Structural Insights into Rational Design of Single-Domain Antibody-Based Antitoxins against Botulinum Neurotoxins.

Botulinum neurotoxin (BoNT) is one of the most acutely lethal toxins known to humans, and effective treatment for BoNT intoxication is urgently needed. Single-domain antibodies (VHH) have been examined as a countermeasure for BoNT because of their high stability and ease of production. Here, we investigate the structures and the neutralization mechanisms for six unique VHHs targeting BoNT/A1 or BoNT/B1. These studies reveal diverse neutralizing mechanisms by which VHHs prevent host receptor binding or block transmembrane delivery of the BoNT protease domain. Guided by this knowledge, we design heterodimeric VHHs by connecting two neutralizing VHHs via a flexible spacer so they can bind simultaneously to the toxin. These bifunctional VHHs display much greater potency in a mouse co-intoxication model than similar heterodimers unable to bind simultaneously. Taken together, our studies offer insight into antibody neutralization of BoNTs and advance our ability to design multivalent anti-pathogen VHHs with improved therapeutic properties

The Wnt Signaling Antagonist Kremen1 is Required for Development of Thymic Architecture

Wnt signaling has been reported to regulate thymocyte proliferation and selection at several stages during T cell ontogeny, as well as the expression of FoxN1 in thymic epithelial cells (TECs). Kremen1 (Krm1) is a negative regulator of the canonical Wnt signaling pathway, and functions together with the secreted Wnt inhibitor Dickkopf (Dkk) by competing for the lipoprotein receptor-related protein (LRP)-6 co-receptor for Wnts. Here krm1 knockout mice were used to examine krm1 expression in the thymus and its function in thymocyte and TEC development. krm1 expression was detected in both cortical and medullary TEC subsets, as well as in immature thymocyte subsets, beginning at the CD25+CD44+ (DN2) stage and continuing until the CD4+CD8+(DP) stage. Neonatal mice show elevated expression of krm1 in all TEC subsets. krm1− / − mice exhibit a severe defect in thymic cortical architecture, including large epithelial free regions. Much of the epithelial component remains at an immature Keratin 5+ (K5) Keratin 8+(K8) stage, with a loss of defined cortical and medullary regions. A TOPFlash assay revealed a 2-fold increase in canonical Wnt signaling in TEC lines derived from krm1− / − mice, when compared with krm1+ / + derived TEC lines. Fluorescence activated cell sorting (FACS) analysis of dissociated thymus revealed a reduced frequency of both cortical (BP1+EpCAM+) and medullary (UEA-1+ EpCAMhi) epithelial subsets, within the krm1− / − thymus. Surprisingly, no change in thymus size, total thymocyte number or the frequency of thymocyte subsets was detected in krm1− / − mice. However, our data suggest that a loss of Krm1 leads to a severe defect in thymic architecture. Taken together, this study revealed a new role for Krm1 in proper development of thymic epithelium

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

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

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