Neuromuscular recovery from botulism involves multiple forms of compensatory plasticity

IntroductionBotulinum 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 resultsMice 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.DiscussionIn 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

Protocadherin-18 Is a Novel Differentiation Marker and an Inhibitory Signaling Receptor for CD8+ Effector Memory T Cells

CD8+ tumor infiltrating T cells (TIL) lack effector-phase functions due to defective proximal TCR-mediated signaling previously shown to result from inactivation of p56lck kinase. We identify a novel interacting partner for p56lck in nonlytic TIL, Protocadherin-18 (‘pcdh18’), and show that pcdh18 is transcribed upon in vitro or in vivo activation of all CD8+ central memory T cells (CD44+CD62LhiCD127+) coincident with conversion into effector memory cells (CD44+CD62LloCD127+). Expression of pcdh18 in primary CD8+ effector cells induces the phenotype of nonlytic TIL: defective proximal TCR signaling, cytokine secretion, and cytolysis, and enhanced AICD. pcdh18 contains a motif (centered at Y842) shared with src kinases (QGQYQP) that is required for the inhibitory phenotype. Thus, pcdh18 is a novel activation marker of CD8+ memory T cells that can function as an inhibitory signaling receptor and restrict the effector phase

Atoxic Derivative of Botulinum Neurotoxin A as a Prototype Molecular Vehicle for Targeted Delivery to the Neuronal Cytoplasm

<div><p>We have previously described genetic constructs and expression systems that enable facile production of recombinant derivatives of botulinum neurotoxins (BoNTs) that retain the structural and trafficking properties of <i>wt</i> BoNTs. In this report we describe the properties of one such derivative, BoNT/A <i>ad</i>, which was rendered atoxic by introducing two amino acid mutations to the light chain (LC) of <i>wt</i> BoNT/A, and which is being developed as a molecular vehicle for delivering drugs to the neuronal cytoplasm. The neuronal binding, internalization, and intracellular trafficking of BoNT/A <i>ad</i> in primary hippocampal cultures was evaluated using three complimentary techniques: flow cytometry, immunohistochemistry, and Western blotting. Neuronal binding of BoNT <i>ad</i> was significantly increased when neurons were incubated in depolarizing medium. Flow cytometry demonstrated that BoNT/A <i>ad</i> internalized into neurons but not glia. After 24 hours, the majority of the neuron-bound BoNT/A <i>ad</i> became internalized, as determined by its resistance to pronase E-induced proteolytic degradation of proteins associated with the plasma membrane of intact cells. Significant amounts of the atoxic LC accumulated in a Triton X-100-extractable fraction of the neurons, and persisted as such for at least 11 days with no evidence of degradation. Immunocytochemical analysis demonstrated that the LC of BoNT/A <i>ad</i> was translocated to the neuronal cytoplasm after uptake and was specifically targeted to SNARE proteins. The atoxic LC consistently co-localized with synaptic markers SNAP-25 and VAMP-2, but was rarely co-localized with markers for early or late endosomes. These data demonstrate that BoNT/A <i>ad</i> mimics the trafficking properties of <i>wt</i> BoNT/A, confirming that our platform for designing and expressing BoNT derivatives provides an accessible system for elucidating the molecular details of BoNT trafficking, and can potentially be used to address multiple medical and biodefense needs.</p></div

Neuronal uptake of BoNT/A <i>ad</i>.

<p>E19 rat hippocampal neurons were cultured in maintenance medium for 10/A <i>ad</i> for 24 hours at 37°C. Cells were analyzed by flow cytometry. <b>Panel </b><b>A:</b> Cells were exposed to 25 nM BoNT/A <i>ad</i>. Plot shows cells stained with F1-40 mAb to detect BoNT/A <i>ad</i> light chain (X-axis) and with anti-GFAP mAb to detect glia (Y-axis). Numbers in each quadrant represent the percentage of cells in that population. <b>Panel </b><b>B:</b> Calculated median fluorescent intensity (MFI) from cell cultures exposed for 24 hr at 37°C to indicated concentrations of BoNT/A <i>ad</i>.</p

Internalized LC <i>ad</i> co-localizes with cytoplasmic/vesicular synaptic markers.

<p>E19 rat hippocampal neurons were cultured in maintenance medium for 10°C to 25 nM BoNT/A <i>ad</i>. After incubation, cells were washed and processed for immunofluorescence (see Materials and Methods). Cells were stained for LC <i>ad</i> and SNAP-25 (<b>Panel </b><b>A</b>), VAMP-2 (<b>Panel </b><b>B</b>), EEA1 (<b>Panel </b><b>C</b>), and Rab5 (<b>Panel </b><b>D</b>). Scale is 10 µm.</p

Intraneuronal persistence of LC <i>ad</i>.

<p>E19 rat hippocampal neurons were cultured in maintenance medium for 10°C to 50 nM BoNT/A <i>ad</i>. After incubation, cells were washed twice with maintenance medium to remove residual BoNT/A <i>ad</i> and chased with the fresh medium for 1 to 11 days. <b>Panel </b><b>A:</b> Western blot analysis of LC <i>ad</i> (mAb F1-40). GAPDH was used as a loading control. <b>Panel </b><b>B:</b> Flow cytometric quantification of the LC <i>ad</i> signal at different days of chase. <b>Panel </b><b>C:</b> Immunostaining for <i>tau</i> (red, anti-<i>tau</i> mouse monoclonal IgG_2b, Cat # 610672, BD Biosciences) and LC <i>ad</i> (white). Scale is 10 µm.</p

Immunofluorescence analysis showing BoNT/A <i>ad</i> binding during neuronal depolarization (active neurons).

<p>E19 rat hippocampal neurons cultured in maintenance medium for 10/A <i>ad</i> for 1 min at 37°C in HEPES Ringer Resting Buffer (top row) or in high K⁺ HEPES Ringer Depolarization Buffer (bottom row). Anti-MAP-2 chicken monoclonal antibody (Cat # PCK-554P, Covance) was used to stain neuronal soma (red), and F1-40 antibody was used for detection of LC <i>ad</i> (green). Scale is 10 µm.</p

Intracellular accumulation of BoNT/A <i>ad</i>.

<p>E19.5/A <i>ad</i> at 37°C. <b>Panel </b><b>A:</b> Western blot analysis after incubation of the cells with BoNT/A <i>ad</i> for 1, 24, or 48 hours, followed by treatment with pronase E or control (see Materials and Methods). Protein concentration was normalized to GAPDH. VAMP-2 loading controls were used to demonstrate absence of synaptic protein degradation in experiments with pronase E treatment. <b>Panel </b><b>B:</b> Western blot of indicated amounts of BoNT/A <i>ad</i> LC developed with F 1-40 mAb. This panel was used to generate the standard curve for LC <i>ad</i> quantification. <b>Panel </b><b>C:</b> Quantification of the amount of LC <i>ad</i> per µg total protein (see Materials and Methods). The experiment was performed in triplicate.</p

Conversion of Cm into Em cells upon activation <i>in vitro</i>.

<p>(4a) FACS purification of spleen CD8⁺ CD44^hiCD62L^loCD127^hi (Effector-memory) and CD44^hiCD62L^hiCD127^hi (Central-memory) cells obtained from young (<5 week) and >50 week old naïve, and 50 week old mice that were previously injected with allogeneic spleen cells as indicated. Following sorting, an aliquot of cells was immediately taken for RNA isolation without activation. Analyses in the left panels are gated on CD8⁺ cells and show the distribution of CD44⁺ cells. Analyses in the right panels show Em and Cm cells within the CD44^lo and CD44^hi populations as indicated. (4b) Cm and Em cells were isolated by FACS from young, old, and memory (<i>L. monocytogenes</i>-infected) mice and activated <i>in vitro</i> with ConA for the indicated times before qRT-PCR. The % of CD8⁺CD44^hi cells as CD62L^loCD127^hi (Em) and CD62L^hiCD127^hi (Cm from the ‘young’ cohort are shown in tabular form in this figure. Flow cytometry analyses of some samples from the kinetic experiment are shown to illustrate gating. (4c) Enriched CD8⁺ spleen cells prior to sorting are shown in the left panels. After labeling Cm were isolated and activated <i>in vitro</i>. The recovery of Em and Cm cells was determined by flow cytometry after re-staining and flow cytometric analysis. Shown are the FACS analyses after the indicated times of Cm activation. Also shown is the number of Cm and Em cells derived from <i>in vitro</i> activation of Cm cells represented as a ratio of Cm to Em cells at the various times of analyses.</p