Purkinje cells (PCs) are primarily affected in neurodegenerative spinocerebellar ataxias (SCAs). For generating animal models for SCAs, genetic regulatory elements specifically targeting PCs are required, thereby linking pathological molecular effects with impaired function and organismic behavior. Because cerebellar anatomy and function are evolutionary conserved, zebrafish represent an excellent model to study SCAs in vivo. We have isolated a 258 bp cross-species PC-specific enhancer element that can be used in a bidirectional manner for bioimaging of transgene-expressing PCs in zebrafish (both sexes) with variable copy numbers for tuning expression strength. Emerging ectopic expression at high copy numbers can be further eliminated by repurposing microRNA-mediated posttranslational mRNA regulation. Subsequently, we generated a transgenic SCA type 13 (SCA13) model, using a zebrafish-variant mimicking a human pathological SCA13 R420H mutation, resulting in cell-autonomous progressive PC degeneration linked to cerebellum-driven eye-movement deficits as observed in SCA patients. This underscores that investigating PC-specific cerebellar neuropathologies in zebrafish allows for interconnecting bioimaging of disease mechanisms with behavioral analysis suitable for therapeutic compound testing.

Antibodies are essential tools for therapy and diagnostics. Yet, production remains expensive as it is mostly done in mammalian expression systems. As most therapeutic IgG require mammalian glycosylation to interact with the human immune system, other expression systems are rarely used for production. However, for neutralizing antibodies that are not required to activate the human immune system as well as antibodies used in diagnostics, a cheaper production system would be advantageous. In our study, we show cost-efficient, easy and high yield production of antibodies as well as various secreted antigens including Interleukins and SARS-CoV-2 related proteins in a baculovirus-free insect cell expression system. To improve yields, we optimized the expression vector, media and feeding strategies. In addition, we showed the feasibility of lyophilization of the insect cell produced antibodies. Furthermore, stability and activity of the antibodies was compared to antibodies produced by Expi293F cells revealing a lower aggregation of antibodies originating from High Five cell production. Finally, the newly established High Five expression system was compared to the Expi293F mammalian expression system in regard of yield and costs. Most interestingly, all tested proteins were producible in our High Five cell expression system what was not the case in the Expi293F system, hinting that the High Five cell system is especially suited to produce difficult-to-express target proteins.

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N-methyl-d-aspartate receptor (NMDAR) encephalitis is the most common subtype of autoimmune encephalitis characterized by a complex neuropsychiatric syndrome usually including memory impairment. Patients develop an intrathecal immune response against NMDARs with antibodies that presumably bind to the amino-terminal domain of the GluN1 subunit. The therapeutic response to immunotherapy is often delayed. Therefore, new therapeutic approaches for fast neutralization of NMDAR antibodies are needed. Here, we developed fusion constructs consisting of the Fc part of immunoglobulin G and the amino-terminal domains of either GluN1 or combinations of GluN1 with GluN2A or GluN2B. Surprisingly, both GluN1 and GluN2 subunits were required to generate high-affinity epitopes. The construct with both subunits efficiently prevented NMDAR binding of patient-derived monoclonal antibodies and of patient CSF containing high-titre NMDAR antibodies. Furthermore, it inhibited the internalization of NMDARs in rodent dissociated neurons and human induced pluripotent stem cell-derived neurons. Finally, the construct stabilized NMDAR currents recorded in rodent neurons and rescued memory defects in passive-transfer mouse models using intrahippocampal injections. Our results demonstrate that both GluN1 and GluN2B subunits contribute to the main immunogenic region of the NMDAR and provide a promising strategy for fast and specific treatment of NMDAR encephalitis, which could complement immunotherapy.