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Memories of fearful events can last a lifetime. The prelimbic (PL) cortex, a subregion of prefrontal cortex, plays a critical role in fear memory retrieval over time. Most studies have focused on acquisition, consolidation, and retrieval of recent memories, but much less is known about the neural mechanisms of remote memory. Using a new knock-in mouse for activity-dependent genetic labeling (TRAP2), we demonstrate that neuronal ensembles in the PL cortex are dynamic. PL neurons TRAPed during later memory retrievals are more likely to be reactivated and make larger behavioral contributions to remote memory retrieval compared to those TRAPed during learning or early memory retrieval. PL activity during learning is required to initiate this time-dependent reorganization in PL ensembles underlying memory retrieval. Finally, while neurons TRAPed during earlier and later retrievals have similar broad projections throughout the brain, PL neurons TRAPed later have a stronger functional recruitment of cortical targets.DeNardo et al. characterize TRAP2, which allows genetic access to neurons based on their activity, and use it to show that neuronal ensembles in prelimbic cortex for remote fear memory undergo dynamic changes during the first 14 days after learning.

A CRISPR/Cas9 genome editing framework has been developed that allows controlled introduction of mono- and bi-allelic sequence changes, and is used to generate induced human pluripotent stem cells with heterozygous and homozygous dominant mutations in amyloid precursor protein and presenilin 1 that have been associated with early onset Alzheimer’s disease. CRISPR/Cas9 editing for stem cells Marc Tessier-Lavigne and colleagues have developed a CRISPR/Cas9-based genome-editing method that allows selective introduction of mono- and bi-allelic sequence changes with high efficiency and accuracy. The authors demonstrate the application of these methods in the generation of human induced pluripotent stem cells (iPS cells) with heterozygous and homozygous dominant mutations in amyloid precursor protein and presenilin that have been associated with early onset Alzheimer's disease. The bacterial CRISPR/Cas9 system allows sequence-specific gene editing in many organisms and holds promise as a tool to generate models of human diseases, for example, in human pluripotent stem cells 1 , 2 . CRISPR/Cas9 introduces targeted double-stranded breaks (DSBs) with high efficiency, which are typically repaired by non-homologous end-joining (NHEJ) resulting in nonspecific insertions, deletions or other mutations (indels) 2 . DSBs may also be repaired by homology-directed repair (HDR) 1 , 2 using a DNA repair template, such as an introduced single-stranded oligo DNA nucleotide (ssODN), allowing knock-in of specific mutations 3 . Although CRISPR/Cas9 is used extensively to engineer gene knockouts through NHEJ, editing by HDR remains inefficient 3 , 4 , 5 , 6 , 7 , 8 and can be corrupted by additional indels 9 , preventing its widespread use for modelling genetic disorders through introducing disease-associated mutations. Furthermore, targeted mutational knock-in at single alleles to model diseases caused by heterozygous mutations has not been reported. Here we describe a CRISPR/Cas9-based genome-editing framework that allows selective introduction of mono- and bi-allelic sequence changes with high efficiency and accuracy. We show that HDR accuracy is increased dramatically by incorporating silent CRISPR/Cas-blocking mutations along with pathogenic mutations, and establish a method termed ‘CORRECT’ for scarless genome editing. By characterizing and exploiting a stereotyped inverse relationship between a mutation’s incorporation rate and its distance to the DSB, we achieve predictable control of zygosity. Homozygous introduction requires a guide RNA targeting close to the intended mutation, whereas heterozygous introduction can be accomplished by distance-dependent suboptimal mutation incorporation or by use of mixed repair templates. Using this approach, we generated human induced pluripotent stem cells with heterozygous and homozygous dominant early onset Alzheimer’s disease-causing mutations in amyloid precursor protein (APP Swe ) 10 and presenilin 1 (PSEN1 M146V ) 11 and derived cortical neurons, which displayed genotype-dependent disease-associated phenotypes. Our findings enable efficient introduction of specific sequence changes with CRISPR/Cas9, facilitating study of human disease.

Blood vessels and nerve fibres course throughout the body in an orderly pattern, often alongside one another. Although superficially distinct, the mechanisms involved in wiring neural and vascular networks seem to share some deep similarities. The discovery of key axon guidance molecules over the past decade has shown that axons are guided to their targets by finely tuned codes of attractive and repulsive cues, and recent studies reveal that these cues also help blood vessels to navigate to their targets. Parallels have also emerged between the actions of growth factors that direct angiogenic sprouting and those that regulate axon terminal arborization. Fellow travellers The past decade has seen rapid advances in the study of the axon guidance mechanisms that shape the nervous system. Now it is emerging that blood vessels, though quite distinct from nerve fibres, use a similar system of molecular signals to guide them to target organs. By the time animals developed a vascular system, nerves had been around for millions of years so in this instance, evolution has adapted an old template for a new purpose.

Hexanucleotide-repeat expansions in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). The nucleotide-repeat expansions are translated into dipeptide-repeat (DPR) proteins, which are aggregation prone and may contribute to neurodegeneration. We used the CRISPR–Cas9 system to perform genome-wide gene-knockout screens for suppressors and enhancers of C9ORF72 DPR toxicity in human cells. We validated hits by performing secondary CRISPR–Cas9 screens in primary mouse neurons. We uncovered potent modifiers of DPR toxicity whose gene products function in nucleocytoplasmic transport, the endoplasmic reticulum (ER), proteasome, RNA-processing pathways, and chromatin modification. One modifier, TMX2 , modulated the ER-stress signature elicited by C9ORF72 DPRs in neurons and improved survival of human induced motor neurons from patients with C9ORF72 ALS. Together, our results demonstrate the promise of CRISPR–Cas9 screens in defining mechanisms of neurodegenerative diseases. A genome-wide CRISPR screen for suppressors and enhancers of C9ORF72 dipeptide-repeat protein toxicity identifies candidate genes involved in nucleocytoplasmic transport and other pathways including RNA processing and chromatin modification.

Cortical neurons are rapidly derived from human pluripotent stem cells using a cocktail of small molecules. Considerable progress has been made in converting human pluripotent stem cells (hPSCs) into functional neurons. However, the protracted timing of human neuron specification and functional maturation remains a key challenge that hampers the routine application of hPSC-derived lineages in disease modeling and regenerative medicine. Using a combinatorial small-molecule screen, we previously identified conditions to rapidly differentiate hPSCs into peripheral sensory neurons. Here we generalize the approach to central nervous system (CNS) fates by developing a small-molecule approach for accelerated induction of early-born cortical neurons. Combinatorial application of six pathway inhibitors induces post-mitotic cortical neurons with functional electrophysiological properties by day 16 of differentiation, in the absence of glial cell co-culture. The resulting neurons, transplanted at 8 d of differentiation into the postnatal mouse cortex, are functional and establish long-distance projections, as shown using iDISCO whole-brain imaging. Accelerated differentiation into cortical neuron fates should facilitate hPSC-based strategies for disease modeling and cell therapy in CNS disorders.

Herpes simplex virus-1 (HSV-1) encephalitis (HSE) is typically sporadic. Inborn errors of TLR3- and DBR1-mediated central nervous system cell-intrinsic immunity can account for forebrain and brainstem HSE, respectively. We report five unrelated patients with forebrain HSE, each heterozygous for one of four rare variants of SNORA31, encoding a small nucleolar RNA of the H/ACA class that are predicted to direct the isomerization of uridine residues to pseudouridine in small nuclear RNA and ribosomal RNA. We show that CRISPR/Cas9-introduced bi- and monoallelic SNORA31 deletions render human pluripotent stem cell (hPSC)-derived cortical neurons susceptible to HSV-1. Accordingly, SNORA31-mutated patient hPSC-derived cortical neurons are susceptible to HSV-1, like those from TLR3- or STAT1-deficient patients. Exogenous interferon (IFN)-β renders SNORA31- and TLR3- but not STAT1-mutated neurons resistant to HSV-1. Finally, transcriptome analysis of SNORA31-mutated neurons revealed normal responses to TLR3 and IFN-α/β stimulation but abnormal responses to HSV-1. Human SNORA31 thus controls central nervous system neuron-intrinsic immunity to HSV-1 by a distinctive mechanism.

Axonal and synaptic degeneration is a hallmark of peripheral neuropathy, brain injury, and neurodegenerative disease. Axonal degeneration has been proposed to be mediated by an active autodestruction program, akin to apoptotic cell death; however, loss-of-function mutations capable of potently blocking axon self-destruction have not been described. Here, we show that loss of the Drosophila Toll receptor adaptor dSarm (sterile α/Armadillo/Toll-Interleukin receptor homology domain protein) cell-autonomously suppresses Wallerian degeneration for weeks after axotomy. Severed mouse Sarm1 null axons exhibit remarkable long-term survival both in vivo and in vitro, indicating that Sarm1 prodegenerative signaling is conserved in mammals. Our results provide direct evidence that axons actively promote their own destruction after injury and identify dSarm/Sarm1 as a member of an ancient axon death signaling pathway.

A major barrier to regenerating axons after injury in the mammalian central nervous system is an unfavorable milieu. Three proteins found in myelin--Nogo, MAG, and OMgp--inhibit axon regeneration in vitro and bind to the glycosylphosphatidylinositol-anchored Nogo receptor (NgR). However, genetic deletion of NgR has only a modest disinhibitory effect, suggesting that other binding receptors for these molecules probably exist. With the use of expression cloning, we have found that paired immunoglobulin-like receptor B (PirB), which has been implicated in nervous system plasticity, is a high-affinity receptor for Nogo, MAG, and OMgp. Interfering with PirB activity, either with antibodies or genetically, partially rescues neurite inhibition by Nogo66, MAG, OMgp, and myelin in cultured neurons. Blocking both PirB and NgR activities leads to near-complete release from myelin inhibition. Our results implicate PirB in mediating regeneration block, identify PirB as a potential target for axon regeneration therapies, and provide an explanation for the similar enhancements of visual system plasticity in PirB and NgR knockout mice.

Actin, spectrin, and associated molecules form a periodic, submembrane cytoskeleton in the axons of neurons. For a better understanding of this membrane-associated periodic skeleton (MPS), it is important to address how prevalent this structure is in different neuronal types, different subcellular compartments, and across different animal species. Here, we investigated the organization of spectrin in a variety of neuronal- and glial-cell types. We observed the presence of MPS in all of the tested neuronal types cultured from mouse central and peripheral nervous systems, including excitatory and inhibitory neurons from several brain regions, as well as sensory and motor neurons. Quantitative analyses show that MPS is preferentially formed in axons in all neuronal types tested here: Spectrin shows a long-range, periodic distribution throughout all axons but appears periodic only in a small fraction of dendrites, typically in the form of isolated patches in subregions of these dendrites. As in dendrites, we also observed patches of periodic spectrin structures in a small fraction of glial-cell processes in four types of glial cells cultured from rodent tissues. Interestingly, despite its strong presence in the axonal shaft, MPS is disrupted in most presynaptic boutons but is present in an appreciable fraction of dendritic spine necks, including some projecting from dendrites where such a periodic structure is not observed in the shaft. Finally, we found that spectrin is capable of adopting a similar periodic organization in neurons of a variety of animal species, including Caenorhabditis elegans, Drosophila, Gallus gallus, Mus musculus, and Homo sapiens.

Axon pathfinding is orchestrated by numerous guidance cues, including Slits and their Robo receptors, but it remains unclear how information from multiple cues is integrated or filtered. Robo3, a Robo family member, allows commissural axons to reach and cross the spinal cord midline by antagonizing Robo1/2–mediated repulsion from midline-expressed Slits and potentiating deleted in colorectal cancer (DCC)–mediated midline attraction to Netrin-1, but without binding either Slits or Netrins. We identified a secreted Robo3 ligand, neural epidermal growth factor-like-like 2 (NELL2), which repels mouse commissural axons through Robo3 and helps steer them to the midline. These findings identify NELL2 as an axon guidance cue and establish Robo3 as a multifunctional regulator of pathfinding that simultaneously mediates NELL2 repulsion, inhibits Slit repulsion, and facilitates Netrin attraction to achieve a common guidance purpose.