Stefano Taverna

Direct generation of functional dopaminergic neurons from mouse and human fibroblasts

Transplantation of dopaminergic neurons can potentially improve the clinical outcome of Parkinson’s disease, a neurological disorder resulting from degeneration of mesencephalic dopaminergic neurons. In particular, transplantation of embryonic-stem-cell-derived dopaminergic neurons has been shown to be efficient in restoring motor symptoms in conditions of dopamine deficiency. However, the use of pluripotent-derived cells might lead to the development of tumours if not properly controlled. Here we identified a minimal set of three transcription factors—Mash1 (also known as Ascl1), Nurr1 (also known as Nr4a2) and Lmx1a—that are able to generate directly functional dopaminergic neurons from mouse and human fibroblasts without reverting to a progenitor cell stage. Induced dopaminergic (iDA) cells release dopamine and show spontaneous electrical activity organized in regular spikes consistent with the pacemaker activity featured by brain dopaminergic neurons. The three factors were able to elicit dopaminergic neuronal conversion in prenatal and adult fibroblasts from healthy donors and Parkinson’s disease patients. Direct generation of iDA cells from somatic cells might have significant implications for understanding critical processes for neuronal development, in vitro disease modelling and cell replacement therapies.

Recurrent collateral connections of striatal medium spiny neurons are disrupted in models of Parkinson's disease.

The principal neurons of the striatum, GABAergic medium spiny neurons (MSNs), are interconnected by local recurrent axon collateral synapses. Although critical to many striatal models, it is not clear whether these connections are random or whether they preferentially link functionally related groups of MSNs. To address this issue, dual whole patch-clamp recordings were made from striatal MSNs in brain slices taken from transgenic mice in which D1 or D2 dopamine receptor expression was reported with EGFP (enhanced green fluorescent protein). These studies revealed that unidirectional connections were common between both D1 receptor-expressing MSN (D1 MSN) pairs (26%) and D2 receptor-expressing MSN (D2 MSN) pairs (36%). D2 MSNs also commonly formed synapses on D1 MSNs (27% of pairs). Conversely, only 6% of the D1 MSNs formed detectable connections with D2 MSNs. Furthermore, synaptic connections formed by D1 MSNs were weaker than those formed by D2 MSNs, a difference that was attributable to fewer GABAA receptors at D1 MSN synapses. The strength of detectable recurrent connections was dramatically reduced in Parkinsons disease models. The studies demonstrate that recurrent collateral connections between MSNs are not random but rather differentially couple D1 and D2 MSNs. Moreover, this recurrent collateral network appears to be disrupted in Parkinsons disease models, potentially contributing to pathological alterations in MSN activity patterns and psychomotor symptoms.

Mode of action of thiocoraline. a natural marine compound with anti-tumour activity

SummaryThiocoraline, a new anticancer agent derived from the marine actinomycete Micromonospora marina, was found to induce profound perturbations of the cell cycle. On both LoVo and SW620 human colon cancer cell lines, thiocoraline caused an arrest in G1 phase of the cell cycle and a decrease in the rate of S phase progression towards G2/M phases, as assessed by using bromodeoxyuridine/DNA biparametric flow cytometric analysis. Thiocoraline does not inhibit DNA-topoisomerase II enzymes in vitro, nor does it induce DNA breakage in cells exposed to effective drug concentrations. The cell cycle effects observed after exposure to thiocoraline appear related to the inhibition of DNA replication. By using a primer extension assay it was found that thiocoraline inhibited DNA elongation by DNA polymerase α at concentrations that inhibited cell cycle progression and clonogenicity. These studies indicate that the new anticancer drug thiocoraline probably acts by inhibiting DNA polymerase α activity.

Rapid Conversion of Fibroblasts into Functional Forebrain GABAergic Interneurons by Direct Genetic Reprogramming

Transplantation of GABAergic interneurons (INs) can provide long-term functional benefits in animal models of epilepsy and other neurological disorders. Whereas GABAergic INs can be differentiated from embryonic stem cells, alternative sources of GABAergic INs may be more tractable for disease modeling and transplantation. We identified five factors (Foxg1, Sox2, Ascl1, Dlx5, and Lhx6) that convert mouse fibroblasts into induced GABAergic INs (iGABA-INs) possessing molecular signatures of telencephalic INs. Factor overexpression activates transcriptional networks required for GABAergic fate specification. iGABA-INs display progressively maturing firing patterns comparable to cortical INs, form functional synapses, and release GABA. Importantly, iGABA-INs survive and mature upon being grafted into mouse hippocampus. Optogenetic stimulation demonstrated functional integration of grafted iGABA-INs into host circuitry, triggering inhibition of host granule neuron activity. These five factors also converted human cells into functional GABAergic INs. These properties suggest that iGABA-INs have potential for disease modeling and cell-based therapeutic approaches to neurological disorders.

Remote control of induced dopaminergic neurons in parkinsonian rats.

Direct lineage reprogramming through genetic-based strategies enables the conversion of differentiated somatic cells into functional neurons and distinct neuronal subtypes. Induced dopaminergic (iDA) neurons can be generated by direct conversion of skin fibroblasts; however, their in vivo phenotypic and functional properties remain incompletely understood, leaving their impact on Parkinsons disease (PD) cell therapy and modeling uncertain. Here, we determined that iDA neurons retain a transgene-independent stable phenotype in culture and in animal models. Furthermore, transplanted iDA neurons functionally integrated into host neuronal tissue, exhibiting electrically excitable membranes, synaptic currents, dopamine release, and substantial reduction of motor symptoms in a PD animal model. Neuronal cell replacement approaches will benefit from a system that allows the activity of transplanted neurons to be controlled remotely and enables modulation depending on the physiological needs of the recipient; therefore, we adapted a DREADD (designer receptor exclusively activated by designer drug) technology for remote and real-time control of grafted iDA neuronal activity in living animals. Remote DREADD-dependent iDA neuron activation markedly enhanced the beneficial effects in transplanted PD animals. These data suggest that iDA neurons have therapeutic potential as a cell replacement approach for PD and highlight the applicability of pharmacogenetics for enhancing cellular signaling in reprogrammed cell-based approaches.

Membrane properties and synaptic connectivity of fast-spiking interneurons in rat ventral striatum

In vitro patch-clamp recordings were made to study the membrane properties and synaptic connectivity of fast-spiking interneurons in rat ventral striatum. Using a whole-cell configuration in acutely prepared slices, fast-spiking interneurons were recognized based on their firing properties and their morphological phenotype was confirmed by immunocytochemistry. Membrane properties of fast-spiking interneurons were distinguished from those of medium-sized spiny neurons by their more depolarized resting membrane potential, lower action potential amplitude and shorter half-width, short spike repolarization time and deep spike afterhyperpolarization. Firing patterns of interneurons could be subdivided in a bursting and non-bursting mode. Simultaneous dual whole-cell recordings revealed a high degree of connectivity of fast-spiking interneurons to medium-sized spiny neurons via unidirectional synapses. Burst firing in fast-spiking interneurons that were presynaptic to medium-sized spiny neurons resulted in barrages of postsynaptic potentials showing an initial amplitude increment, rapidly followed by a decrement. In conclusion, ventral striatal fast-spiking interneurons can be clearly distinguished from medium-sized spiny neurons by their membrane properties and their firing patterns can be subdivided in bursting and non-bursting modes. Their synaptic connectivity to medium-sized spiny neurons is unidirectional and characterized by frequency-dependent, dynamic changes in postsynaptic amplitude.

Selective activation of parvalbumin- or somatostatin-expressing interneurons triggers epileptic seizurelike activity in mouse medial entorhinal cortex.

GABAergic interneurons are thought to play a critical role in eliciting interictal spikes (IICs) and triggering ictal discharges in temporal lobe epilepsy, yet the contribution of different interneuronal subtypes to seizure initiation is still largely unknown. Here we took advantage of optogenetic techniques combined with patch-clamp and field recordings to selectively stimulate parvalbumin (PV)- or somatostatin (SOM)-positive interneurons expressing channelrhodopsin-2 (CHR-2) in layers II-III of adult mouse medial entorhinal cortical slices during extracellular perfusion with the proconvulsive compound 4-aminopyridine (4-AP, 100-200 μM). In control conditions, blue laser photostimulation selectively activated action potential firing in either PV or SOM interneurons and, in both cases, caused a robust GABAA-receptor-mediated inhibition in pyramidal cells (PCs). During perfusion with 4-AP, brief photostimuli (300 ms) activating either PV or SOM interneurons induced patterns of epileptiform activity that closely replicated spontaneously occurring IICs and tonic-clonic ictal discharges. Laser-induced synchronous firing in both interneuronal types elicited large compound GABAergic inhibitory postsynaptic currents (IPSCs) correlating with IICs and preictal spikes. In addition, spontaneous and laser-induced epileptic events were similarly initiated in concurrence with a large increase in extracellular potassium concentration. Finally, interneuron activation was unable to stop or significantly shorten the progression of seizurelike episodes. These results suggest that entorhinal PV and SOM interneurons are nearly equally effective in triggering interictal and ictal discharges that closely resemble human temporal lobe epileptic activity.

Differential Expression of TASK Channels between Horizontal Interneurons and Pyramidal Cells of Rat Hippocampus

Among the electrophysiological properties differentiating stratum oriens horizontal interneurons from pyramidal neurons of the CA1 hippocampal subfield are the more depolarized resting potential and the higher input resistance; additionally, these interneurons are also less sensitive to ischemic damage than pyramidal cells. A differential expression of pH-sensitive leakage potassium channels (TASK) could contribute to all of these differences. To test this hypothesis, we studied the expression and properties of TASK channels in the two cell types. Electrophysiological recordings from acute slices showed that barium- and bupivacaine-sensitive TASK currents were detectable in pyramidal cells but not in interneurons and that extracellular acidification caused a much stronger depolarization in pyramidal cells than in interneurons. This pyramidal cell depolarization was paralleled by an increase of the input resistance, suggesting the blockade of a background conductance. Single-cell reverse transcription-PCR experiments showed that the expression profile of TASK channels differ between the two cell types and suggested that these channels mediate an important share of the leakage current of pyramidal cells. We suggest that the different expression of TASK channels in these cell types contribute to their electrophysiological differences and may result in cell-specific sensitivity to extracellular acidification in conditions such as epilepsy and ischemia.

In vitro large-scale experimental and theoretical studies for the realization of bi-directional brain-prostheses.

Brain-machine interfaces (BMI) were born to control “actions from thoughts” in order to recover motor capability of patients with impaired functional connectivity between the central and peripheral nervous system. The final goal of our studies is the development of a new proof-of-concept BMI—a neuromorphic chip for brain repair—to reproduce the functional organization of a damaged part of the central nervous system. To reach this ambitious goal, we implemented a multidisciplinary “bottom-up” approach in which in vitro networks are the paradigm for the development of an in silico model to be incorporated into a neuromorphic device. In this paper we present the overall strategy and focus on the different building blocks of our studies: (i) the experimental characterization and modeling of “finite size networks” which represent the smallest and most general self-organized circuits capable of generating spontaneous collective dynamics; (ii) the induction of lesions in neuronal networks and the whole brain preparation with special attention on the impact on the functional organization of the circuits; (iii) the first production of a neuromorphic chip able to implement a real-time model of neuronal networks. A dynamical characterization of the finite size circuits with single cell resolution is provided. A neural network model based on Izhikevich neurons was able to replicate the experimental observations. Changes in the dynamics of the neuronal circuits induced by optical and ischemic lesions are presented respectively for in vitro neuronal networks and for a whole brain preparation. Finally the implementation of a neuromorphic chip reproducing the network dynamics in quasi-real time (10 ns precision) is presented.

Synchronous Inhibitory Potentials Precede Seizure-Like Events in Acute Models of Focal Limbic Seizures

Interictal spikes in models of focal seizures and epilepsies are sustained by the synchronous activation of glutamatergic and GABAergic networks. The nature of population spikes associated with seizure initiation (pre-ictal spikes; PSs) is still undetermined. We analyzed the networks involved in the generation of both interictal and PSs in acute models of limbic cortex ictogenesis induced by pharmacological manipulations. Simultaneous extracellular and intracellular recordings from both principal cells and interneurons were performed in the medial entorhinal cortex of the in vitro isolated guinea pig brain during focal interictal and ictal discharges induced in the limbic network by intracortical and brief arterial infusions of either bicuculline methiodide (BMI) or 4-aminopyridine (4AP). Local application of BMI in the entorhinal cortex did not induce seizure-like events (SLEs), but did generate periodic interictal spikes sensitive to the glutamatergic non-NMDA receptor antagonist DNQX. Unlike local applications, arterial perfusion of either BMI or 4AP induced focal limbic SLEs. PSs just ahead of SLE were associated with hyperpolarizing potentials coupled with a complete blockade of firing in principal cells and burst discharges in putative interneurons. Interictal population spikes recorded from principal neurons between two SLEs correlated with a depolarizing potential. We demonstrate in two models of acute limbic SLE that PS events are different from interictal spikes and are sustained by synchronous activation of inhibitory networks. Our findings support a prominent role of synchronous network inhibition in the initiation of a focal seizure.