered the capacitance, the neuronal firing and the AP waveform of CA1 pyramidal neurons. Some of these findings are consistent with previous work on younger PDAPP; they also show important differences that can be potentially ascribed to the interaction between amyloidopathy and ageing. Such a change of IE properties over time underlies that the increased incidence of seizure observed in AD patients might rely on different mechanistic pathways during progression of the disease.

Low Concentrations of the Solvent Dimethyl Sulphoxide Alter Intrinsic Excitability Properties of Cortical and Hippocampal Pyramidal Cells

Dimethylsulfoxide (DMSO) is a widely used solvent in biology. It has many applications perhaps the most common of which is in aiding the preparation of drug solutions from hydrophobic chemical entities. Recent studies have suggested that this molecule may be able to induce apoptosis in neural tissues urging caution regarding its introduction into humans, for example as part of stem cell transplants. Here we have used in vitro electrophysiological methods applied to murine brain slices to examine whether a few hours treatment with 0.05% DMSO (a concentration regarded by many as innocuous) alters intrinsic excitability properties of neurones. We investigated pyramidal neurones in two distinct brain regions, namely area CA1 of the hippocampus and layer 2 of perirhinal cortex. In the former there was no effect on resting potential but input resistance was decreased by DMSO pre-treatment. In line with this action potential count for any level of depolarizing current stimulus was reduced by ∼25% following DMSO treatment. Ih-mediated “sag” was also increased in CA1 pyramids and action potential waveform analysis demonstrated that DMSO treatment moved action potential threshold towards resting potential. In perirhinal cortex a decreased action potential output for various depolarizing current stimuli was also seen. In these cells action potential threshold was unaltered by DMSO but a significant increase in action potential width was apparent. These data indicate that pre-treatment with this widely employed solvent can elicit multifaceted neurophysiological changes in mammalian neurones at concentrations below those frequently encountered in the published literature.

Viscoelasticity of amyloid plaques in transgenic mouse brain studied by Brillouin microspectroscopy and correlative Raman analysis

Amyloidopathy is one of the most prominent hallmarks of Alzheimers disease (AD), the leading cause of dementia worldwide, and is characterized by the accumulation of amyloid plaques in the brain parenchyma. The plaques consist of abnormal deposits mainly composed of an aggregation-prone protein fragment, β-amyloid 1-40/1-42, into the extracellular matrix. Brillouin microspectroscopy is an all-optical contactless technique that is based on the interaction between visible light and longitudinal acoustic waves or phonons, giving access to the viscoelasticity of a sample on a subcellular scale. Here, we describe the first application of micromechanical mapping based on Brillouin scattering spectroscopy to probe the stiffness of individual amyloid plaques in the hippocampal part of the brain of a β-amyloid overexpressing transgenic mouse. Correlative analysis based on Brillouin and Raman microspectroscopy showed that amyloid plaques have a complex structure with a rigid core of β-pleated sheet conformation (β-amyloid) protein surrounded by a softer ring-shaped region richer in lipids and other protein conformations. These preliminary results give a new insight into the plaque biophysics and biomechanics, and a valuable contrast mechanism for the study and diagnosis of amyloidopathy.

Hippocampal neurophysiology is modified by a disease-associated C-terminal fragment of tau protein

The accumulation of cleaved tau fragments in the brain is associated with several tauopathies. For this reason, we recently developed a transgenic mouse that selectively accumulates a C-Terminal 35 kDa human tau fragment (Tau35). These animals develop progressive motor and spatial memory impairment, paralleled by increased hippocampal glycogen synthase kinase 3β activity. In this neurophysiological study, we focused on the CA1 subfield of the hippocampus, a brain area involved in memory encoding. The accumulation of Tau35 results in a significant increase of short-term facilitation of the synaptic response in the theta frequency range (10 Hz), without affecting basal synaptic transmission and long-term synaptic plasticity. Tau35 expression also alters the intrinsic excitability of CA1 pyramidal neurons. Thus, Tau35 presence is associated with increased and decreased excitability at hyperpolarized and depolarized potentials, respectively. These observations are paralleled by a hyperpolarization of the voltage-sensitivity of noninactivating K+ currents. Further investigation is needed to assess the causal link between such functional alterations and the cognitive and motor impairments previously observed in this model.

CA 2+ SIGNALLING IN HUMAN BRAIN PERICYTES: INVESTIGATION OF EFFECTS OF AMYLOID BETA TREATMENT

induce the formation of aggresomes. GFP-tau cells were transduced with NUB1 lentiviral vectors both in the absence and presence of proteasome inhibitor, followed by western blotting, filter trap assays, immunoprecipitation and immunofluorescence. Results: Inhibition of the proteasome induced the formation of GFP-tau aggresomes that were positive for phospho-tau (p-tau) markers (AT8, pS396). Filter trap assays revealed that NUB1 significantly decreased the ratio of p-tau to tau in the total cellular fraction (both soluble and insoluble), as well as in only the detergent insoluble fraction, both with and without proteasome inhibition. However, the ratio of tau and p-tau in the insoluble fraction to that in the total fraction revealed that the NUB1-mediated decrease in the levels of detergent insoluble tau and p-tau exceeded that of the overall decline in total levels, suggesting NUB1 can target aggregationprone tau and p-tau. This effect of NUB1 was more pronounced for p-tau following proteasome inhibition. Blocking the proteasome induced the upregulation of p62 and increased the ratio of LC3BII to LC3B-I. NUB1 induced further upregulation of autophagy following proteasome inhibition as seen by an increase in the LC3B-II/LC3B-I ratio and changes in LC3B positive puncta in cells. Finally, GFP-tau aggresomes were positive for p62 and NUB1 overexpression promoted the interaction between GFP-tau and p62. Conclusions: These data suggest that NUB1 may play a role in the clearance of aggregation-prone tau and p-tau following inhibition of the proteasome via autophagy.