Molecular diversity and evolution of neuron types in the amniote brain.

The existence of evolutionarily conserved regions in the vertebrate brain is well established. The rules and constraints underlying the evolution of neuron types, however, remain poorly understood. To compare neuron types across brain regions and species, we generated a cell type atlas of the brain of a bearded dragon and compared it with mouse datasets. Conserved classes of neurons could be identified from the expression of hundreds of genes, including homeodomain-type transcription factors and genes involved in connectivity. Within these classes, however, there are both conserved and divergent neuron types, precluding a simple categorization of the brain into ancestral and novel areas. In the thalamus, neuronal diversification correlates with the evolution of the cortex, suggesting that developmental origin and circuit allocation are drivers of neuronal identity and evolution.

Multiplex Hextuple Luciferase Assaying.

We recently expanded the commonly used dual luciferase assaying method toward multiplex hextuple luciferase assaying, allowing monitoring the activity of five experimental pathways against one control at the same time. In doing so, while our expanded assay utilizes a total of six orthogonal luciferases instead of two, this assay, conveniently, still utilizes the well-established reagents and principles of the widely used dual luciferase assay. Three quenchable D-luciferin-consuming luciferases are measured after addition of D-Luciferin substrate, followed by quenching of their bioluminescence (BL) and the measurement of three coelenterazine (CTZ)-consuming luciferases after addition of CTZ substrate, all in the same vessel. Here, we provide detailed protocols on how to perform such multiplex hextuple luciferase assaying to monitor cellular signal processing upstream of five transcription factors and their corresponding transcription factor-binding motifs, using a constitutive promoter as normalization control. The first protocol is provided on how to perform cell culture in preparation toward genetic or pharmaceutical perturbations, as well as transfecting a multiplex hextuple luciferase reporter vector encoding all luciferase reporter units needed for multiplex hextuple luciferase assaying. The second protocol details on how to execute multiplex hextuple luciferase assaying using a microplate reader appropriately equipped to detect the different BLs emitted by all six luciferases. Finally, the third protocol provides details on analyzing, plotting, and interpreting the data obtained by the microplate reader.

Simultaneous Examination of Cellular Pathways using Multiplex Hextuple Luciferase Assaying.

Multiplex experimentation that can assay multiple cellular signaling pathways in the same cells requires orthogonal genetically encoded reporters that report over large dynamic ranges. Luciferases are cost-effective, versatile candidates whose output signals can be sensitively detected in a multiplex fashion. Commonly used dual luciferase reporter assays detect one luciferase that is coupled to a single cellular pathway and a second that is coupled to a control pathway for normalization purposes. We have expanded this approach to multiplex hextuple luciferase assays that can report on five cellular signaling pathways and one control, each of which is encoded by a unique luciferase. Light emission by the six luciferases can be distinguished by the use of two distinct substrates, each specific for three luciferases, followed by spectral decomposition of the light emitted by each of the three luciferase enzymes with bandpass filters. Here, we present detailed protocols on how to perform multiplex hextuple luciferase assaying to monitor pathway fluxes through transcriptional response elements for five specific signaling pathways (i.e., c-Myc, NF-κß, TGF-ß, p53, and MAPK/JNK) using the constitutive CMV promoter as normalization control. Protocols are provided for preparing reporter vector plasmids for multiplex reporter assaying, performing cell culture and multiplex luciferase reporter vector plasmid transfection, executing multiplex luciferase assays, and analyzing and interpreting data obtained by a plate reader appropriately equipped to detect the different luminescences. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of vectors for multiplex hextuple luciferase assaying Basic Protocol 2: Cell culture work for multiplex hextuple luciferase assays Basic Protocol 3: Transfection of luciferase reporter plasmids followed by drug and recombinant protein treatments Basic Protocol 4: Performing the multiplex hextuple luciferase assay.

A claustrum in reptiles and its role in slow-wave sleep.

The mammalian claustrum, owing to its widespread connectivity with other forebrain structures, has been hypothesized to mediate functions that range from decision-making to consciousness1. Here we report that a homologue of the claustrum, identified by single-cell transcriptomics and viral tracing of connectivity, also exists in a reptile-the Australian bearded dragon Pogona vitticeps. In Pogona, the claustrum underlies the generation of sharp waves during slow-wave sleep. The sharp waves, together with superimposed high-frequency ripples2, propagate to the entire neighbouring pallial dorsal ventricular ridge (DVR). Unilateral or bilateral lesions of the claustrum suppress the production of sharp-wave ripples during slow-wave sleep in a unilateral or bilateral manner, respectively, but do not affect the regular and rapidly alternating sleep rhythm that is characteristic of sleep in this species3. The claustrum is thus not involved in the generation of the sleep rhythm itself. Tract tracing revealed that the reptilian claustrum projects widely to a variety of forebrain areas, including the cortex, and that it receives converging inputs from, among others, areas of the mid- and hindbrain that are known to be involved in wake-sleep control in mammals4-6. Periodically modulating the concentration of serotonin in the claustrum, for example, caused a matching modulation of sharp-wave production there and in the neighbouring DVR. Using transcriptomic approaches, we also identified a claustrum in the turtle Trachemys scripta, a distant reptilian relative of lizards. The claustrum is therefore an ancient structure that was probably already present in the brain of the common vertebrate ancestor of reptiles and mammals. It may have an important role in the control of brain states owing to the ascending input it receives from the mid- and hindbrain, its widespread projections to the forebrain and its role in sharp-wave generation during slow-wave sleep.

Examining multiple cellular pathways at once using multiplex hextuple luciferase assaying.

Sensitive simultaneous assessment of multiple signaling pathways within the same cells requires orthogonal reporters that can assay over large dynamic ranges. Luciferases are such genetically encoded candidates due to their sensitivity, versatility, and cost-effectiveness. We expand luciferase multiplexing in post-lysis endpoint luciferase assays from two to six. Light emissions are distinguished by a combination of distinct substrates and emission spectra deconvolution. All six luciferase reporter units are stitched together into one plasmid facilitating delivery of all reporter units through a process we termed solotransfection, minimizing experimental errors. We engineer a multiplex hextuple luciferase assay to probe pathway fluxes through five transcriptional response elements against a control constitutive promoter. We can monitor effects of siRNA, ligand, and chemical compound treatments on their target pathways along with the four other probed cellular pathways. We demonstrate the effectiveness and adaptiveness of multiplex luciferase assaying, and its broad application across different research fields.

Cross-species genetic screens to identify kinase targets for APP reduction in Alzheimer's disease.

An early hallmark of Alzheimer's disease is the accumulation of amyloid-ß (Aß), inspiring numerous therapeutic strategies targeting this peptide. An alternative approach is to destabilize the amyloid beta precursor protein (APP) from which Aß is derived. We interrogated innate pathways governing APP stability using a siRNA screen for modifiers whose own reduction diminished APP in human cell lines and transgenic Drosophila. As proof of principle, we validated PKCß-a known modifier identified by the screen-in an APP transgenic mouse model. PKCß was genetically targeted using a novel adeno-associated virus shuttle vector to deliver microRNA-adapted shRNA via intracranial injection. In vivo reduction of PKCß initially diminished APP and delayed plaque formation. Despite persistent PKCß suppression, the effect on APP and amyloid diminished over time. Our study advances this approach for mining druggable modifiers of disease-associated proteins, while cautioning that prolonged in vivo validation may be needed to reveal emergent limitations on efficacy.

High-Throughput Functional Analysis Distinguishes Pathogenic, Nonpathogenic, and Compensatory Transcriptional Changes in Neurodegeneration.

Discriminating transcriptional changes that drive disease pathogenesis from nonpathogenic and compensatory responses is a daunting challenge. This is particularly true for neurodegenerative diseases, which affect the expression of thousands of genes in different brain regions at different disease stages. Here we integrate functional testing and network approaches to analyze previously reported transcriptional alterations in the brains of Huntington disease (HD) patients. We selected 312 genes whose expression is dysregulated both in HD patients and in HD mice and then replicated and/or antagonized each alteration in a Drosophila HD model. High-throughput behavioral testing in this model and controls revealed that transcriptional changes in synaptic biology and calcium signaling are compensatory, whereas alterations involving the actin cytoskeleton and inflammation drive disease. Knockdown of disease-driving genes in HD patient-derived cells lowered mutant Huntingtin levels and activated macroautophagy, suggesting a mechanism for mitigating pathogenesis. Our multilayered approach can thus untangle the wealth of information generated by transcriptomics and identify early therapeutic intervention points.

RAS-MAPK-MSK1 pathway modulates ataxin 1 protein levels and toxicity in SCA1.

Many neurodegenerative disorders, such as Alzheimer's, Parkinson's and polyglutamine diseases, share a common pathogenic mechanism: the abnormal accumulation of disease-causing proteins, due to either the mutant protein's resistance to degradation or overexpression of the wild-type protein. We have developed a strategy to identify therapeutic entry points for such neurodegenerative disorders by screening for genetic networks that influence the levels of disease-driving proteins. We applied this approach, which integrates parallel cell-based and Drosophila genetic screens, to spinocerebellar ataxia type 1 (SCA1), a disease caused by expansion of a polyglutamine tract in ataxin 1 (ATXN1). Our approach revealed that downregulation of several components of the RAS-MAPK-MSK1 pathway decreases ATXN1 levels and suppresses neurodegeneration in Drosophila and mice. Importantly, pharmacological inhibitors of components of this pathway also decrease ATXN1 levels, suggesting that these components represent new therapeutic targets in mitigating SCA1. Collectively, these data reveal new therapeutic entry points for SCA1 and provide a proof-of-principle for tackling other classes of intractable neurodegenerative diseases.

Identification of NUB1 as a suppressor of mutant Huntington toxicity via enhanced protein clearance.

Huntington's disease is caused by expanded CAG repeats in HTT, conferring toxic gain of function on mutant HTT (mHTT) protein. Reducing mHTT amounts is postulated as a strategy for therapeutic intervention. We conducted genome-wide RNA interference screens for genes modifying mHTT abundance and identified 13 hits. We tested 10 in vivo in a Drosophila melanogaster Huntington's disease model, and 6 exhibited activity consistent with the in vitro screening results. Among these, negative regulator of ubiquitin-like protein 1 (NUB1) overexpression lowered mHTT in neuronal models and rescued mHTT-induced death. NUB1 reduces mHTT amounts by enhancing polyubiquitination and proteasomal degradation of mHTT protein. The process requires CUL3 and the ubiquitin-like protein NEDD8 necessary for CUL3 activation. As a potential approach to modulating NUB1 for treatment, interferon-ß lowered mHTT and rescued neuronal toxicity through induction of NUB1. Thus, we have identified genes modifying endogenous mHTT using high-throughput screening and demonstrate NUB1 as an exemplar entry point for therapeutic intervention of Huntington's disease.