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Tuberous sclerosis complex (TSC) is a multisystem developmental disorder caused by mutations in the TSC1 or TSC2 genes, whose protein products are negative regulators of mechanistic target of rapamycin complex 1 signaling. Hallmark pathologies of TSC are cortical tubers—regions of dysmorphic, disorganized neurons and glia in the cortex that are linked to epileptogenesis. To determine the developmental origin of tuber cells, we established human cellular models of TSC by CRISPR–Cas9-mediated gene editing of TSC1 or TSC2 in human pluripotent stem cells (hPSCs). Using heterozygous TSC2 hPSCs with a conditional mutation in the functional allele, we show that mosaic biallelic inactivation during neural progenitor expansion is necessary for the formation of dysplastic cells and increased glia production in three-dimensional cortical spheroids. Our findings provide support for the second-hit model of cortical tuber formation and suggest that variable developmental timing of somatic mutations could contribute to the heterogeneity in the neurological presentation of TSC. CRISPR–Cas9-mediated gene editing of TSC1 and TSC2 in human pluripotent stem cells is used to investigate the contribution of tuberous sclerosis complex–mechanistic target of rapamycin complex 1 signaling to human neural development in two-dimensional monolayer and three-dimensional spheroid models of the neurodevelopmental disorder tuberous sclerosis complex.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder defined by altered social interaction and communication, and repetitive, restricted, inflexible behaviors. Approximately 1.5-2% of the general population meet the diagnostic criteria for ASD and several brain regions including the cortex, amygdala, cerebellum and basal ganglia have been implicated in ASD pathophysiology. The midbrain dopamine system is an important modulator of cellular and synaptic function in multiple ASD-implicated brain regions via anatomically and functionally distinct dopaminergic projections. The dopamine hypothesis of ASD postulates that dysregulation of dopaminergic projection pathways could contribute to the behavioral manifestations of ASD, including altered reward value of social stimuli, changes in sensorimotor processing, and motor stereotypies. In this review, we examine the support for the idea that cell-autonomous changes in dopaminergic function are a core component of ASD pathophysiology. We discuss the human literature supporting the involvement of altered dopamine signaling in ASD including genetic, brain imaging and pharmacologic studies. We then focus on genetic mouse models of syndromic neurodevelopmental disorders in which single gene mutations lead to increased risk for ASD. We highlight studies that have directly examined dopamine neuron number, morphology, physiology, or output in these models. Overall, we find considerable support for the idea that the dopamine system may be dysregulated in syndromic ASDs; however, there does not appear to be a consistent signature and some models show increased dopaminergic function, while others have deficient dopamine signaling. We conclude that dopamine dysregulation is common in syndromic forms of ASD but that the specific changes may be unique to each genetic disorder and may not account for the full spectrum of ASD-related manifestations.

Tuberous Sclerosis Complex (TSC) is a neurodevelopmental disorder caused by mutations in the TSC1 or TSC2 genes, which encode proteins that negatively regulate mTOR complex 1 (mTORC1) signaling. Current treatment strategies focus on mTOR inhibition with rapamycin and its derivatives. While effective at improving some aspects of TSC, chronic rapamycin inhibits both mTORC1 and mTORC2 and is associated with systemic side-effects. It is currently unknown which mTOR complex is most relevant for TSC-related brain phenotypes. Here we used genetic strategies to selectively reduce neuronal mTORC1 or mTORC2 activity in mouse models of TSC. We find that reduction of the mTORC1 component Raptor, but not the mTORC2 component Rictor, rebalanced mTOR signaling in Tsc1 knock-out neurons. Raptor reduction was sufficient to improve several TSC-related phenotypes including neuronal hypertrophy, macrocephaly, impaired myelination, network hyperactivity, and premature mortality. Raptor downregulation represents a promising potential therapeutic intervention for the neurological manifestations of TSC. Karalis et al show that genetic reduction of the mTORC1 component Raptor improves multiple phenotypes in mouse models of TSC. Their findings suggest that Raptor could be a potential therapeutic target for treating the neurological aspects of TSC.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder with increasing prevalence. Over 1,000 risk genes have now been implicated in ASD, suggesting diverse etiology. However, the diagnostic criteria for the disorder still comprise two major behavioral domains - deficits in social communication and interaction, and the presence of restricted and repetitive patterns of behavior (RRBs). The RRBs associated with ASD include both stereotyped repetitive movements and other motor manifestations including changes in gait, balance, coordination, and motor skill learning. In recent years, the striatum, the primary input center of the basal ganglia, has been implicated in these ASD-associated motor behaviors, due to the striatum’s role in action selection, motor learning, and habit formation. Numerous mouse models with mutations in ASD risk genes have been developed and shown to have alterations in ASD-relevant behaviors. One commonly used assay, the accelerating rotarod, allows for assessment of both basic motor coordination and motor skill learning. In this corticostriatal-dependent task, mice walk on a rotating rod that gradually increases in speed. In the extended version of this task, mice engage striatal-dependent learning mechanisms to optimize their motor routine and stay on the rod for longer periods. This review summarizes the findings of studies examining rotarod performance across a range of ASD mouse models, and the resulting implications for the involvement of striatal circuits in ASD-related motor behaviors. While performance in this task is not uniform across mouse models, there is a cohort of models that show increased rotarod performance. A growing number of studies suggest that this increased propensity to learn a fixed motor routine may reflect a common enhancement of corticostriatal drive across a subset of mice with mutations in ASD-risk genes.

The Stimulator of Interferon Genes (STING) pathway initiates potent immune responses upon recognition of DNA. To initiate signaling, serine 365 (S365) in the C-terminal tail (CTT) of STING is phosphorylated, leading to induction of type I interferons (IFNs). Additionally, evolutionary conserved responses such as autophagy also occur downstream of STING, but their relative importance during in vivo infections remains unclear. Here we report that mice harboring a serine 365-to-alanine (S365A) mutation in STING are unexpectedly resistant to Herpes Simplex Virus (HSV)-1, despite lacking STING-induced type I IFN responses. By contrast, resistance to HSV-1 is abolished in mice lacking the STING CTT, suggesting that the STING CTT initiates protective responses against HSV-1, independently of type I IFNs. Interestingly, we find that STING-induced autophagy is a CTT- and TBK1-dependent but IRF3-independent process that is conserved in the STING S365A mice. Thus, interferon-independent functions of STING mediate STING-dependent antiviral responses in vivo. STING is considered central to antiviral immunity via induction of type 1 interferons, but can also activate autophagy. Here the authors made two mice with different STING mutations that differentially affect these antiviral responses, and surprisingly show that inhibiting STING-dependent type 1 interferon production does not affect susceptibility to HSV-1 infection.

The recent development of prime editing (PE) genome engineering technologies has the potential to significantly simplify the generation of human pluripotent stem cell (hPSC)-based disease models. PE is a multicomponent editing system that uses a Cas9-nickase fused to a reverse transcriptase (nCas9-RT) and an extended PE guide RNA (pegRNA). Once reverse transcribed, the pegRNA extension functions as a repair template to introduce precise designer mutations at the target site. Here, we systematically compared the editing efficiencies of PE to conventional gene editing methods in hPSCs. This analysis revealed that PE is overall more efficient and precise than homology-directed repair of site-specific nuclease-induced double-strand breaks. Specifically, PE is more effective in generating heterozygous editing events to create autosomal dominant disease-associated mutations. By stably integrating the nCas9-RT into hPSCs we achieved editing efficiencies equal to those reported for cancer cells, suggesting that the expression of the PE components, rather than cell-intrinsic features, limit PE in hPSCs. To improve the efficiency of PE in hPSCs, we optimized the delivery modalities for the PE components. Delivery of the nCas9-RT as mRNA combined with synthetically generated, chemically-modified pegRNAs and nicking guide RNAs improved editing efficiencies up to 13-fold compared with transfecting the PE components as plasmids or ribonucleoprotein particles. Finally, we demonstrated that this mRNA-based delivery approach can be used repeatedly to yield editing efficiencies exceeding 60% and to correct or introduce familial mutations causing Parkinson’s disease in hPSCs. From muscles to nerves, our body is formed of many kinds of cells which can each respond slightly differently to the same harmful genetic changes. Understanding the exact relationship between mutations and cell-type specific function is essential to better grasp how conditions such as Parkinson’s disease or amyotrophic lateral sclerosis progress and can be treated. Stem cells could be an important tool in that effort, as they can be directed to mature into many cell types in the laboratory. Yet it remains difficult to precisely introduce disease-relevant mutations in these cells. To remove this obstacle, Li et al. focused on prime editing, a cutting-edge ‘search and replace’ approach which can introduce new genetic information into a specific DNA sequence. However, it was unclear whether this technique could be used to efficiently create stem cell models of human diseases. A first set of experiments showed that prime editing is superior to conventional approaches when generating mutated genes in stem cells. Li et al. then further improved the efficiency and precision of the method by tweaking how prime editing components are delivered into the cells. The refined approach could be harnessed to quickly generate large numbers of stem cells carrying mutations associated with Parkinson’s disease; crucially, prime editing could then also be used to revert a mutated gene back to its healthy form. The improved prime editing approach developed by Li et al. removes a major hurdle for scientists hoping to use stem cells to study genetic diseases. This could potentially help to unlock progress in how we understand and ultimately treat these conditions.

Neuropeptides are the largest class of neuromodulators. It has been shown that subpopulations of ventral tegmental area (VTA) dopamine neurons express mRNA for the neuropeptide Gastrin-releasing peptide (GRP); however, its functional relevance in mesolimbic circuits is unknown. Here we report that the GRP receptor (GRPR) is expressed in nucleus accumbens medial shell (NAc MSh) neurons, which are targeted by GRP-expressing inputs from the VTA, hippocampus, and amygdala. We show that NAc MSh GRPR-positive cells represent subpopulations of D2 receptor-expressing neurons, comprising both classical striatal projection neurons (SPNs) and eccentric SPNs. GRPR-expressing neurons have high intrinsic excitability and can be activated by GRP in vivo. NAc-specific deletion of Grpr in mice increases motivation in a progressive ratio test, demonstrating that GRPR regulates motivated behaviors. These experiments establish GRP/GRPR signaling as a potent modulator of mesolimbic circuits and advance our understanding of the diversity of cell types present in the NAc. The nucleus accumbens (NAc) controls reward learning and motivated behaviors. Here, authors show that the neuropeptide gastrin-releasing peptide regulates the excitability of subpopulations of NAc neurons and modulates motivated behavior in mice.

The direct and indirect pathways of the basal ganglia have been proposed to oppositely regulate locomotion and differentially contribute to pathological behaviors. Analysis of the distinct contributions of each pathway to behavior has been a challenge, however, due to the difficulty of selectively investigating the neurons comprising the two pathways using conventional techniques. Here we present two mouse models in which the function of striatonigral or striatopallidal neurons is selectively disrupted due to cell type–specific deletion of the striatal signaling protein dopamine- and cAMP-regulated phosphoprotein Mr 32kDa (DARPP-32). Using these mice, we found that the loss of DARPP-32 in striatonigral neurons decreased basal and cocaine-induced locomotion and abolished dyskinetic behaviors in response to the Parkinson's disease drug L-DOPA. Conversely, the loss of DARPP-32 in striatopallidal neurons produced a robust increase in locomotor activity and a strongly reduced cataleptic response to the antipsychotic drug haloperidol. These findings provide insight into the selective contributions of the direct and indirect pathways to striatal motor behaviors.

Tuberous Sclerosis Complex (TSC) is a neurodevelopmental disorder caused by mutations in TSC1 or TSC2 , which encode proteins that negatively regulate mTOR complex 1 (mTORC1). TSC is associated with significant cognitive, psychiatric, and behavioral problems, collectively termed TSC-Associated Neuropsychiatric Disorders (TAND), and the cell types responsible for these manifestations are largely unknown. Here we use cell type-specific Tsc1 deletion to test whether dopamine neurons, which modulate cognitive, motivational, and affective behaviors, are involved in TAND. We show that loss of Tsc1 and constitutive activation of mTORC1 in dopamine neurons causes somatodendritic hypertrophy, reduces intrinsic excitability, alters axon terminal structure, and impairs striatal dopamine release. These perturbations lead to a selective deficit in cognitive flexibility, preventable by genetic reduction of the mTOR-binding protein Raptor. Our results establish a critical role for Tsc1-mTORC1 signaling in setting the functional properties of dopamine neurons, and indicate that dopaminergic dysfunction may contribute to cognitive inflexibility in TSC. Tuberous Sclerosis Complex (TSC) is a neurodevelopmental disorder caused by mutations in the TSC1 or TSC2 genes, which negatively regulate mTORC1 signalling. Here the authors selectively delete Tsc1 from dopamine neurons in mice and find impairments in striatal dopamine release that are sufficient to reduce cognitive flexibility.

Neurodevelopmental disorders (NDDs) are a collection of diseases with early life onset that often present with developmental delay, cognitive deficits, and behavioral conditions. In some cases, severe outcomes such as brain malformations and intractable epilepsy can occur. The mutations underlying NDDs may be inherited or de novo , can be gain- or loss-of-function, and can affect one or more genes. Recent evidence indicates that brain somatic mutations contribute to several NDDs, in particular malformations of cortical development. While advances in sequencing technologies have enabled the detection of these somatic mutations, the mechanisms by which they alter brain development and function are not well understood due to limited model systems that recapitulate these events. Human brain organoids have emerged as powerful models to study the early developmental events of the human brain. Brain organoids capture the developmental progression of the human brain and contain human-enriched progenitor cell types. Advances in human stem cell and genome engineering provide an opportunity to model NDD-associated somatic mutations in brain organoids. These organoids can be tracked throughout development to understand the impact of somatic mutations on early human brain development and function. In this review, we discuss recent evidence that somatic mutations occur in the developing human brain, that they can lead to NDDs, and discuss how they could be modeled using human brain organoids.