A comparison of biological activity of commercially available purified native botulinum neurotoxin serotypes A1 to F1 in vitro, ex vivo, and in vivo.

Botulinum neurotoxin (BoNT) is a major therapeutic agent. Of seven native BoNT serotypes (A to G), only A and B are currently used in the clinic. Here we compared the potency of commercially available purified native serotypes A1 to F1 across in vitro, ex vivo, and in vivo assays. BoNT potency in vitro was assessed in rat primary cells (target protein cleavage and neurotransmitter release assays) in supraspinal, spinal, and sensory systems. BoNT potency ex vivo was measured in the mouse phrenic nerve hemidiaphragm (PNHD) assay, measuring muscle contractility. In vivo, BoNT-induced muscle relaxation in mice and rats was assessed in the Digit Abduction Score (DAS) test, while effects on body weight (BW) gain were used to assess tolerability. In all assays, all BoNT serotypes were potent toxins, except serotype D1 in vivo which failed to produce significant muscle flaccidity in mice and rats. In rats, all serotypes were well-tolerated, whereas in mice, reductions in BW were detected at high doses. Serotype A1 was the most potent serotype across in vitro, ex vivo, and in vivo assays. The rank order of potency of the serotypes revealed differences among assays. For example, species-specificity was seen for serotype B1, and to a lesser extent for serotype C1. Serotypes F1 and C1, not currently in the clinic, showed preference for sensory over motor models and therefore could be considered for development in conditions involving the somatosensory system.

Purification and Characterization of Recombinant Botulinum Neurotoxin Serotype FA, Also Known as Serotype H.

We have purified and characterized recombinant botulinum neurotoxin serotype FA (BoNT/FA). This protein has also been named as a new serotype (serotype H), but the classification has been controversial. A lack of well-characterized, highly pure material has been a roadblock to study. Here we report purification and characterization of enzymatically active, and of inactive nontoxic, recombinant forms of BoNT/FA as tractable alternatives to purifying this neurotoxin from native Clostridium botulinum. BoNT/FA cleaves the same intracellular target proteins as BoNT/F1 and other F serotype BoNTs; the intracellular targets are vesicle associated membrane proteins (VAMP) 1, 2 and 3. BoNT/FA cleaves the same site in VAMP-2 as BoNT/F5, which is different from the cleavage site of other F serotype BoNTs. BoNT/FA has slower enzyme kinetics than BoNT/F1 in a cell-free protease assay and is less potent at inhibiting ex vivo nerve-stimulated skeletal muscle contraction. In contrast, BoNT/FA is more potent at inhibiting neurotransmitter release from cultured neurons.

Analysis of the hippocampal proteome in ME7 prion disease reveals a predominant astrocytic signature and highlights the brain-restricted production of clusterin in chronic neurodegeneration.

Prion diseases are characterized by accumulation of misfolded protein, gliosis, synaptic dysfunction, and ultimately neuronal loss. This sequence, mirroring key features of Alzheimer disease, is modeled well in ME7 prion disease. We used iTRAQ(TM)/mass spectrometry to compare the hippocampal proteome in control and late-stage ME7 animals. The observed changes associated with reactive glia highlighted some specific proteins that dominate the proteome in late-stage disease. Four of the up-regulated proteins (GFAP, high affinity glutamate transporter (EAAT-2), apo-J (Clusterin), and peroxiredoxin-6) are selectively expressed in astrocytes, but astrocyte proliferation does not contribute to their up-regulation. The known functional role of these proteins suggests this response acts against protein misfolding, excitotoxicity, and neurotoxic reactive oxygen species. A recent convergence of genome-wide association studies and the peripheral measurement of circulating levels of acute phase proteins have focused attention on Clusterin as a modifier of late-stage Alzheimer disease and a biomarker for advanced neurodegeneration. Since ME7 animals allow independent measurement of acute phase proteins in the brain and circulation, we extended our investigation to address whether changes in the brain proteome are detectable in blood. We found no difference in the circulating levels of Clusterin in late-stage prion disease when animals will show behavioral decline, accumulation of misfolded protein, and dramatic synaptic and neuronal loss. This does not preclude an important role of Clusterin in late-stage disease, but it cautions against the assumption that brain levels provide a surrogate peripheral measure for the progression of brain degeneration.

Selective presynaptic degeneration in the synaptopathy associated with ME7-induced hippocampal pathology.

Intrahippocampal injection of the murine modified scrapie (ME7) induces a model of prion disease in vivo. Animals inoculated with ME7 brain homogenate were compared to controls at 8, 12 and 21 weeks. The data show that the accumulation of misfolded prion (PrP(Sc)) coincided with selective reduction in presynaptic protein expression early in disease. This loss is independent of a change in the number of cell bodies in CA3 that provide the major presynaptic input to the stratum radiatum. Electron microscopy of the stratum radiatum independently evidenced a progressive decrease in the number of synapses during disease. Further, the number of postsynaptic specializations lacking an intact presynaptic specialization increased from 12 to 21 weeks. This suggests that the presynaptic compartment is selectively disrupted when the previously reported first behavioural deficits are observed in this model. This synaptic pathology or "synaptopathy" may represent the earliest neuronal dysfunction in this and other protein misfolding induced neurodegenerative diseases.

Aquaporin-4 antibodies in neuromyelitis optica and longitudinally extensive transverse myelitis.

BACKGROUND: There is increasing recognition of antibody-mediated immunotherapy-responsive neurologic diseases and a need for appropriate immunoassays. OBJECTIVES: To develop a clinically applicable quantitative assay to detect the presence of aquaporin-4 (AQP4) antibodies in patients with neuromyelitis optica and to characterize the anti-AQP4 antibodies. DESIGN: We compared a simple new quantitative fluorescence immunoprecipitation assay (FIPA) with both indirect immunofluorescence and an AQP4-transfected cell-based assay, both previously described. We used the cell-based assay to characterize the antibodies for their immunoglobulin class, IgG subclass, and ability to induce complement C3b deposition in vitro. SETTING: United Kingdom and Germany. PARTICIPANTS: Serum samples from patients with neuromyelitis optica (n = 25) or longitudinally extensive transverse myelitis (n = 11) and from relevant controls (n = 78) were studied. MAIN OUTCOME MEASURES: Comparison of different assays for AQP4 antibodies and characterization of anti-AQP4 antibodies in patients with neuromyelitis optica. RESULTS: We found antibodies to AQP4 in 19 of 25 patients with neuromyelitis optica (76%) using FIPA, in 20 of 25 patients with neuromyelitis optica (80%) using the cell-based assay, and in 6 of 11 patients with longitudinally extensive transverse myelitis (55%) with both assays; these assays were more sensitive than indirect immunofluorescence and 100% specific. The antibodies bound to extracellular epitope(s) of AQP4, were predominantly IgG1, and strongly induced C3b deposition. CONCLUSIONS: Aquaporin-4 is a major antigen in neuromyelitis optica, and antibodies can be detected in more than 75% of patients. Further studies on larger samples will show whether this novel FIPA is suitable for clinical use. The IgG1 antibodies bind to AQP4 on the cell surface and can initiate complement deposition. These approaches will be useful for investigation of other antibody-mediated diseases.