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How to extract genomic DNA from larvae or finclips using the HotSHOT method.

SummaryAltered brain network activity and the resulting hypersynchrony are important for the pathogenesis of cognitive decline in Alzheimer’s disease (AD) mouse models. Treatments that reduce epileptiform discharges (EDs) or network hyperactivity improve cognition in AD models and humans. We first show that ketogenic diet, but not fasting, rapidly and persistently reduced EDs in the hAPPJ20 Alzheimer’s mouse model over timescales of hours to months. Then, to identify the specific mechanism of the pleiotropic ketogenic diet, we developed small molecule ketone esters to deliver ketone bodies pharmacologically. Two ketone esters recapitulate ED suppression without other dietary manipulation, over time scales of minutes to one week. This small molecule rescue was associated with reduced low-frequency oscillatory activity similar to the recently reported mechanism of an NMDA receptor modulator molecule in this model. Long-term KD resulted in cognitive improvement and in a sex-stratified analysis also improved survival in the more severely affected hAPPJ20 males. Agents that deliver ketone bodies via small molecules or act on downstream targets may hold therapeutic promise in AD through the mechanism of improved network function and reduced epileptiform activity.

Thousands of long intergenic non-coding RNAs (lincRNAs) are transcribed throughout the vertebrate genome. A subset of lincRNAs enriched in developing brains has recently been found to contain cryptic open reading frames and are speculated to encode micropeptides. However, systematic identification and functional assessment of these transcripts have been hindered by technical challenges caused by their small size. Here we show that two putative lincRNAs (linc-mipep and linc-wrb) encode micropeptides with homology to the vertebrate-specific chromatin architectural protein, Hmgn1, and demonstrate that they are required for development of vertebrate-specific brain cell types. Specifically, we show that NMDA receptor-mediated pathways are dysregulated in zebrafish lacking these micropeptides and that their loss preferentially alters the gene regulatory networks that establish cerebellar cells and oligodendrocytes – evolutionarily newer cell types that develop postnatally in humans. These findings highlight the power of screening for unexplored micropeptide functions by revealing a key missing link in the evolution of vertebrate brain cell development and illustrating a genetic basis for how some neural cell types are more susceptible to chromatin disruptions, with implications for neurodevelopmental disorders and disease.

Please cite 10.7554/eLife.59683 if you use this protocol. Note; our eLife publication used Version 2 of this protocol. Later version are subsequent improvements/simplifications. Get in touch for questions/suggestions twitter –@francois_kroll email –francois@kroll.be

Data and code for preprint Kroll, F. et al.2020. A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes. https://www.biorxiv.org/content/10.1101/2020.06.04.133462v3 You can also navigate the files here:https://github.com/francoiskroll/f0knockout (some of the heavier files are only included in the Zenodo repository) If you use some of the data or code, please cite the preprint! ___ Contact: Twitter: @francois_kroll Email: francois@kroll.be

Hundreds of human genes are associated with neurological diseases, but translation into tractable biological mechanisms is lagging. Larval zebrafish are an attractive model to investigate genetic contributions to neurological diseases. However, current CRISPR-Cas9 methods are difficult to apply to large genetic screens studying behavioural phenotypes. To facilitate rapid genetic screening, we developed a simple sequencing-free tool to validate gRNAs and a highly effective CRISPR-Cas9 method capable of converting >90% of injected embryos directly into F0 biallelic knockouts. We demonstrate that F0 knockouts reliably recapitulate complex mutant phenotypes, such as altered molecular rhythms of the circadian clock, escape responses to irritants, and multi-parameter day-night locomotor behaviours. The technique is sufficiently robust to knockout multiple genes in the same animal, for example to create the transparent triple knockoutcrystalfish for imaging. Our F0 knockout method cuts the experimental time from gene to behavioural phenotype in zebrafish from months to one week.

Remember to cite the publication if you use this protocol! Get in touch for questions twitter –@francois_kroll email –francois@kroll.be

Disruption of normal network activity and associated epileptiform spikes from dysfunctional inhibitory interneurons are important for the pathogenesis of cognitive decline in Alzheimer’s disease mouse models. Treatments that reduce epileptiform spikes improve cognition in these models and represent a promising new therapeutic approach for Alzheimer’s disease. Ketogenic diet and fasting have been used for centuries to treat certain forms of epilepsy, including those mechanistically related to disrupted network activity in Alzheimer’s models like Dravet syndrome. We found that ketogenic diet, but not fasting, consistently reduced epileptiform spikes in the APPJ20 Alzheimer’s mouse model. This reduction in spikes was independent or downstream of inhibitory interneuron function. The effect on spike reduction was sustained through several months of treatment. Long-term treatment resulted in cognitive improvement in the water maze and in habituation to the open field, and in the more severely affected male APPJ20 mice also improved survival. Finally, treatment with a novel compound that is metabolized to the ketone body beta-hydroxybutyrate immediately reduced epileptiform spikes in both APPJ20 and a model of Dravet syndrome to a similar degree as ketogenic diet. We suggest that therapies utilizing such ketomimetic agents, including agents that increase blood levels of beta-hydroxybutyrate or act on downstream targets of beta-hydroxybutyrate, may have therapeutic potential in Alzheimer’s disease through improving network function and ameliorating epileptiform activity.

Links between epilepsy and Alzheimer’s disease (AD) are seen in both human patients and mouse models. Human patients with AD may commonly have subclinical epileptiform spikes (EP spikes)1, and overt epilepsy is associated with more rapid cognitive decline2. Mechanistic studies in mouse models of Alzheimer’s disease (AD) have shown that altered network activity and epileptiform spikes stem from dysfunctional inhibitory interneurons3, which are key elements of cortical circuits underlying cognition4. Treatments that reduce epileptiform spikes improve cognition in these models5,6. Thus, targeting subclinical epileptiform activity may be a promising new therapeutic approach to AD7. Ketogenic diet (KD) has long been used to treat forms of epilepsy8, including Dravet syndrome, a childhood epilepsy caused by mutations in a gene that is critical for inhibitory interneuron function in mouse AD models5,9. However, the concurrent effects of a ketogenic diet on brain electrical activity, cognitive decline, and survival have not been tested, and the translational rationale and feasibility of such an intervention remain uncertain. Here we show that a ketogenic diet reduces epileptiform spikes in the hAPPJ20 mouse model of AD. Similar reduction of EP spikes is observed using a β-hydroxybutyrate (BHB) ester in both AD and Dravet mice. A ketogenic diet improves context-dependent and visuo-spatial learning in hAPPJ20 mice. It also reduces the high seizure-related mortality observed in male mice of this model. Therapies derived from β-hydroxybutyrate may have potential application in ameliorating cognitive dysfunction in AD through reducing subclinical epileptiform activity.