Neuromuscular recovery from botulism involves multiple forms of compensatory plasticity.

Introduction: Botulinum neurotoxin (BoNT) causes neuroparalytic disease and death by blocking neuromuscular transmission. There are no specific therapies for clinical botulism and the only treatment option is supportive care until neuromuscular function spontaneously recovers, which can take weeks or months after exposure. The highly specialized neuromuscular junction (NMJ) between phrenic motor neurons and diaphragm muscle fibers is the main clinical target of BoNT. Due to the difficulty in eliciting respiratory paralysis without a high mortality rate, few studies have characterized the neurophysiological mechanisms involved in diaphragm recovery from intoxication. Here, we develop a mouse model of botulism that involves partial paralysis of respiratory muscles with low mortality rates, allowing for longitudinal analysis of recovery. Methods and results: Mice challenged by systemic administration of 0.7 LD50 BoNT/A developed physiological signs of botulism, such as respiratory depression and reduced voluntary running activity, that persisted for an average of 8-12 d. Studies in isolated hemidiaphragm preparations from intoxicated mice revealed profound reductions in nerve-elicited, tetanic and twitch muscle contraction strengths that recovered to baseline 21 d after intoxication. Despite apparent functional recovery, neurophysiological parameters remained depressed for 28 d, including end plate potential (EPP) amplitude, EPP success rate, quantal content (QC), and miniature EPP (mEPP) frequency. However, QC recovered more quickly than mEPP frequency, which could explain the discrepancy between muscle function studies and neurophysiological recordings. Hypothesizing that differential modulation of voltage-gated calcium channels (VGCC) contributed to the uncoupling of QC from mEPP frequency, pharmacological inhibition studies were used to study the contributions of different VGCCs to neurophysiological function. We found that N-type VGCC and P/Q-type VGCC partially restored QC but not mEPP frequency during recovery from paralysis, potentially explaining the accelerated recovery of evoked release versus spontaneous release. We identified additional changes that presumably compensate for reduced acetylcholine release during recovery, including increased depolarization of muscle fiber resting membrane potential and increased quantal size. Discussion: In addition to identifying multiple forms of compensatory plasticity that occur in response to reduced NMJ function, it is expected that insights into the molecular mechanisms involved in recovery from neuromuscular paralysis will support new host-targeted treatments for multiple neuromuscular diseases.

Direct visualization of the drug release process of non-conductive polymeric implants via molecular imaging.

Long-acting parenteral (LAP) implant has garnered the attraction as a drug delivery technique in recent years. Understanding the drug release process is critical for the study of underlying release mechanism. In this paper, we present a novel application of matrix-assisted laser desorption/ionization-mass spectrometry imaging (MADLI-MSI) for the direct visualization of the drug release process from non-conductive polymeric based LAP implants at molecular level. Custom-made sample holders were designed for LAP sample introduction in place of traditional conductive glass slides. The main technical obstacles of applying MALDI-MSI to study non-conductive materials are surface conductivity which can lead to charge build-up. In order to obtain homogeneous imaging of non-conductive sample surfaces, we developed a new sample surface treatment procedure, which is a critical control step to ensure the data reliability and accuracy in understanding kinetics of drug release process of LAP. Overall, this is the first comprehensive report of a sample preparation methodology tailored for imaging LAP at molecular level, allowing for the direct chemical identification and 2D mapping of an active pharmaceutical ingredient (API) distribution during LAP release process. Furthermore, this work has established the foundation to apply MALDI-MSI to the understanding of LAP implant formulation homogeneity, chemical composition, and degradation. More importantly, this work enabled the extension of MALDI-MSI technique to study a wide range of non-conductive materials.

GDNF revisited: A novel mammalian cell-derived variant form of GDNF increases dopamine turnover and improves brain biodistribution.

Parkinson's disease (PD) is a disorder affecting dopamine neurons for which there is no cure. Glial cell line-derived neurotrophic factor (GDNF) and the closely related protein neurturin are two trophic factors with demonstrated neuroprotective and neurorestorative properties on dopamine neurons in multiple animal species. However, GDNF and neurturin Phase-2 clinical trials have failed to demonstrate a significant level of improvement over placebo controls. Insufficient drug distribution in the brain parenchyma has been proposed as a major contributing factor for the lack of clinical efficacy in the Phase-2 trial patients. To address this issue, a novel mammalian cell-derived variant form of GDNF (GDNFv) was designed to promote better tissue distribution by reducing its heparin binding to the extracellular matrix and key amino acids were substituted to enhance its chemical stability. Administration of this fully glycosylated GDNFv in the normal rat striatum increased dopamine turnover and produced significantly greater brain distribution than E. coli-produced wildtype GDNF (GDNFwt). Intrastriatal GDNFv also protected midbrain dopamine neuron function in 6-hydroxydopamine-lesioned rats. Studies conducted in normal adult rhesus macaques support that GDNFv was well tolerated in all animals and demonstrated a greater volume of distribution than GDNFwt in the brain following intrastriatal infusion. Importantly, favorable physiological activity of potential therapeutic value was maintained in this variant trophic factor with significant target activation in GDNFv recipients as indicated by dopamine turnover modulation. These data suggest that GDNFv may be a promising drug candidate for the treatment of PD. Additional studies are needed in non-human primates with dopamine depletion. This article is part of the Special Issue entitled 'Drug Repurposing: old molecules, new ways to fast track drug discovery and development for CNS disorders'.

Methodology and effects of repeated intranasal delivery of DNSP-11 in a rat model of Parkinson's disease.

BACKGROUND: To circumvent the challenges associated with delivering large compounds directly to the brain for the treatment of Parkinson's disease (PD), non-invasive procedures utilizing smaller molecules with protective and/or restorative actions on dopaminergic neurons are needed. NEW METHOD: We developed a methodology for evaluating the effects of a synthetic neuroactive peptide, DNSP-11, on the nigrostriatal system using repeated intranasal delivery in both normal and a unilateral 6-hydroxydopamine (6-OHDA) lesion rat model of PD. RESULTS: Normal rats repeatedly administered varying doses of DNSP-11 intranasally for 3 weeks exhibited a significant increase in dopamine (DA) turnover in both the striatum and substantia nigra (SN) at 300µg, suggestive of a stimulative effect of the dopaminergic system. Additionally, a protective effect was observed following repeated intranasal administration in 6-OHDA lesioned rats, as suggested by: a significant decrease in d-amphetamine-induced rotation at 2 weeks; a decrease in DA turnover in the lesioned striatum; and an increased sparing of tyrosine hydroxylase (TH) positive (+) neurons in a specific sub-region of the lesioned substantia nigra pars compacta (SNpc). Finally, tracer studies showed (125)I-DNSP-11 distributed diffusely throughout the brain, including the striatum and SN, as quickly as 30min after a single intranasal dose. COMPARISON WITH EXISTING METHODS: The results of bilateral intranasal administration of DNSP-11 are compared to our unilateral single infusion studies to the brain in rats. CONCLUSIONS: These studies support that DNSP-11 can be delivered intranasally and maintain its neuroactive properties in both normal rats and in a unilateral 6-OHDA rat model of PD.