Functional Investigation of a Neuronal Microcircuit in the CA1 Area of the Hippocampus Reveals Synaptic Dysfunction in Dravet Syndrome Mice.

Dravet syndrome is severe childhood-onset epilepsy, caused by loss of function mutations in the SCN1A gene, encoding for the voltage-gated sodium channel NaV1.1. The leading hypothesis is that Dravet is caused by selective reduction in the excitability of inhibitory neurons, due to hampered activity of NaV1.1 channels in these cells. However, these initial neuronal changes can lead to further network alterations. Here, focusing on the CA1 microcircuit in hippocampal brain slices of Dravet syndrome (DS, Scn1a A1783V/WT) and wild-type (WT) mice, we examined the functional response to the application of Hm1a, a specific NaV1.1 activator, in CA1 stratum-oriens (SO) interneurons and CA1 pyramidal excitatory neurons. DS SO interneurons demonstrated reduced firing and depolarized threshold for action potential (AP), indicating impaired activity. Nevertheless, Hm1a induced a similar AP threshold hyperpolarization in WT and DS interneurons. Conversely, a smaller effect of Hm1a was observed in CA1 pyramidal neurons of DS mice. In these excitatory cells, Hm1a application resulted in WT-specific AP threshold hyperpolarization and increased firing probability, with no effect on DS neurons. Additionally, when the firing of SO interneurons was triggered by CA3 stimulation and relayed via activation of CA1 excitatory neurons, the firing probability was similar in WT and DS interneurons, also featuring a comparable increase in the firing probability following Hm1a application. Interestingly, a similar functional response to Hm1a was observed in a second DS mouse model, harboring the nonsense Scn1a R613X mutation. Furthermore, we show homeostatic synaptic alterations in both CA1 pyramidal neurons and SO interneurons, consistent with reduced excitation and inhibition onto CA1 pyramidal neurons and increased release probability in the CA1-SO synapse. Together, these results suggest global neuronal alterations within the CA1 microcircuit extending beyond the direct impact of NaV1.1 dysfunction.

Developmental alterations in firing properties of hippocampal CA1 inhibitory and excitatory neurons in a mouse model of Dravet syndrome.

Dravet syndrome (Dravet) is a rare, severe childhood-onset epilepsy, caused by heterozygous de novo mutations in the SCN1A gene, encoding for the alpha subunit of the voltage-gated sodium channel, NaV1.1. The neuronal basis of Dravet is debated, with evidence favoring reduced function of inhibitory neurons, that might be transient, or enhanced activity of excitatory cells. Here, we utilized Dravet mice to trace developmental changes in the hippocampal CA1 circuit, examining the properties of CA1 horizontal stratum-oriens (SO) interneurons and pyramidal neurons, through the pre-epileptic, severe and stabilization stages of Dravet. Our data indicate that reduced function of SO interneurons persists from the pre-epileptic through the stabilization stages, with the greatest functional impairment observed during the severe stage. In contrast, opposing changes were detected in CA1 excitatory neurons, with a transient increase in their excitability during the pre-epileptic stage, followed by reduced excitability at the severe stage. Interestingly, alterations in the function of both inhibitory and excitatory neurons were more pronounced when the firing was evoked by synaptic stimulation, implying that loss of function of NaV1.1 may also affect somatodendritic functions. These results suggest a complex pathophysiological mechanism and indicate that the developmental trajectory of this disease is governed by reciprocal functional changes in both excitatory and inhibitory neurons.

Alternative Functional rad21 Paralogs in Fusarium oxysporum.

Cohesin, the sister chromatid cohesion complex, is an essential complex that ensures faithful sister chromatid segregation in eukaryotes. It also participates in DNA repair, transcription and maintenance of chromosome structure. Mitotic cohesin is composed of Smc1, Smc3, Scc3, and Rad21/Mcd1. The meiotic cohesin complex contains Rec8, a Rad21 paralog and not Rad21 itself. Very little is known about sister chromatid cohesion in fungal plant pathogens. Fusarium oxysporum is an important fungal plant pathogen without known sexual life cycle. Here, we describe that F. oxysporum encodes for three Rad21 paralogs; Rad21, Rec8, and the first alternative Rad21 paralog in the phylum of ascomycete. This last paralog is found only in several fungal plant pathogens from the Fusarium family and thus termed rad21nc (non-conserved). Conserved rad21 (rad21c), rad21nc, and rec8 genes are expressed in F. oxysporum although the expression of rad21c is much higher than the other paralogs. F. oxysporum strains deleted for the rad21nc or rec8 genes were analyzed for their role in fungal life cycle. Δrad21nc and Δrec8 single mutants were proficient in sporulation, conidia germination, hyphal growth and pathogenicity under optimal growth conditions. Interestingly, Δrad21nc and Δrec8 single mutants germinate less effectively than wild type (WT) strains under DNA replication and mitosis stresses. We provide here the first genetic analysis of alternative rad21nc and rec8 paralogs in filamentous fungi. Our results suggest that rad21nc and rec8 may have a unique role in cell cycle related functions of F. oxysporum.

The response to the DNA damaging agent methyl methanesulfonate in a fungal plant pathogen.

DNA damage can cause mutations that in fungal plant pathogens lead to hypervirulence and resistance to pesticides. Almost nothing is known about the response of these fungi to DNA damage. We performed transcriptomic and phosphoproteomic analyses of Fusarium oxysporum exposed to methyl methanesulfonate (MMS). At the RNA level we observe massive induction of DNA repair pathways including the global genome nucleotide excision. Cul3, Cul4, several Ubiquitin-like ligases and components of the proteasome are significantly induced. In agreement, we observed drug synergism between a proteasome inhibitor and MMS. While our data suggest that Yap1 and Xbp1 networks are similarly activated in response to damage in yeast and F. oxysporum we were able to observe modules that were MMS-responsive in F. oxysporum and not in yeast. These include transcription/splicing modules that are upregulated and respiration that is down-regulated. In agreement, MMS treated cells are much more sensitive to a respiration inhibitor. At the phosphoproteomic level, Adenylate cyclase, which generates cAMP, is phosphorylated in response to MMS and forms a network of phosphorylated proteins that include cell cycle regulators and several MAPKs. Our analysis provides a starting point in understanding how genomic changes in response to DNA damage occur in Fusarium species.

In vivo, in vitro and in silico correlations of four de novo SCN1A missense mutations.

Mutations in the SCN1A gene, which encodes for the voltage-gated sodium channel NaV1.1, cause Dravet syndrome, a severe developmental and epileptic encephalopathy. Genetic testing of this gene is recommended early in life. However, predicting the outcome of de novo missense SCN1A mutations is difficult, since milder epileptic syndromes may also be associated. In this study, we correlated clinical severity with functional in vitro electrophysiological testing of channel activity and bioinformatics prediction of damaging mutational effects. Three patients, bearing the mutations p.Gly177Ala, p.Ser259Arg and p.Glu1923Arg, showed frequent intractable seizures that had started early in life, with cognitive and behavioral deterioration, consistent with classical Dravet phenotypes. These mutations failed to produce measurable sodium currents in a mammalian expression system, indicating complete loss of channel function. A fourth patient, who harbored the mutation p.Met1267Ile, though presenting with seizures early in life, showed lower seizure burden and higher cognitive function, matching borderland Dravet phenotypes. In correlation with this, functional analysis demonstrated the presence of sodium currents, but with partial loss of function. In contrast, six bioinformatics tools for predicting mutational pathogenicity suggested similar impact for all mutations. Likewise, homology modeling of the secondary and tertiary structures failed to reveal misfolding. In conclusion, functional studies using patch clamp are suggested as a prognostic tool, whereby detectable currents imply milder phenotypes and absence of currents indicate an unfavorable prognosis. Future development of automated patch clamp systems will facilitate the inclusion of such functional testing as part of personalized patient diagnostic schemes.

Functional restitution of cardiac control in heart transplant patients.

Cardiovascular control is fundamentally altered after heart transplantation (HT) because of surgical denervation of the heart. The main goal of this work was the noninvasive characterization of cardiac rate control mechanisms after HT and the understanding of their nature. We obtained 25 recordings from 13 male HT patients [age = 28-68 yr, time after transplant (TAT) = 0.5-62.5 mo]. The control group included 14 healthy men (age = 28-59 yr). Electrocardiogram, continuous blood pressure (BP), and respiration were recorded for 45 min in the supine position and then during active change of posture (CP) to standing. The signals were analyzed in the time domain [mean and variance of heart rate (HR) and rise time of HR in response to CP] and the frequency domain [low and high frequency (LF and HF)]. Our principal finding was the consistent pattern of evolution of the HR response to standing: from no response, via a slow response (>40 s, TAT > 6 wk), to a fast increase (<20 s, TAT > 24 mo). HR response correlated with TAT (P < 0.001). LF correlated with HR response to CP (P < 0.0001); HF and HR did not. An important finding was the presence of very-high-frequency peaks in the power spectrum of HR and BP fluctuations. Extensive arrhythmias tended to appear at the TAT that corresponds to the transition from slow to fast HR response to CP. Our results indicate a biphasic evolution in cardiac control mechanisms from lack of control to a first-order control loop followed by partial sympathetic reinnervation and, finally, the direct effect of the old sinoatrial node on the pacemaker cell of the new sinoatrial node. There was no indication of vagal reinnervation.