Multiomics analysis identifies novel facilitators of human dopaminergic neuron differentiation.

Midbrain dopaminergic neurons (mDANs) control voluntary movement, cognition, and reward behavior under physiological conditions and are implicated in human diseases such as Parkinson's disease (PD). Many transcription factors (TFs) controlling human mDAN differentiation during development have been described, but much of the regulatory landscape remains undefined. Using a tyrosine hydroxylase (TH) human iPSC reporter line, we here generate time series transcriptomic and epigenomic profiles of purified mDANs during differentiation. Integrative analysis predicts novel regulators of mDAN differentiation and super-enhancers are used to identify key TFs. We find LBX1, NHLH1 and NR2F1/2 to promote mDAN differentiation and show that overexpression of either LBX1 or NHLH1 can also improve mDAN specification. A more detailed investigation of TF targets reveals that NHLH1 promotes the induction of neuronal miR-124, LBX1 regulates cholesterol biosynthesis, and NR2F1/2 controls neuronal activity.

Low glycemic index diet restrains epileptogenesis in a gender-specific fashion.

Dietary restriction, such as low glycemic index diet (LGID), have been successfully used to treat drug-resistant epilepsy. However, if such diet could also counteract antiepileptogenesis is still unclear. Here, we investigated whether the administration of LGID during the latent pre-epileptic period, prevents or delays the appearance of the overt epileptic phenotype. To this aim, we used the Synapsin II knockout (SynIIKO) mouse, a model of temporal lobe epilepsy in which seizures manifest 2-3 months after birth, offering a temporal window in which LGID may affect epileptogenesis. Pregnant SynIIKO mice were fed with either LGID or standard diet during gestation and lactation. Both diets were maintained in weaned mice up to 5 months of age. LGID delayed the seizure onset and induced a reduction of seizures severity only in female SynIIKO mice. In parallel with the epileptic phenotype, high-density multielectrode array recordings revealed a reduction of frequency, amplitude, duration, velocity of propagation and spread of interictal events by LGID in the hippocampus of SynIIKO females, but not mutant males, confirming the gender-specific effect. ELISA-based analysis revealed that LGID increased cortico-hippocampal allopregnanolone (ALLO) levels only in females, while it was unable to affect ALLO plasma concentrations in either sex. The results indicate that the gender-specific interference of LGID with the epileptogenic process can be ascribed to a gender-specific increase in cortical ALLO, a neurosteroid known to strengthen GABAergic transmission. The study highlights the possibility of developing a personalized gender-based therapy for temporal lobe epilepsy.

REST/NRSF drives homeostatic plasticity of inhibitory synapses in a target-dependent fashion.

The repressor-element 1-silencing transcription/neuron-restrictive silencer factor (REST/NRSF) controls hundreds of neuron-specific genes. We showed that REST/NRSF downregulates glutamatergic transmission in response to hyperactivity, thus contributing to neuronal homeostasis. However, whether GABAergic transmission is also implicated in the homeostatic action of REST/NRSF is unknown. Here, we show that hyperactivity-induced REST/NRSF activation, triggers a homeostatic rearrangement of GABAergic inhibition, with increased frequency of miniature inhibitory postsynaptic currents (IPSCs) and amplitude of evoked IPSCs in mouse cultured hippocampal neurons. Notably, this effect is limited to inhibitory-onto-excitatory neuron synapses, whose density increases at somatic level and decreases in dendritic regions, demonstrating a complex target- and area-selectivity. The upscaling of perisomatic inhibition was occluded by TrkB receptor inhibition and resulted from a coordinated and sequential activation of the Npas4 and Bdnf gene programs. On the opposite, the downscaling of dendritic inhibition was REST-dependent, but BDNF-independent. The findings highlight the central role of REST/NRSF in the complex transcriptional responses aimed at rescuing physiological levels of network activity in front of the ever-changing environment.

PRRT2 modulates presynaptic Ca2+ influx by interacting with P/Q-type channels.

Loss-of-function mutations in proline-rich transmembrane protein-2 (PRRT2) cause paroxysmal disorders associated with defective Ca2+ dependence of glutamatergic transmission. We find that either acute or constitutive PRRT2 deletion induces a significant decrease in the amplitude of evoked excitatory postsynaptic currents (eEPSCs) that is insensitive to extracellular Ca2+ and associated with a reduced contribution of P/Q-type Ca2+ channels to the EPSC amplitude. This synaptic phenotype parallels a decrease in somatic P/Q-type Ca2+ currents due to a decreased membrane targeting of the channel with unchanged total expression levels. Co-immunoprecipitation, pull-down assays, and proteomics reveal a specific and direct interaction of PRRT2 with P/Q-type Ca2+ channels. At presynaptic terminals lacking PRRT2, P/Q-type Ca2+ channels reduce their clustering at the active zone, with a corresponding decrease in the P/Q-dependent presynaptic Ca2+ signal. The data highlight the central role of PRRT2 in ensuring the physiological Ca2+ sensitivity of the release machinery at glutamatergic synapses.

Numerical Evaluation of Human Body Near Field Exposure to a Vehicular Antenna for Military Applications.

BACKGROUND: The use of electromagnetic (EM) technologies for military applications is gaining increasing interest to satisfy different operational needs, such as improving battlefield communications or jamming counterpart's signals. This is achieved by the use of high-power EM waves in several frequency bands (e.g., HF, VHF, and UHF). When considering military vehicles, several antennas are present in close proximity to the crew personnel, which are thus potentially exposed to high EM fields. METHODS: A typical exposure scenario was reproduced numerically to evaluate the EM exposure of the human body in the presence of an HF vehicular antenna (2-30 MHz). The antenna was modeled as a monopole connected to a 3D polygonal structure representing the vehicle. Both the EM field levels in the absence and in the presence of the human body and also the specific absorption rate (SAR) values were calculated. The presence of the operator, partially standing outside the vehicle, was simulated with the virtual human body model Duke (Virtual Population, V.3). Several exposure scenarios were considered. The presence of a protective helmet was modeled as well. RESULTS: In the area usually occupied by the personnel, E-field intensity radiated by the antenna can reach values above the limits settled by international safety guidelines. Nevertheless, local SAR values induced inside the human body reached a maximum value of 14 mW/kg, leading to whole-body averaged and 10-g averaged SAR values well below the corresponding limits. CONCLUSION: A complex and realistic near-field exposure scenario of the crew of a military vehicle was simulated. The obtained E-field values radiated in the free space by a HF vehicular antenna may reach values above the safety guidelines reference levels. Such values are not necessarily meaningful for the exposed subject. Indeed, SAR and E-field values induced inside the body remain well below safety limits.

Proline-rich transmembrane protein 2 (PRRT2) regulates the actin cytoskeleton during synaptogenesis.

Mutations in proline-rich transmembrane protein 2 (PRRT2) have been recently identified as the leading cause of a clinically heterogeneous group of neurological disorders sharing a paroxysmal nature, including paroxysmal kinesigenic dyskinesia and benign familial infantile seizures. To date, studies aimed at understanding its physiological functions in neurons have mainly focused on its ability to regulate neurotransmitter release and neuronal excitability. Here, we show that PRRT2 expression in non-neuronal cell lines inhibits cell motility and focal adhesion turnover, increases cell aggregation propensity, and promotes the protrusion of filopodia, all processes impinging on the actin cytoskeleton. In primary hippocampal neurons, PRRT2 silencing affects the synaptic content of filamentous actin and perturbs actin dynamics. This is accompanied by defects in the density and maturation of dendritic spines. We identified cofilin, an actin-binding protein abundantly expressed at the synaptic level, as the ultimate effector of PRRT2. Indeed, PRRT2 silencing unbalances cofilin activity leading to the formation of cofilin-actin rods along neurites. The expression of a cofilin phospho-mimetic mutant (cof-S3E) is able to rescue PRRT2-dependent defects in synapse density, spine number and morphology, but not the alterations observed in neurotransmitter release. Our data support a novel function of PRRT2 in the regulation of the synaptic actin cytoskeleton and in the formation of synaptic contacts.

TBC1D24 regulates axonal outgrowth and membrane trafficking at the growth cone in rodent and human neurons.

Mutations in TBC1D24 are described in patients with a spectrum of neurological diseases, including mild and severe epilepsies and complex syndromic phenotypes such as Deafness, Onycodystrophy, Osteodystrophy, Mental Retardation and Seizure (DOORS) syndrome. The product of TBC1D24 is a multifunctional protein involved in neuronal development, regulation of synaptic vesicle trafficking, and protection from oxidative stress. Although pathogenic mutations in TBC1D24 span the entire coding sequence, no clear genotype/phenotype correlations have emerged. However most patients bearing predicted loss of function mutations exhibit a severe neurodevelopmental disorder. Aim of the study is to investigate the impact of TBC1D24 knockdown during the first stages of neuronal differentiation when axonal specification and outgrowth take place. In rat cortical primary neurons silenced for TBC1D24, we found defects in axonal specification, the maturation of axonal initial segment and action potential firing. The axonal phenotype was accompanied by an impairment of endocytosis at the growth cone and an altered activation of the TBC1D24 molecular partner ADP ribosylation factor 6. Accordingly, acute knockdown of TBC1D24 in cerebrocortical neurons in vivo analogously impairs callosal projections. The axonal defect was also investigated in human induced pluripotent stem cell-derived neurons from patients carrying TBC1D24 mutations. Reprogrammed neurons from a patient with severe developmental encephalopathy show significant axon formation defect that were absent from reprogrammed neurons of a patient with mild early onset epilepsy. Our data reveal that alterations of membrane trafficking at the growth cone induced by TBC1D24 loss of function cause axonal and excitability defects. The axonal phenotype correlates with the disease severity and highlight an important role for TBC1D24 in connectivity during brain development.

Spike-Related Electrophysiological Identification of Cultured Hippocampal Excitatory and Inhibitory Neurons.

Cultured hippocampal neurons represent the most widely used experimental substrate for in vitro electrophysiological studies. Nevertheless, in most cases, the nature of neuron under study is not identified as excitatory or inhibitory, or even worse, recorded neurons are considered as excitatory because of the paucity of GABAergic interneurons. Thus, the definition of reliable criteria able to guarantee an unequivocal identification of excitatory and inhibitory cultured hippocampal neurons is an unmet need. To reach this goal, we compared the electrophysiological properties and the localization and size of the axon initial segment (AIS) of cultured hippocampal neurons, taking advantage from GAD67-GFP knock-in mice, which expressing green fluorescent protein (GFP) in gamma-aminobutyric acid (GABA)-containing cells, allowed to unambiguously determine the precise nature of the neuron under study. Our results demonstrate that the passive electrophysiological properties, the localization and size of the AIS, and the shape and frequency of the action potential (AP) are not reliable to unequivocally identify neurons as excitatory or inhibitory. The only parameter, related to the shape of the single AP, showing minimal overlap between the sample-point distributions of the two neuronal subpopulations, was the AP half-width. However, the estimation of the AP failure ratio evoked by a short train of high-current steps applied at increasing frequency (40-140 Hz) resulted to be indisputably the safer and faster way to identify the excitatory or inhibitory nature of an unknown neuron. Our findings provide a precise framework for further electrophysiological investigations of in vitro hippocampal neurons.