The investigated CBSDA has a high sensitivity and an acceptable specificity for triggering VNS. Despite the moderate effects on seizure frequency, combined open- and closed-loop VNS may provide valuable improvements in seizure severity and QOL in refractory epilepsy patients.

No abstract

A major challenge to studying Fe-S cluster biosynthesis in higher eukaryotes is the lack of simple tools for imaging metallocluster binding to proteins. We describe the first fluorescent approach for in vivo detection of 2Fe2S clusters that is based upon the complementation of Venus fluorescent protein fragments via human glutaredoxin 2 (GRX2) coordination of a 2Fe2S cluster. We show that Escherichia coli and mammalian cells expressing Venus fragments fused to GRX2 exhibit greater fluorescence than cells expressing fragments fused to a C37A mutant that cannot coordinate a metallocluster. In addition, we find that maximal fluorescence in the cytosol of mammalian cells requires the iron-sulfur cluster assembly proteins ISCU and NFS1. These findings provide evidence that glutaredoxins can dimerize within mammalian cells through coordination of a 2Fe2S cluster as observed with purified recombinant proteins.

Background: Vagus nerve stimulation (VNS) modifies brain rhythms in the locus coeruleus (LC) via the solitary nucleus. Degeneration of the LC in Parkinson's disease (PD) is an early catalyst of the spreading neurodegenerative process, suggesting that stimulating LC output with VNS has the potential to modify disease progression. We previously showed in a lesion PD model that VNS delivered twice daily reduced neuroinflammation and motor deficits, and attenuated tyrosine hydroxylase (TH)-positive cell loss. Objective: The goal of this study was to characterize the differential effects of three clinically-relevant VNS paradigms in a PD lesion model. Methods: Eleven days after DSP-4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine, noradrenergic lesion, administered systemically)/6-OHDA (6-hydroxydopamine, dopaminergic lesion, administered intrastriatally) rats were implanted with VNS devices, and received either low-frequency VNS, standardfrequency VNS, or high-frequency microburst VNS. After 10 days of treatment and behavioral assessment, rats were euthanized, right prefrontal cortex (PFC) was dissected for norepinephrine assessment, and the left striatum, bilateral substantia nigra (SN), and LC were sectioned for immunohistochemical detection of catecholamine neurons, a-synuclein, astrocytes, and microglia.Results: At higher VNS frequencies, specifically microburst VNS, greater improvements occurred in motor function, attenuation of TH-positive cell loss in SN and LC, and norepinephrine concentration in the PFC.Additionally, higher VNS frequencies resulted in lower intrasomal a-synuclein accumulation and glial density in the SN. Conclusions: These data indicate that higher stimulation frequencies provided the greatest attenuation of behavioral and pathological markers in this PD model, indicating therapeutic potential for these VNS paradigms.

The solute carrier transmembrane protein prestin (SLC26A5) drives an active electromechanical transduction process in cochlear outer hair cells that increases hearing sensitivity and frequency discrimination in mammals. A large intramembraneous charge movement, the nonlinear capacitance (NLC), is the electrical signature of prestin function. The transmembrane domain (TMD) helices and residues involved in the intramembrane charge displacement remain unknown. We have performed cysteine-scanning mutagenesis with serine or valine replacement to investigate the importance of cysteine residues to prestin structure and function. The distribution of oligomeric states and membrane abundance of prestin was also probed to investigate whether cysteine residues participate in prestin oligomerization and/or NLC. Our results reveal that 1) Cys-196 (TMD 4) and Cys-415 (TMD 10) do not tolerate serine replacement, and thus maintaining hydrophobicity at these locations is important for the mechanism of charge movement; 2) Cys-260 (TMD 6) and Cys-381 (TMD 9) tolerate serine replacement and are probably water-exposed; and 3) if disulfide bonds are present, they do not serve a functional role as measured via NLC. These novel findings are consistent with a recent structural model, which proposes that prestin contains an occluded aqueous pore, and we posit that the orientations of transmembrane domain helices 4 and 10 are essential for proper prestin function.Prestin is an essential component of the voltage-sensitive molecular motor of outer hair cells that converts changes in transmembrane potential into mechanical forces. This transduction causes outer hair cells to undergo a characteristic length change termed electromotility that enhances sound amplification and frequency selectivity (1, 2). In the absence of prestin, hearing thresholds are effectively elevated 40 -60 dB in mice (3). A large displacement current, analogous to the gating current in ion channels, has been established as a reliable electrical signature for electromotility (4,5). This electrical signature is referred to as the nonlinear capacitance (NLC) 4 and requires the presence of intracellular chloride (6).Very little is known about the structure of prestin and its relationship to prestin function. Several topologies have been proposed based on hydrophobicity and sequence conservation between prestin and the family of solute anion transporters to which it belongs. Proteins of the solute carrier family 26 (SLC26) probably span the membrane 10 -14 times (7-9). Because prestin (SLC26A5) has been shown to have intracellular N and C termini (10), topologies have been proposed having an even number of transmembrane domains. Ten-pass and reentrant loop 12-pass models of prestin secondary structure have been proposed (Fig. 1 and supplemental Table S1) and refined based on knowledge of phosphorylation sites (11), evolutionary analysis (12), and conflicting reports of N-linked glycosylation (13, 14), a result possibly due to expression in different model cell systems. Cur...