Chromothripsis is a recently identified mutational phenomenon, by which a presumably single catastrophic event generates extensive genomic rearrangements of one or a few chromosome(s). Considered as an early event in tumour development, this form of genome instability plays a prominent role in tumour onset. Chromothripsis prevalence might have been underestimated when using low-resolution methods, and pan-cancer studies based on sequencing are rare. Here we analyse chromothripsis in 28 tumour types covering all major adult cancers . We show that chromothripsis affects a substantial proportion of human cancers, with a prevalence of 49% across all cases. Chromothripsis generates entity-specific genomic alterations driving tumour development, including clinically relevant druggable fusions. Chromothripsis is linked with specific telomere patterns and univocal mutational signatures in distinct tumour entities. Longitudinal analysis of chromothriptic patterns in 24 matched tumour pairs reveals insights in the clonal evolution of tumours with chromothripsis. The shattering of chromosomes is a dramatic early event in tumourigenesis and is termed chromothripsis. Here, the authors examine chromothripsis across 28 tumour types and show that 49% of cancers exhibit features of chromothripsis. This catastrophic process drastically contrasts with the classical view of multi-step tumour evolution. In a presumably single event, chromothripsis leads to extensive chromosome rearrangements, fostering the simultaneous acquisition of multiple genomic aberrations3. Following the partial or full shattering of one or a few chromosome(s) via tens to hundreds of DNA double-strand breaks, imperfect repair occurs. DNA fragments not reincorporated into the derivative chromosome are often lost to the cell. Within one or very few cell cycles, tumour suppressor functions are disrupted and/or oncogenic fusions and oncogene amplifications arise3. Cells that survive such a cataclysm likely have gained a strong selection advantage due to their massively rearranged genome - a phenomenon that potentially transforms such cells into cancer cells. Importantly, chromothripsis is linked with aggressive tumour behaviour and poor prognosis for cancer patients6.The development of next-generation sequencing technologies and their applications in cancer genome studies have enabled the discovery of a new form of genome instability called chromothripsis1, chromothripsis is probably much more widespread than originally suspected. As more cancer genomes are being sequenced, more cancer types with high frequencies of chromothriptic events emerge10, suggesting that chromothripsis might play a major role in a substantial number of human cancers. The prevalence varies from zero to 100% across tumour (sub)entities11. However, comparisons between studies and between tumour entities are challenging for several reasons. First, the type of data used for chromothripsis scoring differ, with high-coverage whole-genome sequencing providing the most reliable scoring, but other methods offering lower resolution also being employed . Second, varying definitions and minimal criteria for inferring chromothripsis have been applied, from relatively loose to conservative cutoff values3. Third, visual versus automated scoring both have advantages and weaknesses, with risks of false-positive and false-negative cases for each of the methods. Ideally, automated scoring and manual review used together offer the most reliable chromothripsis inference, as reported in a recent study on melanoma12.Despite initial prevalence estimates in the range of two to three per cent of all cancer cases13. However, this type of data, even though available for large cohorts, does not allow formal testing of all criteria defining chromothripsis for a conclusive identification of bona fide chromothriptic cases3. In the second study, Gröbner and colleagues analysed chromothripsis in paediatric cancer14, focusing on tumour entities that are frequent in children. Cortés-Ciriano and colleagues performed automated scoring of chromothripsis in cancer genomes from the PCAWG consortium15 .To ensure the best comparability across tumour entities, chromothripsis scoring needs to be performed with a standardized workflow. Three previous studies described pan-cancer analyses of chromothripsis. Cai and colleagues performed a comprehensive analysis of chromothriptic-like patterns in large SNP array and comparative genomic hybridization datasetsHere we present a comprehensive sequencing-based analysis of chromothripsis in a pan-cancer cohort of 634 adult tumours comprising 28 histologic cancer types, including rare tumour entities. Using a standardized workflow, we detect a surprisingly high frequency of chromothriptic events and identify marked differences in genomic features between tumours with or without chromothripsis.16) including 316 whole-genome sequences  and 318 whole-exome sequences . Tumour and matched germline samples were processed with standardized pipelines to detect single nucleotide variants (SNVs), short insertions and deletions (indels), copy-number variants (CNVs) and other structural variants. The full tumour cohort is described in Supplementary data We used paired-end Illumina-based sequencing data for 634 tumours from the NCT/DKTK MASTER program . We distinguished canonical chromothripsis involving three or fewer copy-number states from non-canonical chromothripsis involving more than three copy-number states , we also scored intermediate and low-confidence chromothriptic events, with 8–9 and 6–7 switches between copy-number states, respectively.We applied established criteria for inferring chromothripsis in cancer genomestes Fig. . To ensutes Fig. . This con = 318), making use of the off-target reads to maximize the resolution of the CNV calling (see Methods). A similar percentage of cases showed rearrangements consistent with chromothripsis as in the tumours scored with available whole-genome sequences , we analysed the whole-genome and whole-exome cohorts separately. Unless otherwise specified, the results below focus on the high-confidence scoring in whole-genome sequences, with the detailed analyses of the whole-exome sequences shown in the In parallel, we examined a second group of patients belonging to the same cohort but analysed independently, for which we scored chromothripsis based on whole-exome sequences n = 38, makingn = 316 cases with available whole-genome sequencing data, high-confidence scoring, see Fig. 10  not described before, despite the high prevalence of this phenomenon in these entities, or (ii) only reported based on analyses other than whole-genome sequencing . For instance, 100% of the cases showed chromothripsis in malignant peripheral nerve sheath tumours . In germ cell tumours, 71% of the cases showed chromothripsis (n = 9). Therefore, chromothripsis is apparently a key initiating event in a number of tumour entities for which this phenomenon was not suspected to play a major role.We detected chromothripsis in 49% of all cases , the telomere region was not affected by the chromothriptic event. However, chromothriptic events that do not directly affect telomere regions can also result from telomere fusions, since the genomic regions included in chromatin bridges can be distant from the telomeres, depending on the structure of the dicentric chromosomes formed in telomere crisis18. In approximately half of the tumours (55%), the centromere was included in the segment affected by chromothripsis. Altogether, associations between chromothripsis and distinct genomic features support pan-cancer and entity-specific mechanisms leading to chromothripsis.Furthermore, we observed distinct patterns of chromothriptic events across tumour entities Fig. . NotablyTP53 and in ATM are strongly linked with chromothripsis24, suggesting that inactivation of essential checkpoints or DNA repair factors may facilitate chromothripsis occurrence. With the exception of one ATM mutation carrier for which no somatic loss of the wild-type allele was detected, germline mutations in ATM and TP53 were also tightly linked with chromothripsis in this cohort. To find novel germline variants associated with chromothripsis, we systematically assessed pathogenic germline variants across a set of autosomal cancer predisposition genes. We identified pathogenic germline variants across cancer predisposition genes including among others DNA repair genes from mismatch repair  and double-strand break repair . Approximately 40% of the tumours with germline variants exhibited somatic loss of the wild-type allele. Importantly, the chromothripsis prevalence was significantly higher in tumours with somatic loss of the wild-type allele as compared to cases for which no secondary hit was detected , none of the tumours showed chromothripsis, despite a secondary somatic hit in all cases (n = 5). Therefore, this approach may help to identify genes whose inactivation facilitates chromothripsis occurrence.Germline mutations in 11. For each tumour entity, we identified specific chromosomes and chromosome regions significantly more frequently affected by chromothriptic events than expected by chance . We hypothesize that this unequal distribution does not originate from a more frequent occurrence of chromothripsis on specific chromosomes in a given cell type in the first place, but rather from the selection advantage provided by a chromothriptic event when cell type specific drivers are affected. For instance, in adrenal gland adenocarcinomas, chromothriptic events are predominantly detected on chromosomes 17, 19 and 22, affecting driver genes known to play an essential role in this tumour entity, such as PRKAR1A, MLL4, CCNE1 and ZNRF3, respectively  in colorectal cancer, which is the tumour entity with the largest number of cases in this cohort , a mechanism based on DNA recombination of telomeric sequences31.Telomere attrition and breakage-fusion-bridge cycles, which lead to dicentric chromosomes and chromosome bridges, were shown to initiate chromothriptic events in leukaemia and in cell culture modelsTERT gains (n = 80) showed a significantly higher prevalence for chromothripsis  blunt ends and short microhomologies (1 bp), most common after repair by non-homologous end-joining, as well as microhomologies of 3–5 bp, frequent after repair by alternative end-joining, were significantly more enriched in tumours with chromothripsis Fig. . These d33. Across all entities, AC2 and AC13, two base substitution signatures that are closely associated to each other and linked with the activation of AID/APOBEC cytidine deaminases, were more pronounced in tumours with chromothripsis . Importantly, these differences between mutational signatures detected in tumours with or without chromothripsis were not simply due to differences in the mutational burden , as there was no significant difference in the overall SNV or indel count for these tumour entities. Altogether, prevailing mutational signatures in tumours with chromothripsis may shed light on potential internal and external triggers for chromothripsis.Mutational signatures, reflecting mutational processes of both exogenous and endogenous origins, have been previously characterized for most cancer types2. This implies that the chromothriptic chromosome should be detectable in the vast majority of the tumour cells, if chromothripsis generates drivers conferring a selective advantage. However, we and others reported cases with different chromothriptic patterns between matched primary and relapsed tumours, suggesting subclonal evolution34. We systematically analysed the longitudinal evolution of chromothriptic chromosomes for 24 matched pairs including primary and relapsed tumours but also metastases to different sites . Three types of scenarios were observed  and 40% in lung carcinoma (33.3% in our cohort). In a few instances, such as liver adenocarcinoma, the differences in the proportions of tumours with chromothripsis were substantial between both studies (<25 versus 85%), possibly due to different molecular subgroups of patients in both cohorts.Our comprehensive analysis of chromothripsis in cancer showed a considerably higher prevalence of this phenomenon as compared to initial estimates, with 49% of all cases showing chromothripsis. Despite particularities of this pan-cancer cohort outlined above, this discrepancy reveals an under-estimation of chromothriptic events when using scoring on array-based data. Chromothripsis likely initiates a substantial proportion of human cancers. Importantly, another pan-cancer study not focusing on advanced disease patients, with no enrichment for tumour entities such as sarcomas  and with no age selection reported chromothripsis prevalence rates in the same range3. As we used the same sequencing workflow and the same scoring criteria for all cases in this cohort, we were able to identify tissue types that are more or less prone to chromothriptic events, respectively. For instance, we detected chromothripsis in all cases of malignant peripheral nerve sheath tumour (n = 6). In the pancreas, we detected a high chromothripsis prevalence in pancreas carcinomas  but a low prevalence in pancreatic neuroendocrine tumours . This finding suggests different predispositions to chromothriptic events between cell types within one tissue, possibly linked to differences in sensitivity to replication stress, proliferation rate and efficiency of the apoptotic response.To ensure maximal comparability between tumour entities and between studies, it is essential to apply consistent and well-defined criteria to score for chromothripsisWe observed marked differences in chromothriptic patterns between tumour entities, regarding the prevalence of canonical versus non-canonical chromothripsis, involvement of the telomere or centromere in the chromothriptic region, or the number of chromothriptic chromosomes per tumour. This variety of patterns suggests different mechanisms leading to chromothripsis across tumour entities. Entity-specific links between chromothripsis and distinct mutational signatures  may help deciphering the mechanisms leading to chromothripsis in each tumour entity.35. Our study showed that this is a general phenomenon, which plays a role across most tumour entities.In each tumour type, chromothripsis affected different chromosomes, likely due to a selection for entity-specific driver genes (even though we cannot exclude the role of tissue-specific chromosome territories). Interestingly, chromothriptic events generated a substantial proportion of fusion genes, which might have important clinical implications, as fusion genes provide attractive therapeutic targets. Fusion genes generated due to chromothriptic events were reported recently in lung cancer in non-smokersn = 24 pairs with chromothripsis). This analysis revealed that stable chromothriptic patterns between different tumours of the same patient do not represent the prevailing scenario. For a substantial proportion of cases, we detected chromothripsis in the primary tumour, but not in the major clone at relapse, or conversely, only at relapse, but not in the primary tumour. Conceptually, this suggests that the view of chromothripsis as a single early event, with clonal detection of the chromothriptic chromosome, only holds true in a subset of cases. In addition, this finding has potential treatment implications, because targets related to chromothripsis will not necessarily be ubiquitous. Furthermore, potential therapy-induced chromothriptic events or therapies leading to the selection of chromothriptic clones may complicate therapeutic intervention.Importantly, we analysed the longitudinal evolution of chromothriptic patterns in the largest cohort of matched tumour pairs to date  were obtained after receiving a written informed consent under an institutional review board-approved protocol.The whole-genome and whole-exome sequencing data were generated within the NCT/DKTK MASTER program36. The pipeline used BWA-MEM (v0.7.15) for alignment, biobambam (https://github.com/gt1/biobambam) for sorting and sambamba for marking duplication. The tumour-germline paired alignments were then fed to DKFZ indel SNV callers for indel and SNV discovery, as described previously37.Whole-genome sequencing data and whole-exome sequencing data were processed by the DKFZ OTP pipeline38 and SOPHIA v1.2.16 (https://bitbucket.org/utoprak/sophia/src) were used. SvABA is a structural variant caller based on assembly and discordant read based approach. SOPHIA is a structural variant caller based on the supplementary alignment approach. SOPHIA is integrated in the DKFZ OTP pipeline, where the output was used in combination with alignment files for ploidy estimation and copy number calling using ACESeq v1.2.839. SvABA outputs were used for structural variant calling for the analysis of microhomologies at the breakpoints.We performed copy-number analysis and structural variant calling from whole-genome sequencing data. Two structural variant callers, SvABA v13440, which allows hybrid bin size on captured regions and off-target regions (reads available from the sequencing data but not located in exonic regions). The captured regions were plotted as deep blue and off-target regions were represented by cyan colour by the plotting function of EXCAVATOR2  and structural variants from SOPHIA were used as input. We applied the same criteria as previous studies to define a positive call3.In silico chromothripsis scoring was performed by Shatterseek41 and Mutect242. The output of the two tools were intersected to produce a combined set of high-confidence indels. The combined set was further filtered by an in-house blacklist of indels to remove artefacts. The filtered output was converted into 83 indel subclasses by the PCAWG signature preparation tool43. Finally, the Indel exposures were estimated by SigProfiler (v2.5.1)43 on each tumour by indel signatures defined by COSMIC signatures V343.Indels were called by two software tools, platypus44 to perform a mutational signature analysis and retrieve the exposure of 30 SNV signatures from COSMIC V2 (https://cancer.sanger.ac.uk/cosmic/signatures_v2).Somatic high-confidence SNVs were used as input for YAPSAhttps://github.com/suhrig/arriba). Candidate fusions from medium and high confidence were further validated by analysing structural variants from the whole-genome sequencing identified by SOPHIA. These structural variants called by SOPHIA within 200 kb of fusion calls were combined into a high confidence set. We performed a regression analysis to compare the number of fusions per breakpoint in tumours with chromothripsis as compared to tumours without chromothripsis , telomere characteristics were characterized by TelomereHunter45 (1.1.0). Ten telomere variant repeats including TCAGGG, TGAGGG, TTGGGG, TTCGGG, TTTGGG, ATAGGG, CATGGG, CTAGGG, GTAGGG and TAAGGG were quantified and log2 ratio between tumour and germline specimens were computed. SNV calling identified truncating mutations in ATRX and DAXX as well as TERT promoter mutations.For whole-genome sequenced cases (TERT gains are connected to chromothriptic regions by structural variants, genome walking was performed on structural variants upstream and downstream of TERT. At most two upstream and two downstream structural variants of TERT were followed and the walking was restricted to a walking distance of 50 Mb. Each connection was overlapped with the chromothriptic regions to evaluate the prevalence of such links. Chromothriptic cases with TERT connected to chromothriptic regions were contrasted to chromothriptic cases without connection to TERT, by the proportions of TERT gain using chi-square test.To evaluate if 46, an assembly and discordant read based approach for structural variants discovery. The HOMO field was retrieved for each structural variant called by the assembly method of SvABA. To estimate the contribution of different homology sizes, the structural variants with homology information were binned for analysis and visualization. There are 5 bins for homology usage: blunt end to 1, 2, 3–5, 6–9, and >10 bp. The proportions of each bin were normalized by the total number of structural variants, where significance was assessed by beta-regression.Structural variants were called by SvaBASNVs and indels were called in the tumour sample and subsequently annotated as germline variants in case they were detected in the control sample derived from the patient’s whole blood or leukocyte pellet. Rare germline SNVs and indels in a list of cancer predisposition genes were filtered and assessed according to the AMP-ACMG guidelines.47 and ggplot248. For comparison of mutational signatures, Wilcoxon rank-sum test was applied on log2 absolute exposures for statistical testing. The significance for the telomere repeats between groups were assessed by t test on the log2 values between tumour and germline on each telomeric repeats and on total telomere content. Family-wise correction of p values was performed according to Bonferroni on statistic contrasting mutational signatures, microhomologies, chromothripsis occurrence per chromosome, and telomere content.Statistical analyses and visualizations were performed using RFurther information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Reporting Summary


Integral membrane proteins play key functional roles at organelles and the plasma membrane, necessitating their efficient and accurate biogenesis to ensure appropriate targeting and activity. The endoplasmic reticulum membrane protein complex (EMC) has recently emerged as an important eukaryotic complex for biogenesis of integral membrane proteins by promoting insertion and stability of atypical and sub-optimal transmembrane domains (TMDs). Although confirmed as a bona fide complex almost a decade ago, light is just now being shed on the mechanism and selectivity underlying the cellular responsibilities of the EMC. In this Review, we revisit the myriad of functions attributed the EMC through the lens of these new mechanistic insights, to address questions of the cellular and organismal roles the EMC has evolved to undertake. Summary: The EMC is an important factor facilitating membrane protein biogenesis. Here we discuss the broad cellular and organismal responsibilities overseen by client proteins requiring the EMC for maturation. The eukaryotic endoplasmic reticulum (ER) houses the machinery responsible for biogenesis of secreted and integral membrane proteins. Insertion, folding and maturation of nascent polypeptides is coordinated by a collection of ER-resident factors, many of which remain poorly understood. More than a decade ago, a genome-wide screen monitoring the unfolded-protein response (UPR) identified and characterised factors essential for protein folding and ER homeostasis in yeast . Among tThe EMC was first described as an integral membrane protein complex in yeast, with similar genetic interaction patterns and whose six protein products co-precipitated as a hetero-oligomer . Two othIsolation of the mature EMC under stringent solubilisation conditions indicated robust inter-subunit interactions . EMC strThe EMC has few functionally annotated domains or motifs, a fact that has frustrated investigations into its cellular role(s). Aside from hydrophobic TMDs and signal sequences, only EMC1, EMC2 and EMC3 contain annotated domains. EMC1 is by far the largest subunit and harbours pyrroloquinoline quinone (PQQ)-like repeats (aa 21–252) within the region exposed to the ER lumen and a domain of unknown function  that encompasses the TMD-containing C-terminus . PQQ repTail-anchored (TA) proteins contain TMDs typically located within ∼30 aa of the C-terminus, which are completely shielded by the ribosome exit tunnel until release of the full-length protein, preventing recognition by the signal recognition particles (SRPs) and Sec61-mediated insertion. This scenario necessitates insertion of the TA by a post-translational mechanism.Cytosolic ‘pre-targeting complexes’ shield TMDs from aqueous surroundings, enabling TA protein transfer to specific targeting factors for delivery and handover to membrane-embedded insertases  found that endogenous SQS from EMC-deficient cells degraded rapidly; moreover, recombinant SQS inserted inefficiently into microsomes isolated from those cells and formed cytosolic aggregates when overexpressed  eye (D. melanogaster) photoreceptor cells  eye . EMC3 waor cells . These oor cells . EMC engor cells . Yet, whIntegral membrane proteins represent ∼30% of the cellular proteome , with asThe current spectrum of validated EMC clients is limited but includes both TA and SAP-containing proteins . Yet, maQuantitative proteomic methodologies have been powerful predictors of EMC-dependent client proteins by monitoring changes in their abundance within EMC-deficient cells . One limCaenorhabditis elegans were traced back to defective maturation of a nicotinic acetylcholine receptor  to ensure fidelity . While pThrough EMC1, the EMC exhibits considerable exposure to the ER lumen . The glyThe ER-resident Hsp70 family A member 5  also appears to work together with the EMC to deal with improperly inserted clients. Mutation of TMDs in Cx32 is associated with Charcot-Marie-Tooth disease, and leads to binding of these mutants by BiP, followed by ubiquitylation and degradation instead of correct Cx32 membrane insertion aided by the EMC . MoreoveEMC interactions with membrane-embedded chaperones or co-factors represent a second level of synergy. EMC1 binds to DNAJC18 , where tBefore being recognised as part of the yeast EMC , the ER EMC2, EMC8 and EMC9 do not contain ER-targeting signals and, thus, form part of the cytoplasmic interface of the EMC. The TPR repeats within EMC2 are likely to participate in protein–protein interactions – but whether those interactions are with subunits, cofactors or client proteins is not yet established. EMC2 has been identified as a putative Hsp90 interactor in multiple genome-wide screens in yeast as it physically interacts with Hsp90 . FurtherSpatiotemporal constraints imposed by complex folding programmes complicate biogenesis of polytopic membrane proteins and increase the risk of error. This heightened risk for manufacturing defects means cells must remain poised to deal with aberrant aggregation-prone folding intermediates, stoichiometrically surplus complex subunits and terminally misfolded protein species that may arise during EMC-dependent maturation. In that event, clearance from the ER membrane may be required – but how that might occur is an outstanding question. Enforcement of protein quality control would be expected to be through ER-associated degradation (ERAD), the multistep process responsible for aberrant protein feature recognition, polypeptide removal from the lipid bilayer (retrotranslocation and dislocation), covalent modification by polyubiquitin chains and targeting to 26S proteasomes for degradation (reviewed in L90H) used gp78 for ERAD (C341/378S) only conferred partial rescue, other ER-resident ubiquitin ligases (E3s) might be involved. It must also be considered that ERAD factors could be EMC-dependent clients or that unassembled EMC subunits could be ERAD substrates. Both UBAC2 and DERL2 are members of the rhomboid pseudoprotease superfamily with complex polytopic architectures  and coupling of ubiquitin conjugation to ER degradation protein 1 (CUE1) have strong aggravating genetic interactions with the EMC . A chemiNeurological disorders−/− mice exhibit abnormal locomotor behaviour and an increased anxiety response  to restore homeostasis . Some intivation , indicattivation . This inLoss or mutation of core EMC subunits results in embryonic lethality in organismal models. This indicates requirement of the EMC for maturation of key developmentally regulated proteins and hampers investigations into physiological roles of the EMC . Yet, coEMC3 gene correlate closely with degeneration of red-sensitive opsin-expressing cones in zebrafish retina and rhabdomeres in fruit fly  and phosphatidylethanolamine (PtdEtn) between the organelles . Cells ld PtdEtn . Among tMaintenance of sterol homeostasis involves dynamic sensing and response mechanisms located throughout eukaryotic cells, but those concentrated in the ER and endomembrane system responsively control local and global levels of free cholesterol. Recently, the robustness of this homeostatic mechanism was shown to be dependent on the EMC, through insertion of two key enzymes responsible for sterol biosynthesis and storage . The TA Although it has been demonstrated in autonomous cell culture systems, how exactly EMC dysfunction affects tissues or organismal regulation of cholesterol is not yet clear. As both cholesterol and its steroid hormone derivatives play essential roles in membrane integrity as well as signalling, the EMC is likely to influence both these pathways. Moreover, enabling accurate SQS insertion and, hence, activity has indirect consequences for isoprenoid production, post-translational modifications (e.g. dolichol) and their downstream products. To what degree tissues or organs rich in cholesterol (e.g. liver) or cholesterol esters (e.g. myelin) depend on the EMC remains to be determined. In summary, the diverse metabolic changes in lipid homeostasis exhibited by EMC-deficient cells, indicates the EMC plays an important role in optimising the biogenesis and/or the localisation of lipid-modulating enzymes and transporters.Viruses have evolved unique strategies that exploit host biosynthesis and trafficking machinery to promote their own replication. Unbiased, genome-wide forward genetic screens have identified host factors that include signal recognition particles (SRPs), the Sec61 translocon, translocon-associated protein (TRAP), OST complexes as well as ERAD components . This stAbnormalities appearing at early stages of DENV replication have been described in EMC-deficient cell lines. Although these defects were first attributed to impaired virus entry , more reEMC proteins are also harnessed by simian virus 40 (SV40), the archetypic polyomavirus. The SV40 viral particle consists of a non-enveloped capsid that shields the genetic information of the virus. Following receptor-mediated endocytosis, the SV40 virion traffics from endolysosomes to the ER, where it breaches the ER membrane and translocates into the nucleus . The tissue-specific phenotypes apparent with EMC loss might be attributable to as yet unknown TM proteins with restricted expression. Identifying the principal EMC clients present within different tissues will provide greater insight into its influence on physiological functions B.Furthermore, any direct involvement of the EMC in the aetiology of human disease is only beginning to be uncovered. Recent technological advancements enabling genome-wide forward genetic screening approaches revealed the EMC to be a key host factor for flavivirus propagation. Exome sequencing correlated subunit point mutations to severe neurodegenerative syndromes as well as schizophrenia. How and why naturally occurring somatic and inherited mutations in EMC subunits or their clients promote disease development and progression is still poorly understood. However, by understanding the fundamental responsibilities of the EMC, work can now begin to tease apart the complex phenotypes by looking for dysregulation of key clients in affected cell types and the consequences they elicit. Owing to the complexity of reported disease-related EMC phenotypes, efforts to isolate underlying defects have already begun but will require further comprehensive multi-omics as well as targeted biochemical approaches in appropriate model systems.Taken together, these recent advances in our understanding of the EMC have opened the door to an exciting new set of questions. Future studies will not only aim to clarify the mechanism, selectivity and client proteins of the EMC but will also expand our understanding of its role in fundamental processes of protein biogenesis that establishes tissue homeostasis and organismal viability.


In nervous systems, there are two main modes of transmission for the propagation of activity between cells. Synaptic transmission relies on close contact at chemical or electrical synapses while volume transmission is mediated by diffusible chemical signals and does not require direct contact. It is possible to wire complex neuronal networks by both chemical and synaptic transmission. Both types of networks are ubiquitous in nervous systems, leading to the question which of the two appeared first in evolution. This paper explores a scenario where chemically organized cellular networks appeared before synapses in evolution, a possibility supported by the presence of complex peptidergic signalling in all animals except sponges. Small peptides are ideally suited to link up cells into chemical networks. They have unlimited diversity, high diffusivity and high copy numbers derived from repetitive precursors. But chemical signalling is diffusion limited and becomes inefficient in larger bodies. To overcome this, peptidergic cells may have developed projections and formed synaptically connected networks tiling body surfaces and displaying synchronized activity with pulsatile peptide release. The advent of circulatory systems and neurohemal organs further reduced the constraint imposed on chemical signalling by diffusion. This could have contributed to the explosive radiation of peptidergic signalling systems in stem bilaterians. Neurosecretory centres in extant nervous systems are still predominantly chemically wired and coexist with the synaptic brain.This article is part of the theme issue ‘Basal cognition: multicellularity, neurons and the cognitive lens’. The different theories often focus on the evolution of some salient aspects of the brain. These include the evolution of electrical conduction , the oriHere, I approach the question by asking how the first proto-neurons organized into cellular networks with the rapid propagation of excitation. This excludes ionic flows in broad electric fields as occurs in bioelectric signalling during development and regeneration ,13.In neural networks, cellular excitation can propagate between cells by different mechanisms. During synaptic transmission, one cell influences the activity of others through chemical or electrical synapses. In ephaptic coupling, extracellular currents generated by one neuron directly alter the excitability of adjacent neurons (field effects) –16. A thHere I propose a detailed hypothesis, the chemical brain hypothesis for nervous system origins. The theory suggests that the first cellular networks involved in sensing, reacting and coordination of tissue-level and whole-body activity were organized by paracrine signalling. As signalling molecules, I will discuss the potential early origin and function of secreted neuropeptide-like molecules . I will also discuss several predictions of the hypothesis that can be tested experimentally.The general idea that volume transmission may have evolved before synaptic transmission has been proposed by Grundfest, Horridge and others .In §2, I give an overview of peptidergic signalling and the diversity of peptidergic systems in Metazoa. In §3, I define the chemical brain hypothesis and discuss the possibilities and constraints of peptidergic signalling to organize cellular networks. In §4, I discuss cellular transition scenarios for nervous system origins, in light of the hypothesis. Finally, I discuss some predictions of the hypothesis and how they could be tested.. 2Neuropeptides are abundant, diverse secreted intercellular signalling molecules, which are near-ubiquitous in nervous systems. The active signalling peptides are produced in the Golgi from larger propeptides through successive steps of proteolytic cleavages and further chemical modifications. Cleavages generally occur at di- or monobasic cleavage sites (e.g. KR) by prohormone convertases. The cleaved peptides can be further modified, most often by C-terminal α-amidation during which the bifunctional peptidylglycine α-amidating monooxygenase (PAM) enzyme converts a C-terminal glycine into an amide . Mature (a)Clytia hemisphaerica maturation-inducing hormone receptor (MIHR) is sister to a bilaterian clade containing receptors for luqin, NPF, QRFP, tachykinin, FMRFa and NPY peptides  theories are crafted the right way they have utility even if proven wrong, sometimes especially if proven wrong!’ . The sam


Achieving neuroprotection in ischemic stroke patients has been a multidecade medical challenge. Numerous clinical trials were discontinued in futility and many were terminated in response to deleterious treatment effects. Recently, however, several positive reports have generated the much-needed excitement surrounding stroke therapy. In this review, we describe the clinical studies that significantly expanded the time window of eligibility for patients to receive mechanical endovascular thrombectomy. We further summarize the results available thus far for nerinetide, a promising neuroprotective agent for stroke treatment. Lastly, we reflect upon aspects of these impactful trials in our own studies targeting the Kv2.1-mediated cell death pathway in neurons for neuroprotection. We argue that recent changes in the clinical landscape should be adapted by preclinical research in order to continue progressing toward the development of efficacious neuroprotective therapies for ischemic stroke. Stroke is a devastating neuropathology associated with immense comorbidity and mortality . It is tHistorically, neuroprotective agents are notorious for demonstrating efficacy across several in vitro assays and animal models before failing universally in late-stage clinical trials ,4. Many Ischemic stroke, representing 87% of all stroke cases , is clasStroke researchers have committed decades to carefully dissecting the cell death pathways underlying the loss of neuronal tissue within the ischemic penumbra. Indeed, many well-defined molecular mechanisms are activated following the initial metabolic failure resulting from the deprivation of oxygen and glucose. These ischemic penumbra mechanisms include the formation of an excitotoxic environment due to the synaptic accumulation of glutamate and calcium dysregulation , as wellThe mainstay intervention method for ischemic stroke patients is the restoration of blood flow by pharmacological or surgical means. The first effective therapy developed was the administration of intravenous recombinant tissue plasminogen activator (rt-PA), alteplase, which received the US Food and Drug Association (FDA) approval in 1996 . rt-PA aMore recently, ischemic stroke management gained the addition of mechanical thrombectomy with endovascular surgeries. This approach involves a surgeon gaining access to the cerebral vasculature, with a stent retriever, an aspiration device, or a combination tool, and physically removing the occlusive thromboembolism . CriticaDates displayed are based on study start date and publication date of the results. Actual study completion date is typically few months prior to publication. More details can be found on clinicaltrials.gov with their respective NCT number. Mechanical endovascular thrombectomy trials (red shaded) are DAWN, NCT02142283; DEFUSE-3, NCT02586415; DIRECT-MT, NCT03469206; SWIFT DIRECT, NCT03192332; and MR CLEAN-NO IV, ISRCTN80619088 (isrctn.com). Nerinetide trials (blue shaded) are ENACT, NCT00728182; ESCAPE-NA-1, NCT02930018; FRONTIER, NCT02315443; and ESCAPE-NEXT, NCT04462536.n = 107) or standard medical therapy alone (n = 99). Standard medical therapy varied by regional and national guidelines [The efficacy of late time point mechanical thrombolytic reperfusion was evaluated in a landmark clinical trial that took place between 2014 and 2017 . This study selected for patients with high neurological deficits on the NIH Stroke Scale (NIHSS) but relatively small core infarct size at the time from stroke onset of 6–24 h, with the hypothesis that the mismatch between clinical exam and imaging evidence of infarct reflects a high volume of viable penumbra. Three groups of patients were randomized to either mechanical thrombectomy with standard medical therapy  provided a second body of evidence for late time point endovascular thrombectomy in ischemic stroke therapy. In contrast to the DAWN trial, the DEFUSE-3 trial focused on an imaging-based approach for the selection of eligible patients. With evidence from prior studies, the DEFUSE-3 investigators hypothesized that patients with high penumbra-core ratios could benefit from thrombectomy and recanalization at late time points following stroke ,40,41,425 providen = 92) or standard medical therapy alone (n = 90). Critically, they demonstrated that reperfusion was effective and superior when utilized 6–16 h following symptom onset in this patient subset [From 2016 to 2017, the DEFUSE-3 authors used the RAPID neuroimaging platform to identify a total of 182 functionally independent patients with large vessel occlusion (LVO) of the MCA-M1, MCA-M2, or ICA and a viable penumbra that might benefit from delayed reperfusion. Specific neuroimaging criteria included patients with LVO and a main infarct core lesion <70 mL in volume, with mismatch ratio of ischemic tissue to infarct core ≥1.8 and ≥15 mL of mismatched tissue area, representing the ischemic penumbra. Patients were randomized to either standard medical therapy with late endovascular thrombectomy  generation. As a result, many energy-dependent biological functions stall, including the Na minutes ,49. In n2+-permeable N-methyl-d-aspartate (NMDA) receptor [2+-activated enzyme calmodulin. The activation of nNOS releases nitric oxide (NO) and the reaction of NO and superoxide (O−) anions form the highly toxic peroxynitrite (ONOO−). Peroxynitrite has been shown to mediate most of the toxic actions of NO, leading to many mechanisms of cell death, ranging from necrotic to apoptotic [Neuronal excitotoxicity is primarily mediated by the activation of the Careceptor ,51. Downpoptotic .Nerinetide is a neuroprotective agent designed to ameliorate neuronal excitotoxic damage by preventing the activation of nNOS . Previoun = 92 nerinetide; n = 93 saline). An initial estimation of ~400 patients necessary to detect significance was reduced by half with the macaque primate data designed to mimic the clinical presentation of microstrokes after aneurysm repair [In a Phase II clinical trial, the safety and efficacy of nerinetide was evaluated in stroke patients undergoing endovascular aneurysm repair  . Betweenm repair . PatientFurther examination and stratification of the data based on patient status found an interesting caveat. The effect of nerinetide was most significant in patients with ruptured as opposed to unruptured aneurysms. Considering only patients with ruptured aneurysms, both lesion number and volume was significantly decreased in patients treated with nerinetide. This is not the case when only evaluating patients with unruptured aneurysms, in which case, neither the number nor volume differ between the treatment groups. This physiological efficacy is reflected in the patient behavioral assessment, which shows significantly more nerinetide-treated patients with ruptured aneurysms receiving a minimal NIHSS score of 0–1 , compared to saline-treated patients with also ruptured aneurysms . On the other hand, the neurological outcome of patients with unruptured aneurysms was virtually identical in both drug and placebo treatment groups.In a Phase III clinical trial, nerinetide was evaluated for efficacy in patients experiencing ischemic stroke undergoing rapid endovascular thrombectomy  . This muA larger proportion of nerinetide-treated patients who did not receive rt-PA achieved functional independence with a modified Rankin scale of 0–2 . The infarct volume of patients who did not receive rt-PA was significantly reduced by nerinetide treatment (26.7 mL vs. 39.2 mL), as was mortality . In contrast, patients who received rt-PA in this trial did not exhibit any beneficial responses to nerinetide treatment. In fact, rt-PA drastically reduced plasma concentration of nerinetide, perhaps to subtherapeutic levels. It was reported in the authors’ communications that nerinetide contains amino acid sequences known to be cleaved by plasmin  and thatThe FRONTEIR Phase III trial (NCT02315443) began in 2015 to evaluate the use of nerinetide in stroke patients within 3 h of stroke, to be administered IV by first responders. The recruitment for this study is still ongoing; the estimated study completion date is mid-2021.These above clinical data represent rare occasions of potential success in ischemic stroke therapy. We must consider the evolving clinical context driven by these results and continue the momentum in future research on neuroprotective drugs and stroke therapy. A common theme that connects all the clinical trials presented here is in the careful stratification of the patient population that may have better represented a well-controlled scientific experiment. In the DAWN and DEFUSE-3 trials, advances in imaging techniques allowed the identification of the patient population with large stroke penumbra regions, optimizing the risk-reward of an invasive procedure. This provided a criterion that is far more tangible than the previously, almost subjective, estimation of time from “last known well”. These results drastically expanded the patient population eligible for endovascular reperfusion. Because more stroke patients are eligible to receive endovascular thrombectomy in this “age of reperfusion” [This stratification of the patient population in both clinical trials for nerinetide was also the critical factor in unmasking the drug’s effects. The ENACT trial found significant differences in drug effect based on whether the patient undergoing aneurysm repair sustained a ruptured aneurysm. The ESCAPE-NA1 trial found the unexpectedly strong effect of rt-PA reduces blood nerinetide concentrations to below the therapeutic level. These considerations have allowed nerinetide to continue its clinical development, and it has the potential to be the first drug to demonstrate robust neuroprotection—given the optimal conditions. Decades of extensive basic science and highly specific experimental designs in both the preclinical and clinical setting contributed to this potential success and are necessary to continue the momentum. This is clearly demonstrated in the development of the novel emboli stroke macaque model , reflectThese impactful clinical trials provide important hints on what must be accomplished in the development of stroke therapy in the near future, especially in the field of neuroprotection. Encouraged by the positive outlook for nerinetide, we have incorporated elements of these studies in our own research, focusing on the translational targeting of the well-studied neuronal cell death pathway modulated by the voltage-gated potassium channel Kv2.1. We highlight our preclinical progress below.The depletion of intracellular potassium has been shown to be an essential event in the activation of cell death machineries, including APAF-1 apoptosome formation, caspase activation, and nuclease activity . Indeed,Prior to commitment to apoptosis, Kv2.1 is inserted in the plasma membrane via an enhanced interaction with the SNARE protein syntaxin. The Kv2.1–syntaxin interaction appears to not be necessary for the basal trafficking of the Kv2.1 channel. In cells expressing botulinum toxin that totally abrogates SNARE activity, basal Kv2.1 currents can still be observed while the expression of the enhanced proapoptotic current is abolished . Using aUnlike the well-studied interaction between GluN2B and PSD-95, the Kv2.1–syntaxin interaction was not molecularly localized on the target protein. In a bid to further understand the Kv2.1–syntaxin interaction and to extend the effectiveness of our approach, we utilized molecular dynamic simulations to dock C1aB onto syntaxin . We locaRecent advances in our understanding of the cellular microdomains that contain Kv2.1 channels have provided valuable insights into their role in neuronal cell death and have highlighted yet another unique protein–protein interaction that may be targeted pharmacologically for neuroprotection. Curiously, a subpopulation of nonconducting Kv2.1 channels localize to micronsized somatodendritic clusters on the cell surface ,76,77. TIn this work, we similarly identified the critical sequence within the Kv2.2 C-terminus that can disrupt Kv2.1-VAPA association, effectively removing the portal of entry for proapoptotic Kv2.1 channels reaching the membrane. As with the TAT-C1aB peptide, we again created a TAT-linked therapeutic peptide based on this sequence (TAT-DP-2). We showed that this peptide, importantly, induces rapid disruption of Kv2.1 channel clusters in mice in vivo following intraperitoneal injection and demonstrated its neuroprotective efficacy in the context of ischemic stroke D. We shoTaken together, the results of these studies provide promising evidence for the specific targeting of proapoptotic potassium efflux as an ischemic stroke therapy. With late time point reperfusion becoming a mainstay of clinical stroke treatment, targeting mechanisms such as these may aid in penumbral preservation, increasing the number of patients eligible for endovascular thrombectomy and improving the already positive outcomes that this therapy provides.The optimization of late time point reperfusion in ischemic stroke management is an ongoing process with new clinical trials constantly underway. Recently, the DIRECT-MT trial (NCT03469206) found that endovascular thrombectomy alone was not inferior to combinational treatment with rt-PA and endovascular thrombectomy, although there was a slight improvement with the combined treatment in prethrombectomy reperfusion and overall successful reperfusion . WhetherIn the sphere of neuroprotection, NMDA antagonism is one of the most extensively explored strategies, yet many preclinical studies focused on NMDA antagonism have failed to reach clinical translation—often due to their small therapeutic efficacy window, poor safety profile, and failure to target extrasynaptic and prodeath roles of NMDA receptors aside from canonical synaptic ion conduction . In addiAlthough these ischemic stroke trials primarily focus on direct targeting of synaptic NMDA receptors, newer evidence suggests the importance of targeting more nuanced roles of extrasynaptic NMDA receptors. For instance, activation of extrasynaptic NMDA receptors have been indicated in downregulating the ERK pathway, which ultimately leads to the expression of prodeath genes. A recent review article from Wu and Tymianski in 2018  elegantlThe development of an effective and integrated antithrombotic treatment regimen in the clinical treatment of ischemic stroke has been a multidecade effort plagued by challenges and failures, yet highlighted by revolutionary findings that provide meaningful benefits to patients afflicted by this devastating pathology. Nine years after rt-PA was initially approved for thrombolytic use in myocardial infarction, it was finally validated and approved by the US Food and Drug Administration (FDA) for use in ischemic stroke in 1996. After a lull, the advancements in stroke management techniques over the last few years have been accelerating—from the demonstration of the efficacy of endovascular thrombectomy in 2015 to the validation of late time point revascularization up to 24 h poststroke beginning in 2018. Not covered in this review, antiplatelet and antiedema therapies are both massive and immensely promising lines of research. In addition, the diabetes treatments associated with GLP-1 agonism and DPP-4 inhibition have also shown neuroprotective effects in preclinical studies and promising results in reducing major cardiovascular events specifically in type 2 diabetes patients. This literature, including the two recent related clinical trials CARMELINA NCT01897532) and CAROLINA (NCT01243424), have been reviewed by Darsalia and colleagues 7532 and . In this


Polarity is at the core of plant development, as well as that of other multicellular organisms since polarization acts as a driving force to generate a variety of specialized cells. Therefore, unraveling how cells establish polarity is the most fundamental issue toward understanding the principles of plant morphogenesis.Drosophila melanogaster and the orientation of hair follicles in mammalian skin are well-known examples of planar polarity.Cell polarity coordinated within the plane of a single tissue layer is particularly termed “planar polarity ,” which offers an experimentally accessible model system. Therefore, planar polarity is studied in various multicellular organisms. In the animal kingdom, polarized arrangement of ommatidia and bristles in Arabidopsis thaliana, root hair emergence is restricted to close to the basal ends (rootward ends) of specialized cells aligned within a single file, namely the hair-forming cell lineage (trichoblasts), thus manifesting typical planar polarity  small GTPase protein family are implicated in establishing root hair planar polarity. Among the 11 members of the ROP family in ion site . Severe e mutant . More imone cell , indicatone cell , providiAmong phytohormones, auxin and ethylene have been reported to provide a cue for root hair positioning . ExogenoEukaryotic cells possess three types of cytoskeleton, namely, actin microfilaments (MFs), microtubules (MTs), and intermediate filaments; the presence of intermediate filaments in plants is albeit still unresolved . In thisACTIN2 (ACT2) gene encoding globular actin (G-actin) causes an apical shift of root hair positioning in Arabidopsis  is another actin-related key regulator for root hair planar polarity . TWD1 ispe roots . TWD1 inpe roots . The TWDpe roots . TherefoMyosin, a motor protein involved in vesicle trafficking by interacting with actin filaments, is required for tip growth of root hair, based on the finding that the lack of a member of the myosin family results in a shorter root hair phenotype . Howeverclasp mutant exhibits a basal shift of ROP foci , which consist of α-tubulin and β-tubulin, play an essential role in determining plant cell shape, including that of root hair . ImpaireROP foci . Unlike polarity . CLASP hpolarity . It is sKinesins are motor proteins that assist cells with transport of molecules along microtubules. The armadillo domain-containing putative kinesin MRH2 has been reported to control root hair morphology by altering MT organization , but kinRoot hair positioning provides an excellent model for planar polarity, but some important questions and points are still open as follows: 1) how does the cytoskeleton place ROPs at the future initiation site? The cytoskeleton possibly acts as a scaffold of ROP proteins, constraining ROP subcellular localization at the initiation site. To support this view, in some cases, the cytoskeleton affects polarity establishment by functioning as a scaffold-like structure required for polarization of key molecules . For exaAll authors listed have made a substantial and direct contribution to the work, and approved it for publication.This work was supported by JSPS KAKENHI Grant Number 19K05951 and the 2017 Inamori Research Grant Program to HT.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Flavivirus genus that is spread by Culex mosquitos. It is maintained in an enzootic cycle in pigs and wild birds in which humans are dead-end hosts [Japanese encephalitis virus (JEV) is a positive-sense single-stranded RNA virus of the nd hosts . Despitend hosts . As a nend hosts . There aThe central nervous system (CNS) relies on the BBB, a tightly regulated barrier between the peripheral circulation and the CNS, to prevent entry of pathogens, including viruses. Yet, JEV and other neuroinvasive viruses can overcome the BBB, which usually excludes foreign substances. It is formed primarily by tight junctions (TJ) between endothelial cells, comprised of proteins such as claudin-5, zonula occludens (ZO)-1, and occludin. The BBB is sustained by supporting cell types, including astrocytes, pericytes, microglia, and mast cells (MCs) . TogetheNeuroinvasive viruses use several mechanisms to access the CNS: (1) direct infection of endothelial cells and subsequent transcellular release of virus into the brain parenchyma, (2) infection of peripheral immune cells that enter the CNS in a “Trojan Horse” mechanism, (3) paracellular entry following breakdown of the BBB, (4) retrograde transport of virus from the peripheral nervous system (PNS) into the CNS, and 5) translocation from the blood to the cerebral spinal fluid (CSF)  transloc Fig 1)..Fig 1). Some neurotropic viruses directly infect endothelial cells to reach the brain from the circulation and travel transcellularly to release viruses into the brain parenchyma . JEV hasNeurotropic viruses can gain entry into the brain through compromising the BBB. This occurs when TJs between endothelial cells become leaky, often due to the proinflammatory response. That JEV gains entry into the brain through cleaved TJs is supported by reduced resistance across JEV-exposed cultured brain-endothelial-cell membranes in vitro  and incrAnother potential pathway establishing CNS infection is through infection of peripheral immune cells that can subsequently gain entry into the CNS in a “Trojan Horse” mechanism. For JEV infection, macrophages  and DCs Once present in the CNS, inflammation due to JEV amplifies BBB breakdown. Pericytes, which are situated within the basement membrane next to endothelial cells, aid in breakdown of the BBB through release of IL-6, which leads to ZO-1 degradation . CocultuAnother factor that has been implicated in accentuating BBB breakdown during JEV infection are interferons (IFNs). Quick induction of type I IFNs has been associated with protection of astrocytes from apoptosis  and protJEV causes high morbidity and mortality in humans, leading to permanent neurological deficits, even in those who survive. Recent reports have advanced our understanding of the pathophysiological events that allow JEV to traverse the BBB and cause encephalitis. Although multiple routes of CNS entry are plausible, paracellular penetration of viruses resulting from protease and cytokine-driven breakdown of the BBB appears to be the dominant mechanism for JEV neuropenetration. This new knowledge may aid to the development of therapeutics for the treatment of virally induced encephalitis.


When Rebecca Medrano, a Washington D.C. resident who tested positive for SARS-CoV-2 infection, first started feeling unwell with viral symptoms, she noticed an additional symptom—she had lost her sense of smell NPR.org, . WorldwiUnderlying mechanisms for these observations remain unclear. In addition, they raise important questions: Does SARS-CoV-2 infect olfactory tissue and if so, which olfactory cell types do they infect and how do viral mechanisms in these cells modulate smell function? Does diabetes exacerbate viral infection or does virus-mediated dysregulation of diabetic mechanisms further aggravate symptoms in diabetics? These are critical questions to focus on amidst the tremendous increase in research efforts related to this pandemic.Within just few months, the number of SARS-CoV-2 cases has crossed 40 million with over one million deaths reported around the world .While many COVID-19 patients have reported a loss in their ability to smell, it is unclear how this virus mediates smell loss. Is there SARS-CoV-2 activity in olfactory tissue or could loss in smell simply be a byproduct of an overactive host immune response to the virus? Ample recent evidence supports the idea that viral mechanisms play a role in olfactory tissue.SARS-CoV-2 uses the receptor, Angiotensin-converting enzyme 2 (ACE2) to attach to a host cell. ACE2, a metalloprotease is found in cells of several tissues including olfactory tissue  or of neighboring cells is responsible for smell loss. One possibility is that smell loss may be due to death of OSNs  (Cornell et al., Drosophila (Kim et al., Drosophila olfactory cells have been described and are available in public collections. Considering olfactory tissue offers a convenient venue to explore interactions between viral and diabetic mechanisms as well as to evaluate them at the level of function, we cannot ignore the possibility that olfactory research will lead to insights that may aid our fight against this virus. Few other tissues harbor both ACE2 and insulin mechanisms and even when they do, such as in lungs and liver, studying functional interactions in these tissues can be challenging. We therefore urge research agencies to consider funding basic research focused on viral mechanisms in olfactory tissue.Olfactory systems, especially in genetically tractable model organisms like mice and Drosophila offers a different approach. Such an approach will not only lead to new insights about SARS-CoV-2 mechanisms but may also help identify potential drug targets primarily relevant to disease progression in diabetics. Identifying viable drug candidates will greatly impact our collective capability of fighting this disease over the next 5–10 years. If scientists and funding agencies consider novel but thoughtful actions right now, they will help change course of events and thus make a significant impact in the long term.SARS-CoV-2 has negatively impacted lives around the world. Amidst growing concerns of its impact, federal and private agencies are rushing to develop effective cures and vaccines. While there is a need for such solutions, there is also a need for basic research into underlying viral mechanisms and thereby generate more innovative solutions. One area that needs more basic research is the relationship between viral and diabetic mechanisms. Could abnormal insulin signaling in olfactory neurons of diabetics exacerbate SARS-CoV-2-mediated mechanisms and lead to loss in smell in Covid-19 patients? This relationship can no longer be ignored, especially since diabetics infected with SARS-CoV-2 have up to 3.5 times the odds of serious complications and death. Fortunately, stories of Rebecca Medrano and others offer us an opportunity to tackle this enemy from a different perspective and eventually defeat it. Studying interactions between viral and diabetic mechanisms in olfactory tissue of genetically tractable model organisms such as mice and DM conceived and wrote this opinion piece.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mental health disorders are a leading cause of disability worldwide. Challenges such as disease heterogeneity, incomplete characterization of the targets of existing drugs and a limited understanding of functional interactions of complex genetic risk loci and environmental factors have compromised the identification of novel drug candidates. There is a pressing clinical need for drugs with new mechanisms of action which address the lack of efficacy and debilitating side effects of current medications. Here we discuss a novel strategy for neuropsychiatric drug discovery which aims to address these limitations by identifying disease-related functional responses  in a variety of patient-derived cells, such as induced pluripotent stem cell (iPSC)-derived neurons and organoids or peripheral blood mononuclear cells (PBMCs). Disease-specific alterations in cellular responses can subsequently yield novel drug screening targets and drug candidates. We discuss the potential of this approach in the context of recent advances in patient-derived cellular models, high-content single-cell screening of cellular networks and changes in the diagnostic framework of neuropsychiatric disorders. They are associated with significant comorbidities including cardiovascular disease, suicide, substance abuse, immune disorders, obesity and diabetes3. Current treatments are effective in only 40–60% of individuals6, providing symptomatic relief as opposed to a cure. Other limitations include debilitating side effects, such as over-sedation and delayed-onset of therapeutic efficacy6. Despite this urgent medical need, no drugs with fundamentally new mechanisms of action have emerged for over two decades7 and many pharmaceutical companies have abandoned their neuropsychiatric R&D initiatives altogether7.Major neuropsychiatric disorders represent a substantial burden on worldwide health, accounting for 31% of years lived with disability (YLD)7. The major neuropsychiatric medications share mechanisms of action, including effects on monoaminergic neurotransmission7, with compounds that were discovered serendipitously in the 1950s and 1960s7. Since then the pharmaceutical industry has focused on the development of a vast array of monoaminergic drug derivatives with improved efficacy, safety or administration profiles8. However, because the fundamental mechanisms of drug actions have remained similar, specific patient subgroups and symptom spectra (such as negative symptoms in schizophrenia) which were refractory to first generation drugs have not been addressed by newer generation monoaminergic drugs7. Likewise, the tenuous relationship between behavioral traits in preclinical animal models and neuropsychiatric symptoms in humans is often validated using existing monoaminergic drugs7, further precluding any mechanistically novel pharmacophores. Finally, the full mechanisms of action of many of the monoaminergic drugs and non-specific binding to off-target receptors are yet to be characterized9.This is largely because there is a fundamental lack of understanding with regards to the pathophysiology of neuropsychiatric disorders which has compromised the identification of novel drug targetsDRD2) or glutamate receptor subunits  in schizophrenia, been linked to genetic risk of disease at the population level through large-scale genome-wide association studies 10. However, polygenic risk scores explain only a fraction of genetic disease liability, for example 7% in schizophrenia3 relative to 64–81% heritability derived from family and twin studies11. Moreover, putative individual GWAS risk alleles account only for a marginal increase in disease risk with odds ratios typically under 1.1 and differences in allele frequencies between cases and controls often less than 2%12. The concept that each neuropsychiatric patient presents with a different combination of multiple common but weak, or in some cases rare but penetrant, risk alleles3 has led to the use of in silico pathway analyses to identify cellular pathways which may represent convergent drug targets at the population level13. However, this approach is hindered by the fact that expression quantitative trait loci (eQTL), protein function and pathway analysis databases are insufficiently annotated to provide meaningful functional analyses relative to the molecular and cellular complexity of the human brain. Moreover, these resources often implicate non-specific pathophysiological alterations such as cell motility, glycolysis, synaptic plasticity or differentiation13, which are too general to represent ‘druggable’ targets. This is compounded by a limited understanding of how complex environmental risk factors, such as childhood social adversity, maternal infection, urbanicity, migration status or substance abuse, interact with genetic risk loci (gene-environment interactions) to impact disease etiology, onset and progression15. Thus, despite the wealth of molecular profiling data accrued in recent years it is very hard to translate these insights into functional target-based drug discovery  using post-mortem brain tissue, neuroimaging or animal model paradigms. While these approaches have added to our understanding of the disorders, they lack the vital feature of being able to assess dynamic cellular changes in relevant human tissue. However, the emerging concept that neuropsychiatric disorders are systemic disorders with corresponding manifestations in the brain and peripheral tissuesInduced pluripotent stem cells (iPSCs), created by introducing key pluripotency genes into adult somatic cells, have received considerable attention in recent years as a potential source of patient-derived cellular models for neuropsychiatric disorders, including schizophrenia, bipolar disorder, autism spectrum condition, Timothy syndrome, Fragile X syndrome and major depressive disorder22. iPSCs have been reprogrammed into a variety of different brain cell lineages, including cortical-excitatory, hippocampal and inhibitory neurons, microglia, oligodendrocytes and astrocytes22. Importantly they have demonstrated putative disease hallmarks, such as altered neuronal connectivity in schizophrenia or neuronal hyperexcitability in bipolar disorder, which were reversed by antipsychotic and mood-stabilizing medications, respectively, suggesting that they could potentially predict clinical drug efficacy. Recent developments in this field have concentrated on scaling-up iPSC-derived cultures to form more complex multi-dimensional cell networks which enable spatial interactions between different cell-types to be explored. These include co-cultures of microglia-mediated synaptic pruning23, microfluidic hippocampal synapses24, neural spheroids and brain organoids. Brain organoids have furthermore displayed a diversity of brain cell types, photosensitivity and complex cortical-like features26 and have been used to study complex developmental processes such as neuronal progenitor proliferation, interneuron migration and cortical layer formation21. The use of brain organoids is still in early stages for neuropsychiatric disorders. For example, one single-cell RNA sequencing study reported altered GABAergic specification and Wnt signaling in brain organoids derived from monozygotic twins discordant for schizophrenia27. Another study reported downregulation of pathways involved in synaptic biology, neurodevelopment and cell adhesion, concurrent with reduced stimulation and depolarization responses in brain organoids from individuals with bipolar disorder28. Nevertheless, organoids have been successfully employed in a number of other disease indications such as drug repurposing screens against Zika virus, SARS-CoV-2 infection modeling and precision medicine for cystic fibrosis and a range of cancers29.22. These are compounded in the case of organoids by differences in intrinsic versus directed patterning and the inability to mature to postnatal stages, potentially due to lack of vascularization21. Together, these features have meant that this approach remains relatively high-cost, variable, and low-throughput.While these iPSC-derived models represent an unprecedented opportunity to explore neuropsychiatric cellular alterations in relevant CNS tissue with the genetic background of patients, they continue to face several limitations. These include difficulties in selection of the iPSC colonies, specificity of end fate differentiation, intra-patient variability of iPSC clones, karyotypic instability across passages and differential power requirements for idiopathic versus monogenic gene variants30, microglial-like cells from peripheral monocytes31 and olfactory neurosphere-derived cells32. Finally, CNS cell lines or cells from control donors can be cultured in patient-specific body fluids, for example using patient-derived serum or cerebrospinal fluid, to investigate the effects of disease-associated secreted factors33.Cells which share many of the characteristics of brain cell lineages can also be induced directly from primary patient tissue without the need for reprogramming, including neuronal-like cells from fibroblastsPeripheral blood mononuclear cells (PBMCs) are possibly the best example of this application. They are both accessible for sampling and amenable to high-content screening in suspension34. Consequently, they represent a scalable model with the potential to satisfy the power requirements of neuropsychiatric disease investigations whilst facilitating the depth of cellular exploration necessary to reveal complex disease processes in their native state. The majority of investigations using PBMCs in neuropsychiatry have focused on determining the relative proportions of different cell subsets, their activation status or their cytokine secretion profiles36, consistent with hypotheses of immunological dysfunction in these disorders, and more recently on interactions with the human microbiome19. However, recent data suggests that PBMCs can also provide a surrogate model for exploring systemic alterations in a subset of CNS drug targets. Subtypes of CNS receptors  and their cell signaling substrates  have been shown to be altered in the brain, as well as PBMC subsets of neuropsychiatric patients and correlated with therapeutic efficacy or disease severity39. GWAS data also suggests the enrichment of single nucleotide polymorphisms associated with neuropsychiatric (schizophrenia) risk loci within PBMC subtype-specific gene expression enhancers10. Moreover, PBMCs have shown preliminary evidence of parallel epigenetic changes to those observed in the brain following exposure to environmental stressors, such as early life social adversity40, raising the possibility of exploring drug-target interactions which are specific to environmental risk factors. Although many of the pathways which are shared between PBMCs and CNS cells are likely to respond differently and the degree of functional overlap between lineages remains to be fully determined, recent evidence suggests that subsets of pathways  or even individual protein-protein interactions, which do overlap, might serve as a proxy for clinically relevant targets which are otherwise inaccessible in primary patient samples41. Likewise, it is possible that, at least in a subpopulation of patients, targeting proteins which mitigate immune dysfunction may contribute to symptom remission, as exemplified by the modest efficacy of celecoxib in clinical trials involving first-episode schizophrenia patients with predominantly positive symptoms42.It is also possible to use primary peripheral cells in their native state. functional cellular endophenotype (see Text box) strategy aims to directly identify abnormal functional responses in patient-derived live cells, relative to healthy individuals, and subsequently use these responses as novel drug screening targets , which are shared by subgroups of patients despite divergent genetic backgrounds, represent an opportunity to summarize genetic heterogeneity, in addition to environmental risk factors, at a time when functional interactions between risk variants are currently too complicated to model or even unknown. Examples of functional endophenotypes include altered calcium responses in T cells at PLC−γ1 linked to ATP2A2 polymorphisms in schizophrenia41 or spontaneous calcium hyperexcitability in dentate gyrus-like neurons derived from iPSCs in bipolar disorder45. Moreover, the use of functional testing in live cells allows the elucidation of relevant disease-specific alterations in cellular networks (or pathways) which are not reflected by quantitative changes in mRNA or protein levels in their basal state, as demonstrated by glycolytic pathway alterations following antigenic stimulation in schizophrenia patient PBMCs46. This includes perturbations in homeostatic and regulatory mechanisms consistent with the concept of altered ‘cellular coping’47.The application of this strategy is particularly relevant for tackling complex disorders, such as neuropsychiatric conditions. The use of patient-derived cells provides a unique opportunity to model the genomic and epigenomic complexity of neuropsychiatric disorders in a physiologically relevant context. Recent data suggests that the genetic architecture of neuropsychiatric disorders consists of multiple common but weak or rare but penetrant genetic risk factors, some of which are inherited while others may be sporadic (or ‘de novo’)48, mass cytometry34, high-throughput microscopy49, and single-cell RNA sequencing50, enable the depth of functional exploration necessary to identify endophenotypes in neuropsychiatric patient-derived cells41. The simultaneous detection of multiple readouts  in individual cells following diverse ligand stimulation allows the readouts to be correlated across thousands of single-cell measurements in each sample. This can serve to generate hypotheses as to causative signaling relationships and alterations in network connectivity associated with disease at the target discovery stage, for example increased negative regulation within the Akt1 pathway in CD4+ T cells from autism spectrum condition and schizophrenia patients51. Moreover, changes in the phosphorylation activation status of key therapeutic targets can be normalized relative to total protein abundance or mRNA expression, a feature that has recently revealed novel mechanisms of action for the mood stabilizer lithium in iPSC-derived neurons from patients with bipolar disorder52. The ability to measure multiple markers at the single-cell level also affords the statistical power necessary to identify clinically relevant functional phenotypes in minority cell sub-populations within a heterogeneous patient-derived cell sample and define functional overlap between cells from divergent lineages . In this respect, computational approaches  which provide high-dimensional representations of deep lineage phenotyping combined with multiple functional measurements represent a valuable means for extracting disease-associated cellular phenotypes from high-content data without relying on prior knowledge. This has been applied to identify cellular phenotypes relevant to prognosis in other disease indications including acute myeloid leukemia (AML)56. Such an approach has particular potential, although as yet unapplied, for neuropsychiatric disorders as it is unclear which cell subtypes represent the best functional surrogates for different aspects of CNS pathology or drug discovery indications.High-content screening technologies, such as flow cytometry57, especially for neuropsychiatric disorders, consistent with their roles as key cellular functional executioners. Thus, targeting these proteins in the drug target discovery phase represents a heuristic means for screening the most ‘druggable’ part of the genome. Importantly, while many of these highly functional cellular proteins, for example GPCRs, are not easily detectable by traditional proteomic screens, an amplified signaling event downstream of these low abundance proteins can be accurately measured using fluorescence flow cytometry or mass cytometry34. Furthermore, technologies such as cellular barcoding58, which permit multiplexing of the ligand treatments, can be employed to increase the number of functional conditions analyzed in a limited clinical sample, for example 64 concurrent ligand conditions applied to schizophrenia PBMCs41. Finally, at the drug discovery stage, candidate compounds can be screened to identify multi-target efficacies, a feature common to existing neuropsychiatric drugs, or potentially toxic off-target interactions directly in patient samples at early stages in the drug development pipeline. The importance of characterizing neuropsychiatric drug interactions outside of conventional targets is poignantly illustrated by the association of TREK-2 potassium channel binding with antidepressant efficacy59 or histamine H1 receptor affinity with the side-effects of antipsychotic-induced weight gain and sedation9. High-content resolution of cellular responses can also be used to explore synergistic interactions between highly specific ligands acting at different sites in the cellular network, a strategy which has shown the potential for overcoming treatment resistance related to genetic heterogeneity in other disease indications such as oncology60.An essential feature of the high-content functional screening approach is the ability to tailor the ligands and cellular readouts used for high-content exploration of patient samples to increase the likelihood of relevant drug target identification. Collectively, G-protein-coupled receptors (GPCRs), ion channels and protein kinases and phosphatases represent the targets for the vast majority of currently approved medications61. While correlation does not necessarily imply causation, these criteria can serve to prioritize nodes which are more likely to represent causative variants and thus, potentially relevant therapeutic targets. As a final consideration, nodes of comparable significance across these criteria may be chosen based on their amenability to high-throughput drug screening. For example, this may include nodes with a higher signal to noise ratio (Z-prime test), expression in cell-types which are more easily scaled-up in a cost-effective manner and more specific readouts  relative to generalized responses .One of the major challenges, having identified relevant functional endophenotypes in neuropsychiatric patient samples, is the prioritization of pathway responses with potentially causal disease influences for subsequent drug screening. In this respect, a multi-tiered approach may be useful. First, given the possibility of multiple hits arising from high-content screening (described below) it is important to statistically adjust for false discoveries and extensively cross-validate the findings using techniques which take into account the structure of the data, such as non-parametric permutation procedures and nested cross-validation, as well as to consider primarily functional nodes with exceptional significance in drug-naïve patient vs. control comparisons. Second, target nodes for which activity is correlated to disease severity at baseline (before treatment) or to improvements in symptomatology over the course of efficacious treatment, if longitudinal follow-up samples are available, are more likely to be related to active psychopathology. Third, if genotyping data is available for the same samples, the nodes which correlate to polygenic risk scores, summarizing known genetic risk, or individual risk variants might be suggestive of targets which are supported by parallel genetic evidence, at least in subgroups of patients, and could offer mechanistic insights underlying the endophenotype. Fourth, expression of the target node in brain tissue and/or recapitulation of the target response in brain cell lineages, although not essential, can serve to prioritize targets with CNS activity. This can be further supported by evidence of behavioral abnormalities in animal models, in which the target node has been knocked-out or knocked-in, or developmental changes in transgenic model organisms such as zebrafish41. This study prioritized an abnormal response to thapsigargin at PLC-γ1 as the most relevant drug target based on being the most significant node in the drug-naïve patient vs. control comparison, normalization over the course of efficacious clinical antipsychotic therapy, correlation to schizophrenia risk allele loading at the sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (ATP2A2) risk locus62, concurrent activity in neuronal SH-SY5Y cells and parallel evidence of schizophrenia-like behavioral changes in animal models following forebrain-specific ablation of PLC-γ163.A recent study using this approach for drug target discovery in schizophrenia, assessed 3696 cell signaling responses in PBMCs from individuals with schizophrenia and matched controls with a six-week longitudinal follow up64, a disorder caused by a missense mutation in L-type CaV 1.2 calcium channels and associated with developmental delay and autism spectrum condition, showed abnormalities in action potential firing and calcium signaling using patch clamp recording and calcium imaging in iPSC-derived neurons from patients relative to controls. This was further characterized to show differences in calcium-dependent gene expression following depolarization, including tyrosine hydroxylase, with concurrent increases in dopamine and noradrenaline secretion. The authors then screened different L-type calcium channel blockers to show that the tyrosine hydroxylase endophenotype could be improved using roscovitine, a cyclin-dependent kinase inhibitor and atypical L-type channel blocker. Interestingly, in the aforementioned study relating to functional endophenotypes in schizophrenia PBMCs, screening of an FDA-approved compound library (n = 786) identified different subsets of L-type calcium channel blockers  capable of reversing calcium signaling deficits in response to thapsigargin at PLC-γ141. This highlights this compound class as potentially worthy of follow up across different neuropsychiatric indications, a feature supported by the genetic association of L-type calcium channel subunits  across several major neuropsychiatric disorders65.Having prioritized the relevant drug targets from patient-derived cellular models, phenotypic drug screening can be used to identify compounds which normalize these pathway responses and could serve as potential novel drug candidates. This provides a means to identify novel drug candidates even before the full spectrum and functional interactions of putative risk alleles and environmental stressors are defined. For example, one study focused on Timothy syndrome41. Second, functional endophenotype strategies to date have been modest in terms of sample numbers (discussed below) and validation in larger patient cohorts is necessary to determine whether the target response and drug candidates are reproducible and whether there might be heterogeneity in terms of drug response in the target population. Third, given the overlap in genetic risk factors between different neuropsychiatric disorders, it is important to determine target specificity by comparing target activity in different neuropsychiatric disorders. Previous studies have shown that subsets of abnormalities in cell signaling responses can be shared between different neuropsychiatric disorders while others are unique51. Furthermore, this heterogeneity manifests at the individual level whereby individuals with different diagnoses can have partially overlapping signaling profiles. Given the changing diagnostic landscape of neuropsychiatric disorders, it is plausible that targets related to symptom subtypes which extend across diagnostic boundaries could find utility in multiple indications. For example, one study reported that alterations in phosphorylation responses at proinflammatory proteins NF-κB p65 (pS529) and Stat3 (pS727) were shared between conditions with negative symptomatology (schizophrenia and major depression) while aberrant responses to phosphatase inhibitor calyculin A at S6 (pS235/pS236) were shared between conditions with potential psychotic symptomatology (schizophrenia and bipolar disorder)51. Conversely, disorders which do not share the same targets can represent relevant exclusion criteria for future clinical trials. Fourth, novel compounds still need to undergo preclinical trials to determine efficacy, toxicity and pharmacokinetics. Despite the limitations of current preclinical models in terms of equating behavioral changes to complex psychiatric symptoms and the reliance on existing treatments as gold standards, functional endophenotypes at least offer the alternative to genetically engineer the target response instead of using acute pharmacological interventions to precipitate symptom-like behaviors. An alternative to the reliance on animal models is the screening of approved medications (drug repurposing) whereby the well documented toxicology, pharmacokinetic, dosing and medicinal chemistry profiles of these compounds could serve to expedite their clinical application to neuropsychiatric indications at a lower cost relative to new chemical entities67. Finally, in terms of clinical trial design, the same functional endophenotypes used for drug discovery have the potential to serve as ex vivo treatment response predictors, which could stratify patients during clinical drug development to overcome the heterogeneous results of previous clinical trials. Examples include ex-vivo calcium responses at PLC-γ1 in T cells41, glucocorticoid sensitivity in whole blood68, or CRMP2 phosphorylation in iPSC-derived neurons52 correlated with in vivo clinical efficacy in schizophrenia, major depression and bipolar disorder, respectively. In this regard, an increase in the proportion of clinical trials which focus on drug-naïve or recent-onset patients relative to chronic treatment-resistant patients would help to improve the development of effective early intervention strategies. Moreover, where the functional target is sensitive to clinically approved drugs ex vivo, response prediction can be used to validate the target and support the potential in vivo efficacy of novel drugs41.While this strategy represents a means to rapidly generate early stage candidates, several subsequent steps are relevant when translating these findings towards potential clinical trials. First, novel drug candidates can be directly compared within the same cellular model to established treatments, or to each other, to identify lead compounds which show putative enhanced target specificity, cellular potency or brain penetrance. For example, this has been demonstrated for subtypes of 1,4-dihydropyridines within the L-type calcium channel blocker class in phenotypic screening of functional cellular endophenotypes in schizophreniaThe functional cellular endophenotype strategy in patient-derived cellular models represents a reverse engineering approach. Traditional target-based, or ‘rational’, drug discovery aims to quantify pathologically-linked gene products and propose a mechanistic drug target using in silico pathway analysis, followed by screening for new drugs in a purpose-built reporter system  and inferring clinical relevance. In contrast, the functional endophenotype strategy, proposed here, aims to identify compounds with differential activity directly in physiologically relevant patient-derived cells, relative to healthy individuals, and subsequently dissect their mechanisms of action and underlying genetic targets. Despite the progress made, there are several limitations and key features worth considering to optimize its future utility.70 and up to several dozen samples using PBMCs68, suggesting that they are likely underpowered relative to the complexity of neuropsychiatric phenotypes. The power requirements for target definition using this approach therefore remain to be accurately determined. However, the fact that relatively small endophenotype strategies in schizophrenia PBMCs 41 have identified similar lead compounds  as suggested by much larger GWAS studies 66 raises the possibility that they might have lower power requirements as a result of summarizing genetic risk at the pathway level, a feature echoed by studies using patient iPSCs and cerebral organoids69. Nevertheless, the increased cost of functional studies on live cells and the possibility of expectancy bias, means that it is important to cross-reference cellular responses with large-scale genetic and proteomic studies such that emerging functional targets might be interpreted in light of better-powered existing studies as the field develops. The effect of cost in limiting sample size is particularly relevant for iPSC-based and organoid studies, where extended culture protocols are needed to reprogram and differentiate cells towards neuronal lineages. In these studies the trade-off between increasing the total number of donors and increasing the number of independent iPSC clones per donor is critical to determining statistical power69. Although independent iPSC clones from the same donor are vital to quantifying intra-patient variability (derived from the transformation and differentiation processes), it has been suggested that the use of single iPSC lines for each donor, while maximizing the number of donors, may be the most efficient strategy to maximize statistical power in light of false discovery constraints69. Moreover, it is recognized that decreasing inter-patient heterogeneity by focusing on more genetically homogenous patient and control groups might further improve statistical power. This can take the form of selecting patients with highly-penetrant rare genetic variants with a large effect size, patients with high polygenic risk scores based on common variants or gene-editing  to introduce specific risk alleles in isogenic iPSC lines71. A final consideration in terms of cost is that while cellular assays may initially be more expensive than genotyping or steady-state protein profiling, the resulting functional endophenotypes are more directly amenable to drug screening. In contrast, the interpretation and engineering of genomic or proteomic targets into cellular systems can represent a considerable additional cost beyond the initial target identification.First, obtaining large sample numbers of clinically well-characterized neuropsychiatric patients and sufficient volumes of viable patient-derived cells is a major challenge logistically and in terms of cost. Functional endophenotype studies to date have used relatively few samples, generally less than ten samples for iPSC-based studies72, suggest that the same upscaling may be possible in the field of mental health. In this respect, composite workflows starting with more accessible cell types  followed by more resource-intensive cellular systems , or vice versa, may prove efficient and cost effective. This will likely be complemented by recent efforts to scale-up iPSC-derived cell types for high-throughput compound-screening22, inclusion of a greater number of iPSCs from complex idiopathic vs. monogenic disorders and direct comparisons of target overlap between different cellular models from the same individuals. Greater numbers of valuable drug-naïve samples might also be facilitated by including high-risk individuals  or patient groups where the disease often remains undiagnosed . In line with increasing the power of functional endophenotype strategies, it will also be crucial to leverage available data to control for false discoveries and expectancy bias using statistical methods such as non-parametric permutation procedures and nested cross-validation, which take into account the data structure.While sample numbers remain low for patient-derived cellular models in neuropsychiatry, initiatives like the NextGen Genetic Association Studies Consortium, which integrated data from over 2000 iPSC lines with GWAS and QTL data to identify functional cellular phenotypes for cardiovascular diseaseSecond, comparing cohorts with high and low polygenic risk profile scores, or with and without rare penetrant risk variants, across key environmental risk factors is an essential step in understanding disease heterogeneity and targeting treatments to specific disease aetiologies. Third, as the diagnostic framework of neuropsychiatric disorders evolves beyond DSM-5 and ICD-10, it will be important to incorporate cellular responses, in addition to other biomarker strategies, to help predict response to clinical treatment on an individual basis and define diagnostic categories which align more closely with therapeutic indications. Fourth, while cellular responses to existing neuropsychiatric treatments can be helpful to validate functional endophenotypes, establish relevant drug discovery workflows and provide clinical correlates for predicted efficacy, the field must eventually depart from the reliance on existing medications in order to identify mechanistically novel drugs which target resistant symptom spectra and avoid the ‘catch-22 scenario’ which has limited the scope of animal models to date.73 or GSK-3β animal models of bipolar disorder74. Fluorescence-activated cell sorting of cells from the same patient and cell subtype which differentially exhibit the putative pathological response can also enable characterization of genomic or proteomic readouts whilst controlling for molecular variation between sample donors and cell lineages. Lastly, the combination of technologies such a single-cell RNA sequencing with multiplexed ion beam imaging75 in patient-derived brain organoids could provide spatial resolution for understanding the functional interactions between cells which drive neuropsychiatric disease in a physiologically relevant context.Finally, disease mechanisms underlying functional cellular endophenotypes require further dissection. Complementary screening technologies such as siRNA, CRISPR-Cas9 genome editing, or protein-specific inhibitors provide opportunities to systematically knock-out or knock-in the function of network proteins to gauge their influence on the target response, as demonstrated in DISC1 iPSC-neuronal models of schizophreniaIn conclusion, the presented approach is not the sole solution for addressing the paucity of novel therapeutic options for neuropsychiatric disorders. Its wider applicability, including the pharmacokinetic, brain penetrance and safety profiles of the candidate compounds, remains to be determined in addition to better understanding which neuropsychiatric conditions are likely to best be served by this approach. However, in a field where primary disease tissue is scarcely accessible and genetic complexity is daunting, relative to the magnitude of the public health burden, this approach could offer a complementary strategy to expedite the identification of relevant drug candidates and personalized treatment response predictors.