Autoantibodies against leucine-rich glioma inactivated 1 (LGI1) are found in patients with limbic encephalitis and focal seizures. Here, we generate patient-derived monoclonal antibodies (mAbs) against LGI1. We explore their sequences and binding characteristics, plus their pathogenic potential using transfected HEK293T cells, rodent neuronal preparations, and behavioural and electrophysiological assessments in vivo after mAb injections into the rodent hippocampus. In live cell-based assays, LGI1 epitope recognition was examined with patient sera (n = 31), CSFs (n = 11), longitudinal serum samples (n = 15), and using mAbs (n = 14) generated from peripheral B cells of two patients. All sera and 9/11 CSFs bound both the leucine-rich repeat (LRR) and the epitempin repeat (EPTP) domains of LGI1, with stable ratios of LRR:EPTP antibody levels over time. By contrast, the mAbs derived from both patients recognized either the LRR or EPTP domain. mAbs against both domain specificities showed varied binding strengths, and marked genetic heterogeneity, with high mutation frequencies. LRR-specific mAbs recognized LGI1 docked to its interaction partners, ADAM22 and ADAM23, bound to rodent brain sections, and induced internalization of the LGI1-ADAM22/23 complex in both HEK293T cells and live hippocampal neurons. By contrast, few EPTP-specific mAbs bound to rodent brain sections or ADAM22/23-docked LGI1, but all inhibited the docking of LGI1 to ADAM22/23. After intrahippocampal injection, and by contrast to the LRR-directed mAbs, the EPTP-directed mAbs showed far less avid binding to brain tissue and were consistently detected in the serum. Post-injection, both domain-specific mAbs abrogated long-term potentiation induction, and LRR-directed antibodies with higher binding strengths induced memory impairment. Taken together, two largely dichotomous populations of LGI1 mAbs with distinct domain binding characteristics exist in the affinity matured peripheral autoantigen-specific memory pools of individuals, both of which have pathogenic potential. In human autoantibody-mediated diseases, the detailed characterization of patient mAbs provides a valuable method to dissect the molecular mechanisms within polyclonal populations.
The neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is essential for synaptic vesicle exocytosis, but its study has been limited by the neonatal lethality of murine SNARE knockouts. Here, we describe a viable mouse line carrying a mutation in the b-isoform of neuronal SNARE synaptosomal-associated protein of 25 kDa (SNAP-25). The causative I67T missense mutation results in increased binding affinities within the SNARE complex, impaired exocytotic vesicle recycling and granule exocytosis in pancreatic -cells, and a reduction in the amplitude of evoked cortical excitatory postsynaptic potentials. The mice also display ataxia and impaired sensorimotor gating, a phenotype which has been associated with psychiatric disorders in humans. These studies therefore provide insights into the role of the SNARE complex in both diabetes and psychiatric disease.diabetes ͉ mutagenesis ͉ soluble N-ethylmaleimide-sensitive factor attachment protein receptor ͉ schizophrenia ͉ exocytosis I ntracellular membrane fusion events within eukaryotic cells are mediated by proteins of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family. The prototypic SNARE-mediated fusion event is synaptic vesicle exocytosis, in which the membrane-associated SNAP-25 and syntaxin-1A interact with the vesicle-associated membrane protein (VAMP) to create a stable ternary core complex with 1:1:1 stoichiometry (1). This interaction has been extensively studied in various systems including neurons, neuroendocrine chromaffin cells, and pancreatic -islet cells, all of which use the neuronal core complex proteins to regulate secretion (2, 3). In vivo knockout and mutagenesis studies in invertebrate systems have allowed SNARE function to be analyzed at the level of the synapse (4), but their scope has been limited by the failure of these organisms to model higher neurological functions in which SNARE function is strongly implicated. A major goal, therefore, has been the generation of a mammalian model of SNARE dysfunction in which the effects on complex neurological and behavioral phenotypes can be assessed.Both Vamp2 and Snap25 knockouts are perinatally lethal in homozygous mice, and heterozygotes show no apparent phenotype, so detailed analysis of viable affected individuals has not been possible (5, 6). The spontaneous mouse mutant coloboma carries a heterozygous deletion spanning four genes including Snap25 (7), but its locomotor hyperactivity phenotype is likely to depend on the additional loss of one or more of the other genes within the deleted region. Most recently, a viable syntaxin-1a knockout mouse has been described with subtle defects in long-term potentiation and fear-memory behavior, although normal synaptic transmission is unaffected likely because of functional compensation by syntaxin-1b (8). Existing mammalian SNARE mutations have therefore offered only very limited insights into the consequences of SNARE dysfunction in an adult system and have yielded no informat...
The robotic mouse is an autosomal dominant mutant that arose from a large-scale chemical mutagenesis program. It has a jerky, ataxic gait and develops adult-onset Purkinje cell loss in the cerebellum in a striking region-specific pattern, as well as cataracts. Genetic and physical mapping of the disease locus led to the identification of a missense mutation in a highly conserved region of Af4, a putative transcription factor that has been previously implicated in leukemogenesis. We demonstrate that Af4 is specifically expressed in Purkinje cells, and we hypothesize that the expression of mutant Af4 leads to neurodegeneration. This function was not identified through knock-out studies, highlighting the power of phenotype-driven mutagenesis in the mouse to identify new pathways involved in neurological disease.
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