Seth L. Shipman

Functional dependence of neuroligin on a new non-PDZ intracellular domain.

Neuroligins, a family of postsynaptic adhesion molecules, are important in synaptogenesis through a well-characterized trans-synaptic interaction with neurexin. In addition, neuroligins are thought to drive postsynaptic assembly through binding of their intracellular domain to PSD-95. However, there is little direct evidence to support the functional necessity of the neuroligin intracellular domain in postsynaptic development. We found that presence of endogenous neuroligin obscured the study of exogenous mutated neuroligin. We therefore used chained microRNAs in rat organotypic hippocampal slices to generate a reduced background of endogenous neuroligin. On this reduced background, we found that neuroligin function was critically dependent on the cytoplasmic tail. However, this function required neither the PDZ ligand nor any other previously described cytoplasmic binding domain, but rather required a previously unknown conserved region. Mutation of a single critical residue in this region inhibited neuroligin-mediated excitatory synaptic potentiation. Finally, we found a functional distinction between neuroligins 1 and 3.

Rapid neurogenesis through transcriptional activation in human stem cells

Advances in cellular reprogramming and stem cell differentiation now enable ex vivo studies of human neuronal differentiation. However, it remains challenging to elucidate the underlying regulatory programs because differentiation protocols are laborious and often result in low neuron yields. Here, we overexpressed two Neurogenin transcription factors in human‐induced pluripotent stem cells and obtained neurons with bipolar morphology in 4 days, at greater than 90% purity. The high purity enabled mRNA and microRNA expression profiling during neurogenesis, thus revealing the genetic programs involved in the rapid transition from stem cell to neuron. The resulting cells exhibited transcriptional, morphological and functional signatures of differentiated neurons, with greatest transcriptional similarity to prenatal human brain samples. Our analysis revealed a network of key transcription factors and microRNAs that promoted loss of pluripotency and rapid neurogenesis via progenitor states. Perturbations of key transcription factors affected homogeneity and phenotypic properties of the resulting neurons, suggesting that a systems‐level view of the molecular biology of differentiation may guide subsequent manipulation of human stem cells to rapidly obtain diverse neuronal types.

Factors affecting the hippocampal BOLD response during spatial memory.

The hippocampus has long been implicated in spatial memory, from work in rodents to imaging and brain lesion studies in humans. However, recent evidence has pointed to the recruitment of areas outside the hippocampus proper on spatial memory tasks, including the parahippocampal gyrus and precuneus, possibly suggesting a more focused role for the hippocampus proper. In this study, a virtual version of the standard rodent spatial memory assessment, the Morris water task, has been employed during fMRI to investigate the differential involvement of these distinct brain areas. Twenty-eight healthy participants completed a block designed version of the virtual Morris water task (vMWT) which consisted of three conditions: (1) a hippocampal dependent condition during which the participants were forced to use distal room cues in the virtual environment to navigate to a hidden platform; (2) a non-hippocampal dependent condition during which participants were to navigate to a visible platform; (3) a fixation period. Activations of the BOLD signal were evident in the hidden condition as compared to the visible condition in the parahippocampal gyrus, precuneus, and fusiform when analyzed using to a blocked analysis. Moreover, this blocked analysis revealed increases in the right hippocampal BOLD signal during fixation. However, when hidden trials were compared to visible trials using a post hoc event-related analysis focused on the beginning of each trial, activations of the right hippocampus are evident. These results support the theory that extra-hippocampal structures contribute to spatial memory behavior and identify a temporally specific involvement of the hippocampus. Furthermore, they substantiate previous results reporting hippocampal BOLD increases during fixation.

The cellular and molecular landscape of neuroligins

A fundamental physical interaction exists across the synapse. It is mediated by synaptic adhesion molecules, and is among the earliest and most indispensable of molecular events occurring during synaptogenesis. The regulation of adhesion molecules and their interactions with other synaptic proteins likely affect not only on synapse formation but also on ongoing synaptic function. We review research on one major family of postsynaptic adhesion molecules, neuroligins, which bind to their presynaptic partner neurexin across the synaptic cleft. We move from a structural overview to the broad cellular and synaptic context of neuroligins, intermolecular interactions, and molecular modifications that occur within a synapse. Finally, we examine evidence concerning the physiological functions of neuroligin in a cell and highlight areas requiring further investigation.

Molecular recordings by directed CRISPR spacer acquisition

INTRODUCTION Although recent advances in DNA synthesis and sequencing technologies have made practical the writing and readout of arbitrary data in the form of synthetic DNA, still lacking are the robust tools necessary to generate a dynamic record of such information within the genomes of living cells. An in vivo system, built out of biological parts with large storage capacity, would enable the recording of defined biological events into stable genetic memory and facilitate the tracking of long molecular and cellular histories. RATIONALE The CRISPR (clustered regularly interspaced short palindromic repeats)–Cas system is a prokaryotic type of immunological memory. Foreign DNA sequences originating from viral infections are stored within genome-based arrays in the form of short sequences—called spacers—that confer sequence-specific resistance to the invading nucleic acids. These arrays not only preserve the spacer sequences but also record the order in which the sequences are acquired, generating a temporal record of acquisition events. We harnessed this system to record arbitrary DNA sequences into a genomic CRISPR array in the form of spacers acquired from synthetic oligonucleotides electroporated into a population of cells overexpressing the CRISPR adaptation proteins Cas1 and Cas2. This enabled the recording of defined molecular events into a stable genomic locus over time and the storage of arbitrary information across a population of cells. RESULTS We show that the Cas1-Cas2 complex can be used in vivo to integrate synthetic DNA of a defined sequence into the Escherichia coli genome. We used this feature to examine the type I-E CRISPR-Cas spacer acquisition process and optimized the synthetic spacer design to achieve higher acquisition efficiency and specific integration orientation through the addition of an AAG protospacer adjacent motif (PAM). We then generated stable genomic recordings of multiple molecular events by electroporating sets of oligonucleotides over several days. These molecular records were read out with high-throughput sequencing and then decoded with a program that identified and faithfully reconstructed the temporal event order. Last, we used directed evolution to generate many Cas1-Cas2 mutants with modified PAM specificity (PAMNC). By modulating expression of these mutant and wild-type Cas1-Cas2 complexes, we could dynamically control the orientation of spacer integration. This enabled us to record acquisition events in multiple modes. That is, information was encoded in both the temporal order of the spacers and the orientation in which they were integrated. CONCLUSION Our results establish a recording system that uses the nucleotide content, temporal ordering, and orientation of defined DNA sequences within a CRISPR array in order to encode arbitrary information within the genomes of a population of cells. Because information can be encoded in spacer nucleotide space (up to two bits per base) and in alternate modes, the system has the potential to record and permanently store higher capacities of information than any other synthetic biological system to date. This lays the foundation for an in vivo recording device that could be coupled with diverse molecular phenomena and used for applications that require tracing of long molecular histories. We also demonstrate that delivery of synthetic DNA substrates to a CRISPR-Cas adaptation system in vivo is a practical method to probe and adapt the system. Two modes of encoding information into the CRISPR locus. (A) Oligonucleotides containing an AAG PAM and 32 variable bases were electroporated into cells overexpressing Cas1-Cas2 and inserted into the genomic CRISPR array. Delivery of oligos with distinct sequence over time generates a molecular record. (B) Cas1-Cas2 mutants identified through directed evolution alter the orientation of acquisition. Varying expression ratios of wild-type and mutant Cas1-Cas2 over time generates a record encoded in spacer orientation. The ability to write a stable record of identified molecular events into a specific genomic locus would enable the examination of long cellular histories and have many applications, ranging from developmental biology to synthetic devices. We show that the type I-E CRISPR (clustered regularly interspaced short palindromic repeats)–Cas system of Escherichia coli can mediate acquisition of defined pieces of synthetic DNA. We harnessed this feature to generate records of specific DNA sequences into a population of bacterial genomes. We then applied directed evolution so as to alter the recognition of a protospacer adjacent motif by the Cas1-Cas2 complex, which enabled recording in two modes simultaneously. We used this system to reveal aspects of spacer acquisition, fundamental to the CRISPR-Cas adaptation process. These results lay the foundations of a multimodal intracellular recording device.

CRISPR–Cas encoding of a digital movie into the genomes of a population of living bacteria

DNA is an excellent medium for archiving data. Recent efforts have illustrated the potential for information storage in DNA using synthesized oligonucleotides assembled in vitro. A relatively unexplored avenue of information storage in DNA is the ability to write information into the genome of a living cell by the addition of nucleotides over time. Using the Cas1–Cas2 integrase, the CRISPR–Cas microbial immune system stores the nucleotide content of invading viruses to confer adaptive immunity. When harnessed, this system has the potential to write arbitrary information into the genome. Here we use the CRISPR–Cas system to encode the pixel values of black and white images and a short movie into the genomes of a population of living bacteria. In doing so, we push the technical limits of this information storage system and optimize strategies to minimize those limitations. We also uncover underlying principles of the CRISPR–Cas adaptation system, including sequence determinants of spacer acquisition that are relevant for understanding both the basic biology of bacterial adaptation and its technological applications. This work demonstrates that this system can capture and stably store practical amounts of real data within the genomes of populations of living cells.

CaMKII phosphorylation of neuroligin-1 regulates excitatory synapses

Neuroligins are postsynaptic cell adhesion molecules that are important for synaptic function through their trans-synaptic interaction with neurexins (NRXNs). The localization and synaptic effects of neuroligin-1 (NL-1, also called NLGN1) are specific to excitatory synapses with the capacity to enhance excitatory synapses dependent on synaptic activity or Ca2+/calmodulin kinase II (CaMKII). Here we report that CaMKII robustly phosphorylates the intracellular domain of NL-1. We show that T739 is the dominant CaMKII site on NL-1 and is phosphorylated in response to synaptic activity in cultured rodent neurons and sensory experience in vivo. Furthermore, a phosphodeficient mutant (NL-1 T739A) reduces the basal and activity-driven surface expression of NL-1, leading to a reduction in neuroligin-mediated excitatory synaptic potentiation. To the best of our knowledge, our results are the first to demonstrate a direct functional interaction between CaMKII and NL-1, two primary components of excitatory synapses.

Dimerization of postsynaptic neuroligin drives synaptic assembly via transsynaptic clustering of neurexin

The transsynaptic complex of neuroligin (NLGN) and neurexin forms a physical connection between pre- and postsynaptic neurons that occurs early in the course of new synapse assembly. Both neuroligin and neurexin have, indeed, been proposed to exhibit active, instructive roles in the formation of synapses. However, the process by which these instructive roles play out during synaptogenesis is not well understood. Here, we examine one aspect of postsynaptic neuroligin with regard to its synaptogenic properties: its basal state as a constitutive dimer. We show that dimerization is required for the synaptogenic properties of neuroligin and likely serves to induce presynaptic differentiation via a transsynaptic clustering of neurexin. Further, we introduce chemically inducible, exogenous dimerization domains to the neuroligin molecule, effectively bestowing chemical control of neuroligin dimerization. This allows us to identify the acute requirements of neuroligin dimerization by chemically manipulating the monomeric-to-dimeric conversion of neuroligin. Based on the results of the inducible dimerization experiments, we propose a model in which dimerized neuroligin induces the mechanical clustering of presynaptic molecules as part of a requisite step in the coordinated assembly of a chemical synapse.

Distance-dependent scaling of AMPARs is cell-autonomous and GluA2 dependent.

The extensive dendritic arbor of a pyramidal cell introduces considerable complexity to the integration of synaptic potentials. Propagation of dendritic potentials is largely passive, in contrast to regenerative axonal potentials that are maintained by voltage-gated sodium channels, leading to a declination in amplitude as dendritic potentials travel toward the soma in a manner that disproportionally affects distal synaptic inputs. To counteract this amplitude filtering, Schaffer collateral synapses onto CA1 pyramidal cells contain a varying number of AMPA receptors (AMPARs) per synapse that increases with distance from the soma, a phenomenon known as distance-dependent scaling. Here, we undertake an investigation into the molecular mechanisms of distance-dependent scaling. Using dendritic recordings from rat pyramidal neurons, we confirm the basic scaling phenomenon and find that it is expressed and can be manipulated cell autonomously. Finally, we show that it depends on the presence of both a reserve pool of AMPARs and the AMPAR subunit GluA2.

Conneconomics: The Economics of Large-Scale Neural Connectomics

We analyze the scaling and cost-performance characteristics of current and projected connectomics approaches, with reference to the potential implications of recent advances in diverse contributing fields. This analysis suggests potential cost-effective strategies for dense connectivity mapping at the scale of whole mammalian brains.