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Humans can remember specific events without acting on them and can influence which memories are retrieved based on internal goals. However, current animal models of memory typically present sensory cues to trigger retrieval and assess retrieval based on action 1-5 . As a result, it is difficult to determine whether measured patterns of neural activity relate to the cue(s), the retrieved memory, or the behavior. We therefore asked whether we could develop a paradigm to isolate retrieval-related neural activity in animals without retrieval cues or the requirement of a behavioral report. To do this, we focused on hippocampal "place cells." These cells primarily emit spiking patterns that represent the animal's current location (local representations), but they can also generate representations of previously visited locations distant from the animal's current location (remote representations) 6-13 . It is not known whether animals can deliberately engage specific remote representations, and if so, whether this engagement would occur during specific brain states. So, we used a closed-loop neurofeedback system to reward expression of remote representations that corresponded to uncued, experimenter-selected locations, and found that rats could increase the prevalence of these specific remote representations over time; thus, demonstrating memory retrieval modulated by internal goals in an animal model. These representations occurred predominately during periods of immobility but outside of hippocampal sharp-wave ripple (SWR) 13-15 events. This paradigm enables future direct studies of memory retrieval mechanisms in the healthy brain and in models of neurological disorders.

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Scientific progress depends on reliable and reproducible results. Progress can also be accelerated when data are shared and re-analyzed to address new questions. Current approaches to storing and analyzing neural data typically involve bespoke formats and software that make replication, as well as the subsequent reuse of data, difficult if not impossible. To address these challenges, we created Spyglass, an open-source software framework that enables reproducible analyses and sharing of data and both intermediate and final results within and across labs. Spyglass uses the Neurodata Without Borders (NWB) standard and includes pipelines for several core analyses in neuroscience, including spectral filtering, spike sorting, pose tracking, and neural decoding. It can be easily extended to apply both existing and newly developed pipelines to datasets from multiple sources. We demonstrate these features in the context of a cross-laboratory replication by applying advanced state space decoding algorithms to publicly available data. New users can try out Spyglass on a Jupyter Hub hosted by HHMI and 2i2c: https://spyglass.hhmi.2i2c.cloud/.

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A brain-machine interface demonstrates volitional control of hippocampal activity.

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Representations related to past experiences play a critical role in memory and decision-making processes. The rat hippocampus expresses these types of representations during sharp-wave ripple (SWR) events, and previous work identified a minority of SWRs that contain 'replay' of spatial trajectories at ∼20x the movement speed of the animal. Efforts to understand replay typically make multiple assumptions about which events to examine and what sorts of representations constitute replay. We therefore lack a clear understanding of both the prevalence and the range of representational dynamics associated with replay. Here, we develop a state space model that uses a combination of movement dynamics of different speeds to capture the spatial content and time evolution of replay during SWRs. Using this model, we find that the large majority of replay events contain spatially coherent, interpretable content. Furthermore, many events progress at real-world, rather than accelerated, movement speeds, consistent with actual experiences.

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Executing memory-guided behavior requires storage of information about experience and later recall of that information to inform choices. Awake hippocampal replay, when hippocampal neural ensembles briefly reactivate a representation related to prior experience, has been proposed to critically contribute to these memory-related processes. However, it remains unclear whether awake replay contributes to memory function by promoting the storage of past experiences, facilitating planning based on evaluation of those experiences, or both. We designed a dynamic spatial task that promotes replay before a memory-based choice and assessed how the content of replay related to past and future behavior. We found that replay content was decoupled from subsequent choice and instead was enriched for representations of previously rewarded locations and places that had not been visited recently, indicating a role in memory storage rather than in directly guiding subsequent behavior.

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RATIONALE: As a new approach to DNA adductomics, we directly reacted intact, double-stranded (ds)-DNA under warm conditions with an alkylating mass tag followed by analysis by liquid chromatography/mass spectrometry. This method is based on the tendency of adducted nucleobases to locally disrupt the DNA structure (forming a "DNA bubble") potentially increasing exposure of their nucleophilic (including active hydrogen) sites for preferential alkylation. Also encouraging this strategy is that the scope of nucleotide excision repair is very broad, and this system primarily recognizes DNA bubbles. METHODS: A cationic xylyl (CAX) mass tag with limited nonpolarity was selected to increase the retention of polar adducts in reversed-phase high-performance liquid chromatography (HPLC) for more detectability while maintaining resolution. We thereby detected a diversity of DNA adducts (mostly polar) by the following sequence of steps: (1) react DNA at 45°C for 2 h under aqueous conditions with CAX-B (has a benzyl bromide functional group to label active hydrogen sites) in the presence of triethylamine; (2) remove residual reagents by precipitating and washing the DNA (a convenient step); (3) digest the DNA enzymatically to nucleotides and remove unlabeled nucleotides by nonpolar solid-phase extraction (also a convenient step); and (4) detect CAX-labeled, adducted nucleotides by LC/MS2 or a matrix-assisted laser desorption/ionization (MALDI)-MS technique. RESULTS: Examples of the 42 DNA or RNA adducts detected, or tentatively so based on accurate mass and fragmentation data, are as follows: 8-oxo-dGMP, ethyl-dGMP, hydroxyethyl-dGMP (four isomers, all HPLC-resolved), uracil-glycol, apurinic/apyrimidinic sites, benzo[a]pyrene-dGMP, and, for the first time, benzoquinone-hydroxymethyl-dCMP. Importantly, these adducts are detected in a single procedure under a single set of conditions. Sensitivity, however, is only defined in a preliminary way, namely the latter adduct seems to be detected at a level of about 4 adducts in 109 nucleotides (S/N ~30). CONCLUSIONS: CAX-Prelabeling is an emerging new technique for DNA adductomics, providing polar DNA adductomics in a practical way for the first time. Further study of the method is encouraged to better characterize and extend its performance, especially in scope and sensitivity.

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While MALDI-MS of intact genomic DNA is unheard of, actually many DNA adducts can be detected in this way under certain MALDI conditions: relatively high molar ratio of DNA nucleobases to matrix (0.01 to 0.3), hot matrix (CCA), and high laser fluence. This is because many DNA adducts create "bubbles" on dsDNA (disruption of base pairing), making it easier for these adducts as modified nucleobases to be jettisoned by the laser-derived energy of MALDI (jettison mass spectrometry or JeMS). The method also works for other nucleic acid species, namely nucleobases, nucleosides, nucleotides, and RNA. Examples of what we have detected in this way are as follows: methyladenine in E. coli DNA, 5-hydroxymethylcytosine in human brain DNA, melphalan-adenine in leukocyte DNA from patients on corresponding chemotherapy, wybutosine in tRNA, benzyl DNA adducts in E. coli cell culture treated with benzyl bromide, and various DNA adducts formed in test tube exposure experiments with calf thymus DNA. Noteworthy, in the chemotherapy study, principle component analysis of the data encourages the hypothesis that patient DNA undergoes much more damage than just melphalan adducts. Overall, our work leads to the preliminary generalization that about 5 fmol of a nucleobase deficient in base pairing, and present in a MALDI spot, will be detected by JeMS (on the equipment that we used), irrespective of the type of nucleic acid species which houses it, as long as the nucleobase is relatively basic such as A, C, or G.

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PURPOSE: The exocyst complex is a conserved protein complex that mediates fusion of intracellular vesicles to the plasma membrane and is implicated in processes including cell polarity, cell migration, ciliogenesis, cytokinesis, autophagy, and fusion of secretory vesicles. The essential role of these genes in human genetic disorders, however, is unknown. METHODS: We performed homozygosity mapping and exome sequencing of consanguineous families with recessively inherited brain development disorders. We modeled an EXOC7 splice variant in vitro and examined EXOC7 messenger RNA (mRNA) expression in developing mouse and human cortex. We modeled exoc7 loss-of-function in a zebrafish knockout. RESULTS: We report variants in exocyst complex members, EXOC7 and EXOC8, in a novel disorder of cerebral cortex development. In EXOC7, we identified four independent partial loss-of-function (LOF) variants in a recessively inherited disorder characterized by brain atrophy, seizures, and developmental delay, and in severe cases, microcephaly and infantile death. In EXOC8, we found a homozygous truncating variant in a family with a similar clinical disorder. We modeled exoc7 deficiency in zebrafish and found the absence of exoc7 causes microcephaly. CONCLUSION: Our results highlight the essential role of the exocyst pathway in normal cortical development and how its perturbation causes complex brain disorders.

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Whole-genome sequencing of DNA from single cells has the potential to reshape our understanding of mutational heterogeneity in normal and diseased tissues. However, a major difficulty is distinguishing amplification artifacts from biologically derived somatic mutations. Here, we describe linked-read analysis (LiRA), a method that accurately identifies somatic single-nucleotide variants (sSNVs) by using read-level phasing with nearby germline heterozygous polymorphisms, thereby enabling the characterization of mutational signatures and estimation of somatic mutation rates in single cells.

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Endosomal sorting complex required for transport (ESCRT) complex proteins regulate biogenesis and release of extracellular vesicles (EVs), which enable cell-to-cell communication in the nervous system essential for development and adult function. We recently showed human loss-of-function (LOF) mutations in ESCRT-III member CHMP1A cause autosomal recessive microcephaly with pontocerebellar hypoplasia, but its mechanism was unclear. Here, we show Chmp1a is required for progenitor proliferation in mouse cortex and cerebellum and progenitor maintenance in human cerebral organoids. In Chmp1a null mice, this defect is associated with impaired sonic hedgehog (Shh) secretion and intraluminal vesicle (ILV) formation in multivesicular bodies (MVBs). Furthermore, we show CHMP1A is important for release of an EV subtype that contains AXL, RAB18, and TMED10 (ART) and SHH. Our findings show CHMP1A loss impairs secretion of SHH on ART-EVs, providing molecular mechanistic insights into the role of ESCRT proteins and EVs in the brain.

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Single cell whole-genome sequencing (scWGS) is providing novel insights into the nature of genetic heterogeneity in normal and diseased cells. However, the whole-genome amplification process required for scWGS introduces biases into the resulting sequencing that can confound downstream analysis. Here, we present a statistical method, with an accompanying package PaSD-qc (Power Spectral Density-qc), that evaluates the properties and quality of single cell libraries. It uses a modified power spectral density to assess amplification uniformity, amplicon size distribution, autocovariance and inter-sample consistency as well as to identify chromosomes with aberrant read-density profiles due either to copy alterations or poor amplification. These metrics provide a standard way to compare the quality of single cell samples as well as yield information necessary to improve variant calling strategies. We demonstrate the usefulness of this tool in comparing the properties of scWGS protocols, identifying potential chromosomal copy number variation, determining chromosomal and subchromosomal regions of poor amplification, and selecting high-quality libraries from low-coverage data for deep sequencing. The software is available free and open-source at https://github.com/parklab/PaSDqc.

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It has long been hypothesized that aging and neurodegeneration are associated with somatic mutation in neurons; however, methodological hurdles have prevented testing this hypothesis directly. We used single-cell whole-genome sequencing to perform genome-wide somatic single-nucleotide variant (sSNV) identification on DNA from 161 single neurons from the prefrontal cortex and hippocampus of 15 normal individuals (aged 4 months to 82 years), as well as 9 individuals affected by early-onset neurodegeneration due to genetic disorders of DNA repair (Cockayne syndrome and xeroderma pigmentosum). sSNVs increased approximately linearly with age in both areas (with a higher rate in hippocampus) and were more abundant in neurodegenerative disease. The accumulation of somatic mutations with age-which we term genosenium-shows age-related, region-related, and disease-related molecular signatures and may be important in other human age-associated conditions.

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While next-generation sequencing has accelerated the discovery of human disease genes, progress has been largely limited to the "low hanging fruit" of mutations with obvious exonic coding or canonical splice site impact. In contrast, the lack of high-throughput, unbiased approaches for functional assessment of most noncoding variants has bottlenecked gene discovery. We report the integration of transcriptome sequencing (RNA-seq), which surveys all mRNAs to reveal functional impacts of variants at the transcription level, into the gene discovery framework for a unique human disease, microcephaly-micromelia syndrome (MMS). MMS is an autosomal recessive condition described thus far in only a single First Nations population and causes intrauterine growth restriction, severe microcephaly, craniofacial anomalies, skeletal dysplasia, and neonatal lethality. Linkage analysis of affected families, including a very large pedigree, identified a single locus on Chromosome 21 linked to the disease (LOD > 9). Comprehensive genome sequencing did not reveal any pathogenic coding or canonical splicing mutations within the linkage region but identified several nonconserved noncoding variants. RNA-seq analysis detected aberrant splicing in DONSON due to one of these noncoding variants, showing a causative role for DONSON disruption in MMS. We show that DONSON is expressed in progenitor cells of embryonic human brain and other proliferating tissues, is co-expressed with components of the DNA replication machinery, and that Donson is essential for early embryonic development in mice as well, suggesting an essential conserved role for DONSON in the cell cycle. Our results demonstrate the utility of integrating transcriptomics into the study of human genetic disease when DNA sequencing alone is not sufficient to reveal the underlying pathogenic mutation.

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Despite recent advances in understanding the genetic bases of microcephaly, a large number of cases of microcephaly remain unexplained, suggesting that many microcephaly syndromes and associated genes have yet to be identified. Here, we report mutations in PYCR2, which encodes an enzyme in the proline biosynthesis pathway, as the cause of a unique syndrome characterized by postnatal microcephaly, hypomyelination, and reduced cerebral white-matter volume. Linkage mapping and whole-exome sequencing identified homozygous mutations (c.355C>T [p.Arg119Cys] and c.751C>T [p.Arg251Cys]) in PYCR2 in the affected individuals of two consanguineous families. A lymphoblastoid cell line from one affected individual showed a strong reduction in the amount of PYCR2. When mutant cDNAs were transfected into HEK293FT cells, both variant proteins retained normal mitochondrial localization but had lower amounts than the wild-type protein, suggesting that the variant proteins were less stable. A PYCR2-deficient HEK293FT cell line generated by genome editing with the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system showed that PYCR2 loss of function led to decreased mitochondrial membrane potential and increased susceptibility to apoptosis under oxidative stress. Morpholino-based knockdown of a zebrafish PYCR2 ortholog, pycr1b, recapitulated the human microcephaly phenotype, which was rescued by wild-type human PYCR2 mRNA, but not by mutant mRNAs, further supporting the pathogenicity of the identified variants. Hypomyelination and the absence of lax, wrinkly skin distinguishes this condition from that caused by previously reported mutations in the gene encoding PYCR2's isozyme, PYCR1, suggesting a unique and indispensable role for PYCR2 in the human CNS during development.

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Whereas many genes associated with intellectual disability (ID) encode synaptic proteins, transcriptional defects leading to ID are less well understood. We studied a large, consanguineous pedigree of Arab origin with seven members affected with ID and mild dysmorphic features. Homozygosity mapping and linkage analysis identified a candidate region on chromosome 17 with a maximum multipoint logarithm of odds score of 6.01. Targeted high-throughput sequencing of the exons in the candidate region identified a homozygous 4-bp deletion (c.169_172delCACT) in the METTL23 (methyltransferase like 23) gene, which is predicted to result in a frameshift and premature truncation (p.His57Valfs*11). Overexpressed METTL23 protein localized to both nucleus and cytoplasm, and physically interacted with GABPA (GA-binding protein transcription factor, alpha subunit). GABP, of which GABPA is a component, is known to regulate the expression of genes such as THPO (thrombopoietin) and ATP5B (ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide) and is implicated in a wide variety of important cellular functions. Overexpression of METTL23 resulted in increased transcriptional activity at the THPO promoter, whereas knockdown of METTL23 with siRNA resulted in decreased expression of ATP5B, thus revealing the importance of METTL23 as a regulator of GABPA function. The METTL23 mutation highlights a new transcriptional pathway underlying human intellectual function.

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Despite significant heritability of autism spectrum disorders (ASDs), their extreme genetic heterogeneity has proven challenging for gene discovery. Studies of primarily simplex families have implicated de novo copy number changes and point mutations, but are not optimally designed to identify inherited risk alleles. We apply whole-exome sequencing (WES) to ASD families enriched for inherited causes due to consanguinity and find familial ASD associated with biallelic mutations in disease genes (AMT, PEX7, SYNE1, VPS13B, PAH, and POMGNT1). At least some of these genes show biallelic mutations in nonconsanguineous families as well. These mutations are often only partially disabling or present atypically, with patients lacking diagnostic features of the Mendelian disorders with which these genes are classically associated. Our study shows the utility of WES for identifying specific genetic conditions not clinically suspected and the importance of partial loss of gene function in ASDs.

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Although autism has a clear genetic component, the high genetic heterogeneity of the disorder has been a challenge for the identification of causative genes. We used homozygosity analysis to identify probands from nonconsanguineous families that showed evidence of distant shared ancestry, suggesting potentially recessive mutations. Whole-exome sequencing of 16 probands revealed validated homozygous, potentially pathogenic recessive mutations that segregated perfectly with disease in 4/16 families. The candidate genes (UBE3B, CLTCL1, NCKAP5L, ZNF18) encode proteins involved in proteolysis, GTPase-mediated signaling, cytoskeletal organization, and other pathways. Furthermore, neuronal depolarization regulated the transcription of these genes, suggesting potential activity-dependent roles in neurons. We present a multidimensional strategy for filtering whole-exome sequence data to find candidate recessive mutations in autism, which may have broader applicability to other complex, heterogeneous disorders.

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PURPOSE: Chromosomal microarray (CMA) testing provides the highest diagnostic yield for clinical testing of patients with developmental delay (DD), intellectual disability (ID), multiple congenital anomalies (MCA), and autism spectrum disorders (ASD). Despite improved diagnostic yield and studies to support cost-effectiveness, concerns regarding the cost and reimbursement for CMA have been raised because it is perceived that CMA results do not influence medical management. METHODS: We conducted a retrospective chart review of CMA testing performed during a 12-month period on patients with DD/ID, ASD, and congenital anomalies to determine the proportion of cases where abnormal CMA results impacted recommendations for clinical action. RESULTS: Among 1792 patients, 13.1% had clinically relevant results, either abnormal (n = 131; 7.3%) or variants of possible significance (VPS; n = 104; 5.8%). Abnormal variants generated a higher rate of recommendation for clinical action (54%) compared with VPS (34%; Fisher exact test, P = 0.01). CMA results influenced medical care by precipitating medical referrals, diagnostic imaging, or specific laboratory testing. CONCLUSIONS: For all test indications, CMA results influenced medical management in a majority of patients with abnormal variants and a substantial proportion of those with VPS. These results support the use of CMA as a clinical diagnostic test that influences medical management for this patient population.

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