A conserved molecular logic for neurogenesis to gliogenesis switch in the cerebral cortex.

During development, neural stem cells in the cerebral cortex, also known as radial glial cells (RGCs), generate excitatory neurons, followed by production of cortical macroglia and inhibitory neurons that migrate to the olfactory bulb (OB). Understanding the mechanisms for this lineage switch is fundamental for unraveling how proper numbers of diverse neuronal and glial cell types are controlled. We and others recently showed that Sonic Hedgehog (Shh) signaling promotes the cortical RGC lineage switch to generate cortical oligodendrocytes and OB interneurons. During this process, cortical RGCs generate intermediate progenitor cells that express critical gliogenesis genes Ascl1, Egfr, and Olig2. The increased Ascl1 expression and appearance of Egfr+ and Olig2+ cortical progenitors are concurrent with the switch from excitatory neurogenesis to gliogenesis and OB interneuron neurogenesis in the cortex. While Shh signaling promotes Olig2 expression in the developing spinal cord, the exact mechanism for this transcriptional regulation is not known. Furthermore, the transcriptional regulation of Olig2 and Egfr has not been explored. Here, we show that in cortical progenitor cells, multiple regulatory programs, including Pax6 and Gli3, prevent precocious expression of Olig2, a gene essential for production of cortical oligodendrocytes and astrocytes. We identify multiple enhancers that control Olig2 expression in cortical progenitors and show that the mechanisms for regulating Olig2 expression are conserved between the mouse and human. Our study reveals evolutionarily conserved regulatory logic controlling the lineage switch of cortical neural stem cells.

Sortilin inhibition treats multiple neurodegenerative lysosomal storage disorders.

Lysosomal storage disorders (LSDs) are a genetically and clinically diverse group of diseases characterized by lysosomal dysfunction. Batten disease is a family of severe LSDs primarily impacting the central nervous system. Here we show that AF38469, a small molecule inhibitor of sortilin, improves lysosomal and glial pathology across multiple LSD models. Live-cell imaging and comparative transcriptomics demonstrates that the transcription factor EB (TFEB), an upstream regulator of lysosomal biogenesis, is activated upon treatment with AF38469. Utilizing CLN2 and CLN3 Batten disease mouse models, we performed a short-term efficacy study and show that treatment with AF38469 prevents the accumulation of lysosomal storage material and the development of neuroinflammation, key disease associated pathologies. Tremor phenotypes, an early behavioral phenotype in the CLN2 disease model, were also completely rescued. These findings reveal sortilin inhibition as a novel and highly efficacious therapeutic modality for the treatment of multiple forms of Batten disease.

Early postnatal administration of an AAV9 gene therapy is safe and efficacious in CLN3 disease.

CLN3 disease, caused by biallelic mutations in the CLN3 gene, is a rare pediatric neurodegenerative disease that has no cure or disease modifying treatment. The development of effective treatments has been hindered by a lack of etiological knowledge, but gene replacement has emerged as a promising therapeutic platform for such disorders. Here, we utilize a mouse model of CLN3 disease to test the safety and efficacy of a cerebrospinal fluid-delivered AAV9 gene therapy with a study design optimized for translatability. In this model, postnatal day one administration of the gene therapy virus resulted in robust expression of human CLN3 throughout the CNS over the 24-month duration of the study. A range of histopathological and behavioral parameters were assayed, with the therapy consistently and persistently rescuing a number of hallmarks of disease while being safe and well-tolerated. Together, the results show great promise for translation of the therapy into the clinic, prompting the launch of a first-in-human clinical trial (NCT03770572).

Breasi-CRISPR: an efficient genome-editing method to interrogate protein localization and protein-protein interactions in the embryonic mouse cortex.

In developing tissues, knowing the localization and interactors of proteins of interest is key to understanding their function. Here, we describe the Breasi-CRISPR approach (Brain Easi-CRISPR), combining Easi-CRISPR with in utero electroporation to tag endogenous proteins within embryonic mouse brains. Breasi-CRISPR enables knock-in of both short and long epitope tag sequences with high efficiency. We visualized epitope-tagged proteins with varied expression levels, such as ACTB, LMNB1, EMD, FMRP, NOTCH1 and RPL22. Detection was possible by immunohistochemistry as soon as 1 day after electroporation and we observed efficient gene editing in up to 50% of electroporated cells. Moreover, tagged proteins could be detected by immunoblotting in lysates from individual cortices. Next, we demonstrated that Breasi-CRISPR enables the tagging of proteins with fluorophores, allowing visualization of endogenous proteins by live imaging in organotypic brain slices. Finally, we used Breasi-CRISPR to perform co-immunoprecipitation mass-spectrometry analyses of the autism-related protein FMRP to discover its interactome in the embryonic cortex. Together, these data demonstrate that Breasi-CRISPR is a powerful tool with diverse applications that will propel the understanding of protein function in neurodevelopment.

Transmembrane Batten Disease Proteins Interact With a Shared Network of Vesicle Sorting Proteins, Impacting Their Synaptic Enrichment.

Batten disease is unique among lysosomal storage disorders for the early and profound manifestation in the central nervous system, but little is known regarding potential neuron-specific roles for the disease-associated proteins. We demonstrate substantial overlap in the protein interactomes of three transmembrane Batten proteins (CLN3, CLN6, and CLN8), and that their absence leads to synaptic depletion of key partners (i.e., SNAREs and tethers) and altered synaptic SNARE complexing in vivo, demonstrating a novel shared etiology.

Orexin 1 Receptor Antagonism in the Basolateral Amygdala Shifts the Balance From Pro- to Antistress Signaling and Behavior.

BACKGROUND: Stress produces differential behavioral responses through select molecular modifications to specific neurocircuitry elements. The orexin (Orx) system targets key components of this neurocircuitry in the basolateral amygdala (BLA). METHODS: We assessed the contribution of intra-BLA Orx1 receptors (Orx1Rs) in the expression of stress-induced phenotypes of mice. Using the Stress Alternatives Model, a social stress paradigm that produces two behavioral phenotypes, we characterized the role of intra-BLA Orx1R using acute pharmacological inhibition (SB-674042) and genetic knockdown (AAV-U6-Orx1R-shRNA) strategies. RESULTS: In the BLA, we observed that Orx1R (Hcrtr1) messenger RNA is predominantly expressed in CamKIIα+ glutamatergic neurons and rarely in GABAergic (gamma-aminobutyric acidergic) cells. While there is a slight overlap in Hcrtr1 and Orx2 receptor (Hcrtr2) messenger RNA expression in the BLA, we find that these receptors are most often expressed in separate cells. Antagonism of intra-BLA Orx1R after phenotype formation shifted behavioral expression from stress-sensitive (Stay) to stress-resilient (Escape) responses, an effect that was mimicked by genetic knockdown. Acute inhibition of Orx1R in the BLA also reduced contextual and cued fear freezing responses in Stay animals. This phenotype-specific behavioral change was accompanied by biased molecular transcription favoring Hcrtr2 over Hcrtr1 and Mapk3 over Plcb1 cell signaling cascades and enhanced Bdnf messenger RNA. CONCLUSIONS: Functional reorganization of intra-BLA gene expression is produced by antagonism of Orx1R, which promotes elevated Hcrtr2, greater Mapk3, and increased Bdnf expression. Together, these results provide evidence for a receptor-driven mechanism that balances pro- and antistress responses within the BLA.

AAV9 Gene Therapy Increases Lifespan and Treats Pathological and Behavioral Abnormalities in a Mouse Model of CLN8-Batten Disease.

CLN8 disease is a rare form of neuronal ceroid lipofuscinosis caused by biallelic mutations in the CLN8 gene, which encodes a transmembrane endoplasmic reticulum protein involved in trafficking of lysosomal enzymes. CLN8 disease patients present with myoclonus, tonic-clonic seizures, and progressive declines in cognitive and motor function, with many cases resulting in premature death early in life. There are currently no treatments that can cure the disease or substantially slow disease progression. Using a mouse model of CLN8 disease, we tested the safety and efficacy of an intracerebroventricularly (i.c.v.) delivered self-complementary adeno-associated virus serotype 9 (scAAV9) gene therapy vector driving expression of human CLN8. A single neonatal injection was safe and well tolerated, resulting in robust transgene expression throughout the CNS from 4 to 24 months, reducing histopathological and behavioral hallmarks of the disease and restoring lifespan from 10 months in untreated animals to beyond 24 months of age in treated animals. While it is unclear whether some of these behavioral improvements relate to preserved visual function, improvements in learning/memory, or other central or peripheral benefits, these results demonstrate, by far, the most successful degree of rescue reported in an animal model of CLN8 disease, and they support further development of gene therapy for this disorder.

Ariadne's Thread in the Developing Cerebral Cortex: Mechanisms Enabling the Guiding Role of the Radial Glia Basal Process during Neuron Migration.

Radial neuron migration in the developing cerebral cortex is a complex journey, starting in the germinal zones and ending in the cortical plate. In mice, migratory distances can reach several hundreds of microns, or millimeters in humans. Along the migratory path, radially migrating neurons slither through cellularly dense and complex territories before they reach their final destination in the cortical plate. This task is facilitated by radial glia, the neural stem cells of the developing cortex. Indeed, radial glia have a unique bipolar morphology, enabling them to serve as guides for neuronal migration. The key guiding structure of radial glia is the basal process, which traverses the entire thickness of the developing cortex. Neurons recognize the basal process as their guide and maintain physical interactions with this structure until the end of migration. Thus, the radial glia basal process plays a key role during radial migration. In this review, we highlight the pathways enabling neuron-basal process interactions during migration, as well as the known mechanisms regulating the morphology of the radial glia basal process. Throughout, we describe how dysregulation of these interactions and of basal process morphology can have profound effects on cortical development, and therefore lead to neurodevelopmental diseases.

Learning and CRF-Induced Indecision during Escape and Submission in Rainbow Trout during Socially Aggressive Interactions in the Stress-Alternatives Model.

Socially stressful environments induce a phenotypic dichotomy of coping measures for populations in response to a dominant aggressor and given a route of egress. This submission- (Stay) or escape-oriented (Escape) dichotomy represents individual decision-making under the stressful influence of hostile social environments. We utilized the Stress-Alternatives Model (SAM) to explore behavioral factors which might predict behavioral phenotype in rainbow trout. The SAM is a compartmentalized tank, with smaller and larger trout separated by an opaque divider until social interaction, and another divider occluding a safety zone, accessible by way of an escape route only large enough for the smaller fish. We hypothesized that distinctive behavioral responses during the first social interaction would indicate a predisposition for one of the behavioral phenotypes in the subsequent interactions. Surprisingly, increased amount or intensity of aggression received had no significant effect on promoting escape in test fish. In fact, during the first day of interaction, fish that turned toward their larger opponent during attack eventually learned to escape. Escaping fish also learn to monitor the patrolling behavior of aggressors, and eventually escape primarily when they are not being observed. Escape per se, was also predicted in trout exhibiting increased movements directed toward the escape route. By contrast, fish that consistently remained in the tank with the aggressor (Stay) showed significantly higher frequency of swimming in subordinate positions, at the top or the bottom of the water column, as well as sitting at the bottom. In addition, a corticotropin-releasing factor (CRF)-induced behavior, snap-shake, was also displayed in untreated fish during aggressive social interaction, and blocked by a CRF1 receptor antagonist. Especially prevalent among the Stay phenotype, snap-shake indicates indecision regarding escape-related behaviors. Snap-shake was also exhibited by fish of the Escape phenotype, showing a positive correlation with latency to escape. These results demonstrate adaptive responses to stress that reflect evolutionarily conserved stress neurocircuitry which may translate to psychological disorders and decision-making across vertebrate taxa.