Differential effects of vagus nerve stimulation paradigms guide clinical development for Parkinson's disease.

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, standard-frequency 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, α-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 α-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.

A prospective, multicenter study of cardiac-based seizure detection to activate vagus nerve stimulation.

PURPOSE: This study investigates the performance of a cardiac-based seizure detection algorithm (CBSDA) that automatically triggers VNS (NCT01325623). METHODS: Thirty-one patients with drug resistant epilepsy were evaluated in an epilepsy monitoring unit (EMU) to assess algorithm performance and near-term clinical benefit. Long-term efficacy and safety were evaluated with combined open and closed-loop VNS. RESULTS: Sixty-six seizures (n=16 patients) were available from the EMU for analysis. In 37 seizures (n=14 patients) a ≥ 20% heart rate increase was found and 11 (n=5 patients) were associated with ictal tachycardia (iTC, 55% or 35 bpm heart rate increase, minimum of 100 bpm). Multiple CBSDA settings achieved a sensitivity of ≥ 80%. False positives ranged from 0.5 to 7.2/h. 27/66 seizures were stimulated within ± 2 min of seizure onset. In 10/17 of these seizures, where triggered VNS overlapped with ongoing seizure activity, seizure activity stopped during stimulation. Physician-scored seizure severity (NHS3-scale) showed significant improvement for complex partial seizures (CPS) at EMU discharge and through 12 months (p<0.05). Patient-scored seizure severity (total SSQ score) showed significant improvement at 3 and 6 months. Quality of life (total QOLIE-31-P score) showed significant improvement at 12 months. The responder rate (≥ 50% reduction in seizure frequency) at 12 months was 29.6% (n=8/27). Safety profiles were comparable to prior VNS trials. CONCLUSIONS: 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.

Head bobber: an insertional mutation causes inner ear defects, hyperactive circling, and deafness.

The head bobber transgenic mouse line, produced by pronuclear integration, exhibits repetitive head tilting, circling behavior, and severe hearing loss. Transmitted as an autosomal recessive trait, the homozygote has vestibular and cochlea inner ear defects. The space between the semicircular canals is enclosed within the otic capsule creating a vacuous chamber with remnants of the semicircular canals, associated cristae, and vestibular organs. A poorly developed stria vascularis and endolymphatic duct is likely the cause for Reissner's membrane to collapse post-natally onto the organ of Corti in the cochlea. Molecular analyses identified a single integration of ~3 tandemly repeated copies of the transgene, a short duplicated segment of chromosome X and a 648 kb deletion of chromosome 7(F3). The three known genes (Gpr26, Cpxm2, and Chst15) in the deleted region are conserved in mammals and expressed in the wild-type inner ear during vestibular and cochlea development but are absent in homozygous mutant ears. We propose that genes critical for inner ear patterning and differentiation are lost at the head bobber locus and are candidate genes for human deafness and vestibular disorders.

Selective cell-surface labeling of the molecular motor protein prestin.

Prestin, a multipass transmembrane protein whose N- and C-termini are localized to the cytoplasm, must be trafficked to the plasma membrane to fulfill its cellular function as a molecular motor. One challenge in studying prestin sequence-function relationships within living cells is separating the effects of amino acid substitutions on prestin trafficking, plasma membrane localization and function. To develop an approach for directly assessing prestin levels at the plasma membrane, we have investigated whether fusion of prestin to a single pass transmembrane protein results in a functional fusion protein with a surface-exposed N-terminal tag that can be detected in living cells. We find that fusion of the biotin-acceptor peptide (BAP) and transmembrane domain of the platelet-derived growth factor receptor (PDGFR) to the N-terminus of prestin-GFP yields a membrane protein that can be metabolically-labeled with biotin, trafficked to the plasma membrane, and selectively detected at the plasma membrane using fluorescently-tagged streptavidin. Furthermore, we show that the addition of a surface detectable tag and a single-pass transmembrane domain to prestin does not disrupt its voltage-sensitive activity.

Cysteine mutagenesis reveals transmembrane residues associated with charge translocation in prestin.

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.

In vivo fluorescent detection of Fe-S clusters coordinated by human GRX2.

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.