A causal role for TRESK loss of function in migraine mechanisms

The two-pore potassium channel, TRESK has been implicated in nociception and pain disorders. We have for the first time investigated TRESK function in human nociceptive neurons using induced pluripotent stem cell-based models. Nociceptors from migraine patients with the F139WfsX2 mutation show loss of functional TRESK at the membrane, with a corresponding significant increase in neuronal excitability. Furthermore, using CRISPR-Cas9 engineering to correct the F139WfsX2 mutation, we show a reversal of the heightened neuronal excitability, linking the phenotype to the mutation. In contrast we find no change in excitability in induced pluripotent stem cell derived nociceptors with the C110R mutation and preserved TRESK current; thereby confirming that only the frameshift mutation is associated with loss of function and a migraine relevant cellular phenotype. We then demonstrate the importance of TRESK to pain states by showing that the TRESK activator, cloxyquin, can reduce the spontaneous firing of nociceptors in an in vitro human pain model. Using the chronic nitroglycerine rodent migraine model, we demonstrate that mice lacking TRESK develop exaggerated nitroglycerine-induced mechanical and thermal hyperalgesia, and furthermore, show that cloxyquin conversely is able to prevent sensitization. Collectively, our findings provide evidence for a role of TRESK in migraine pathogenesis and its suitability as a therapeutic target

Immune or genetic-mediated disruption of CASPR2 causes pain hypersensitivity due to enhanced primary afferent excitability

Human autoantibodies to contactin-associated protein-like 2 (CASPR2) are often associated with neuropathic pain, and CASPR2 mutations have been linked to autism spectrum disorders, in which sensory dysfunction is increasingly recognized. Human CASPR2 autoantibodies, when injected into mice, were peripherally restricted and resulted in mechanical pain-related hypersensitivity in the absence of neural injury. We therefore investigated the mechanism by which CASPR2 modulates nociceptive function. Mice lacking CASPR2 (Cntnap2 ) demonstrated enhanced pain-related hypersensitivity to noxious mechanical stimuli, heat, and algogens. Both primary afferent excitability and subsequent nociceptive transmission within the dorsal horn were increased in Cntnap2 mice. Either immune or genetic-mediated ablation of CASPR2 enhanced the excitability of DRG neurons in a cell-autonomous fashion through regulation of Kv1 channel expression at the soma membrane. This is the first example of passive transfer of an autoimmune peripheral neuropathic pain disorder and demonstrates that CASPR2 has a key role in regulating cell-intrinsic dorsal root ganglion (DRG) neuron excitability

Antibodies to Kv1 potassium channel-complex proteins leucine-rich, glioma inactivated 1 protein and contactin-associated protein-2 in limbic encephalitis, Morvan’s syndrome and acquired neuromyotonia

Antibodies that immunoprecipitate 125I-α-dendrotoxin-labelled voltage-gated potassium channels extracted from mammalian brain tissue have been identified in patients with neuromyotonia, Morvan’s syndrome, limbic encephalitis and a few cases of adult-onset epilepsy. These conditions often improve following immunomodulatory therapies. However, the proportions of the different syndromes, the numbers with associated tumours and the relationships with potassium channel subunit antibody specificities have been unclear. We documented the clinical phenotype and tumour associations in 96 potassium channel antibody positive patients (titres >400 pM). Five had thymomas and one had an endometrial adenocarcinoma. To define the antibody specificities, we looked for binding of serum antibodies and their effects on potassium channel currents using human embryonic kidney cells expressing the potassium channel subunits. Surprisingly, only three of the patients had antibodies directed against the potassium channel subunits. By contrast, we found antibodies to three proteins that are complexed with 125I-α-dendrotoxin-labelled potassium channels in brain extracts: (i) contactin-associated protein-2 that is localized at the juxtaparanodes in myelinated axons; (ii) leucine-rich, glioma inactivated 1 protein that is most strongly expressed in the hippocampus; and (iii) Tag-1/contactin-2 that associates with contactin-associated protein-2. Antibodies to Kv1 subunits were found in three sera, to contactin-associated protein-2 in 19 sera, to leucine-rich, glioma inactivated 1 protein in 55 sera and to contactin-2 in five sera, four of which were also positive for the other antibodies. The remaining 18 sera were negative for potassium channel subunits and associated proteins by the methods employed. Of the 19 patients with contactin-associated protein-antibody-2, 10 had neuromyotonia or Morvan’s syndrome, compared with only 3 of the 55 leucine-rich, glioma inactivated 1 protein-antibody positive patients (P < 0.0001), who predominantly had limbic encephalitis. The responses to immunomodulatory therapies, defined by changes in modified Rankin scores, were good except in the patients with tumours, who all had contactin-associated-2 protein antibodies. This study confirms that the majority of patients with high potassium channel antibodies have limbic encephalitis without tumours. The identification of leucine-rich, glioma inactivated 1 protein and contactin-associated protein-2 as the major targets of potassium channel antibodies, and their associations with different clinical features, begins to explain the diversity of these syndromes; furthermore, detection of contactin-associated protein-2 antibodies should help identify the risk of an underlying tumour and a poor prognosis in future patients

Investigating the pathogenic effects of antibodies against the VGKC-complex and N-methyl-D-aspartate receptor in CNS disorders

Over the last few years, antibody-mediated disorders of the central nervous system (CNS) have been a rapidly expanding field in neurology. The antibodies bind to extracellular domains of proteins expressed in the CNS and impair neuronal function. Patients with limbic encephalitis, Morvan’s syndrome have antibodies that bind to the voltage gated potassium channel (VGKC)-complex proteins, LGI1 and CASPR2, while patients with antibodies to the NMDAR have more complex encephalopathies. Clinical evidence suggests that these antibodies are pathogenic as immunotherapy reduces antibody levels, which correlate closely with clinical improvement. Antibodies can be detected in patient sera using human embryonic kidney (HEK) cells transfected with CASPR2, LGI1 and NMDAR subunits, or by binding to rodent brain sections and primary neuronal cultures. In vitro NMDAR-antibodies have been shown to cross-link synaptic NMDARs and cause their subsequent internalisation from the cell membrane. It is not known if passive transfer of NMDAR-antibodies can recapitulate features of disease or if these pathogenic mechanisms occur with VGKC-complex antibodies. The characteristics of LGI1 and CASPR2 antibodies were explored in vitro. IgG1 and IgG4 were identified as the predominant antibody subclasses, with all CASPR2 sera containing IgG1 antibodies and all LGI1 sera containing IgG4. On transfected cells both LGI1 and CASPR2 antibodies activated the classical pathway of the complement cascade. Furthermore CASPR2 antibody binding to transfected HEK cells showed a reduction in intensity over time which was most likely a result of internalisation of the CASPR2-antibody bound complex. Both CASPR2 and LGI1 antibodies bound strongly to the surface of live hippocampal neurons in culture, and this staining was reduced 24 hours after the removal of IgG, corresponding to a loss of antigen expressed at the cell surface. Patients with NMDAR-encephalitis present with a psychotic disturbance, memory loss and seizures, usually progressing to a reduced level of consciousness and a marked movement disorder. A single injection of IgG, purified from patients with NMDAR-encephalitis was administered into the lateral ventricle of mice. Animals were observed for 40 days and showed significant behavioural changes, including a deficit in spontaneous alternation, dyskinetic hind-limb clasping and visual signs of spontaneous seizures, which are reminiscent to features of the disease. Behavioural changes were not observed in animals injected with IgG from healthy individuals. These behavioural alterations appeared to be reversible and were no longer present at day 40, which was supported by protein analysis showing equivalent levels of the NMDAR expressed in the hippocampus in animals receiving patient and control IgG. In summary, these findings provide further evidence that supports a direct role of autoantibodies in NMDAR-encephalitis, limbic encephalitis and Morvan’s syndrome.</p

Investigating the pathogenic effects of antibodies against the VGKC-complex and N-methyl-D-aspartate receptor in CNS disorders

Over the last few years, antibody-mediated disorders of the central nervous system (CNS) have been a rapidly expanding field in neurology. The antibodies bind to extracellular domains of proteins expressed in the CNS and impair neuronal function. Patients with limbic encephalitis, Morvan’s syndrome have antibodies that bind to the voltage gated potassium channel (VGKC)-complex proteins, LGI1 and CASPR2, while patients with antibodies to the NMDAR have more complex encephalopathies. Clinical evidence suggests that these antibodies are pathogenic as immunotherapy reduces antibody levels, which correlate closely with clinical improvement. Antibodies can be detected in patient sera using human embryonic kidney (HEK) cells transfected with CASPR2, LGI1 and NMDAR subunits, or by binding to rodent brain sections and primary neuronal cultures. In vitro NMDAR-antibodies have been shown to cross-link synaptic NMDARs and cause their subsequent internalisation from the cell membrane. It is not known if passive transfer of NMDAR-antibodies can recapitulate features of disease or if these pathogenic mechanisms occur with VGKC-complex antibodies. The characteristics of LGI1 and CASPR2 antibodies were explored in vitro. IgG1 and IgG4 were identified as the predominant antibody subclasses, with all CASPR2 sera containing IgG1 antibodies and all LGI1 sera containing IgG4. On transfected cells both LGI1 and CASPR2 antibodies activated the classical pathway of the complement cascade. Furthermore CASPR2 antibody binding to transfected HEK cells showed a reduction in intensity over time which was most likely a result of internalisation of the CASPR2-antibody bound complex. Both CASPR2 and LGI1 antibodies bound strongly to the surface of live hippocampal neurons in culture, and this staining was reduced 24 hours after the removal of IgG, corresponding to a loss of antigen expressed at the cell surface. Patients with NMDAR-encephalitis present with a psychotic disturbance, memory loss and seizures, usually progressing to a reduced level of consciousness and a marked movement disorder. A single injection of IgG, purified from patients with NMDAR-encephalitis was administered into the lateral ventricle of mice. Animals were observed for 40 days and showed significant behavioural changes, including a deficit in spontaneous alternation, dyskinetic hind-limb clasping and visual signs of spontaneous seizures, which are reminiscent to features of the disease. Behavioural changes were not observed in animals injected with IgG from healthy individuals. These behavioural alterations appeared to be reversible and were no longer present at day 40, which was supported by protein analysis showing equivalent levels of the NMDAR expressed in the hippocampus in animals receiving patient and control IgG. In summary, these findings provide further evidence that supports a direct role of autoantibodies in NMDAR-encephalitis, limbic encephalitis and Morvan’s syndrome.This thesis is not currently available in ORA

Staining for mouse IgG and T cell infiltrates in mouse brain following immunization with rhGAD65 or PBS.

<p>A. Coronal sections through hippocampus (Hp) and thalamus, striatum (CPu) and fourth ventricle (4V) were stained with anti-mouse IgG antibodies. Note the strong immune reaction (brown deposit) in the hippocampus, thalamus, septum, brainstem, adjacent to the ventricles and the lateral access of the fourth ventricle. Intensity of immunoreactivity appeared greater in GAD65 immunized mice brains compared to PBS immunized mice brains but this was not quantified further. LV = lateral ventricle. B. Sagittal section of a rodent brain showing the five regions investigated for cellular immunopathology. C. Confocal images showing T cells (CD3, red filter) and leucocytes (CD45, blue filter) in the brain regions. Some cells were identified in the subcortex, in the thalamus, the lateral ventricles and the lateral globus pallidus, but there were very scarce infiltrates in the cortex and hippocampus. D. Higher power images through the cerebellum and brainstem of the one GAD-immunized mouse with T cell (CD3, red filter) and leucocyte (CD45, blue filter) infiltrates in the brainstem, which were not found in the cerebellum. Note that the brainstem but not cerebellum was positive for IgG deposits (A). BS = brainstem; Cb = cerebellum. Scale bar = 25 µm.</p

Neuronal counts and analysis following immunization A.

<p>Regions selected for neuronal counting (numbered 1 to 5), in the hippocampus (Hp), cerebellum (Cb) and brainstem (BS) are shown on a sagittal rodent brain section and in the grey matter on a coronal section of the spinal cord. B. EGFP-positive and total neuronal counts (calbindin+) in cerebellum, and (NeuN+) in two of the three spinal cord regions sampled. No differences were found between test and control mice. C. Representative images from region 5 of the brainstem demonstrating reduced numbers of EGFP+ neurons in the GAD65-immunized mice, compared with PBS-immunized controls, with some apparent reduction in the NeuN+ neurons. D. Counts obtained from the blinded analysis of all five brain stem regions. Although there was no significant reduction in total neurons, the EGFP+ cells showed a trend towards lower numbers in the brainstem (unpaired t test). E. To investigate further, the immunization and neuronal counting was repeated on three further GAD65 and PBS immunized mice, analyzing a larger number of neurons in the same regions of the brainstem with similar results. All neuronal counting was performed on coded sections by an independent investigator. Scale bar = 100 µm. F. To normalize between the results of D. and E., the EGFP-positive neurons were expressed as a percentage of the NeuN positive neurons for each slice.</p

Behavioral analyses on GAD-immunized (n = 9; 6F, 3M) or PBS-immunized (n = 12; 8F, 4M) mice.

<p>Boosts were performed at days (D) D28, D52 and D82, and LPS given at D62 or D64 and D85. The results are given at baseline (B) and the different time points after the first immunization: D59, 1 week after the second boost; D65, 1 day and D75, 11 or 13 days after first LPS; and D92, 1 week after second LPS following third boost. The means and SEMs are shown at each time point for rhGAD65 (▪) and PBS (<b>○</b>) immunized mice. Although both females and males are included, neither these results nor analysis of the females only (6 Test and 8 Controls) revealed any differences between the two groups at any of the time points (2-way ANOVA).</p