Efficient Derivation of Excitatory and Inhibitory Neurons from Human Pluripotent Stem Cells Stably Expressing Direct Reprogramming Factors.

It is essential to generate isolated populations of human neuronal subtypes in order to understand cell-type-specific roles in brain function and susceptibility to disease pathology. Here we describe a protocol for in-parallel generation of cortical glutamatergic (excitatory) and GABAergic (inhibitory) neurons from human pluripotent stem cells (hPSCs) by using the neurogenic transcription factors Ngn2 and a combination of Ascl1 and Dlx2, respectively. In contrast to the majority of neural transdifferentiation protocols that use transient lentiviral infection, the use of stable hPSC lines carrying doxycycline-inducible transcription factors allows neuronal differentiation to be initiated by addition of doxycycline and neural medium. This article presents a method to generate lentivirus from cultured mammalian cells and establish stable transcription factor-expressing cell lines (Basic Protocol 1), followed by a method for monolayer excitatory and inhibitory neuronal differentiation from the established lines (Basic Protocol 2). The resulting neurons reproducibly exhibit properties consistent with human cortical neurons, including the expected morphologies, expression of glutamatergic and GABAergic genes, and functional properties. Our approach enables the scalable and rapid production of human neurons suitable for modeling human brain diseases in a subtype-specific manner and examination of differential cellular vulnerability. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Lentivirus production and generation of stable hPSC lines Support Protocol 1: Expansion and maintenance of hPSCs Basic Protocol 2: Differentiation of EX- and IN-neurons Support Protocol 2: Experimental methods for validation of EX- and IN-neurons.

Intersection of diverse neuronal genomes and neuropsychiatric disease: The Brain Somatic Mosaicism Network.

Neuropsychiatric disorders have a complex genetic architecture. Human genetic population-based studies have identified numerous heritable sequence and structural genomic variants associated with susceptibility to neuropsychiatric disease. However, these germline variants do not fully account for disease risk. During brain development, progenitor cells undergo billions of cell divisions to generate the ~80 billion neurons in the brain. The failure to accurately repair DNA damage arising during replication, transcription, and cellular metabolism amid this dramatic cellular expansion can lead to somatic mutations. Somatic mutations that alter subsets of neuronal transcriptomes and proteomes can, in turn, affect cell proliferation and survival and lead to neurodevelopmental disorders. The long life span of individual neurons and the direct relationship between neural circuits and behavior suggest that somatic mutations in small populations of neurons can significantly affect individual neurodevelopment. The Brain Somatic Mosaicism Network has been founded to study somatic mosaicism both in neurotypical human brains and in the context of complex neuropsychiatric disorders.

Interaction between RING1 (R1) and the Ubiquitin-like (UBL) Domains Is Critical for the Regulation of Parkin Activity.

Parkin is an E3 ligase that contains a ubiquitin-like (UBL) domain in the N terminus and an R1-in-between-ring-RING2 motif in the C terminus. We showed that the UBL domain specifically interacts with the R1 domain and negatively regulates Parkin E3 ligase activity, Parkin-dependent mitophagy, and Parkin translocation to the mitochondria. The binding between the UBL domain and the R1 domain was suppressed by carbonyl cyanide m-chlorophenyl hydrazone treatment or by expression of PTEN-induced putative kinase 1 (PINK1), an upstream kinase that phosphorylates Parkin at the Ser-65 residue of the UBL domain. Moreover, we demonstrated that phosphorylation of the UBL domain at Ser-65 prevents its binding to the R1 domain and promotes Parkin activities. We further showed that mitochondrial translocation of Parkin, which depends on phosphorylation at Ser-65, and interaction between the R1 domain and a mitochondrial outer membrane protein, VDAC1, are suppressed by binding of the UBL domain to the R1 domain. Interestingly, Parkin with missense mutations associated with Parkinson disease (PD) in the UBL domain, such as K27N, R33Q, and A46P, did not translocate to the mitochondria and induce E3 ligase activity by m-chlorophenyl hydrazone treatment, which correlated with the interaction between the R1 domain and the UBL domain with those PD mutations. These findings provide a molecular mechanism of how Parkin recruitment to the mitochondria and Parkin activation as an E3 ubiquitin ligase are regulated by PINK1 and explain the previously unknown mechanism of how Parkin mutations in the UBL domain cause PD pathogenesis.

An AKT3-FOXG1-reelin network underlies defective migration in human focal malformations of cortical development.

Focal malformations of cortical development (FMCDs) account for the majority of drug-resistant pediatric epilepsy. Postzygotic somatic mutations activating the phosphatidylinositol-4,5-bisphosphate-3-kinase (PI3K)-protein kinase B (AKT)-mammalian target of rapamycin (mTOR) pathway are found in a wide range of brain diseases, including FMCDs. It remains unclear how a mutation in a small fraction of cells disrupts the architecture of the entire hemisphere. Within human FMCD-affected brain, we found that cells showing activation of the PI3K-AKT-mTOR pathway were enriched for the AKT3(E17K) mutation. Introducing the FMCD-causing mutation into mouse brain resulted in electrographic seizures and impaired hemispheric architecture. Mutation-expressing neural progenitors showed misexpression of reelin, which led to a non-cell autonomous migration defect in neighboring cells, due at least in part to derepression of reelin transcription in a manner dependent on the forkhead box (FOX) transcription factor FOXG1. Treatments aimed at either blocking downstream AKT signaling or inactivating reelin restored migration. These findings suggest a central AKT-FOXG1-reelin signaling pathway in FMCD and support pathway inhibitors as potential treatments or therapies for some forms of focal epilepsy.

Drosophila DJ-1 decreases neural sensitivity to stress by negatively regulating Daxx-like protein through dFOXO.

DJ-1, a Parkinson's disease (PD)-associated gene, has been shown to protect against oxidative stress in Drosophila. However, the molecular mechanism underlying oxidative stress-induced phenotypes, including apoptosis, locomotive defects, and lethality, in DJ-1-deficient flies is not fully understood. Here we showed that Daxx-like protein (DLP), a Drosophila homologue of the mammalian Death domain-associated protein (Daxx), was upregulated under oxidative stress conditions in the loss-of-function mutants of Drosophila DJ-1ß, a Drosophila homologue of DJ-1. DLP overexpression induced apoptosis via the c-Jun N-terminal kinase (JNK)/Drosophila forkhead box subgroup O (dFOXO) pathway, whereas loss of DLP increased resistance to oxidative stress and UV irradiation. Moreover, the oxidative stress-induced phenotypes of DJ-1ß mutants were dramatically rescued by DLP deficiency, suggesting that enhanced expression of DLP contributes to the DJ-1ß mutant phenotypes. Interestingly, we found that dFOXO was required for the increase in DLP expression in DJ-1ß mutants and that dFOXO activity was increased in the heads of DJ-1ß mutants. In addition, subcellular localization of DLP appeared to be influenced by DJ-1 expression so that cytosolic DLP was increased in DJ-1ß mutants. Similarly, in mammalian cells, Daxx translocation from the nucleus to the cytosol was suppressed by overexpressed DJ-1ß under oxidative stress conditions; and, furthermore, targeted expression of DJ-1ß to mitochondria efficiently inhibited the Daxx translocation. Taken together, our findings demonstrate that DJ-1ß protects flies against oxidative stress- and UV-induced apoptosis by regulating the subcellular localization and gene expression of DLP, thus implying that Daxx-induced apoptosis is involved in the pathogenesis of DJ-1-associated PD.

Characterization of PINK1 (PTEN-induced putative kinase 1) mutations associated with Parkinson disease in mammalian cells and Drosophila.

Mutations in PINK1 (PTEN-induced putative kinase 1) are tightly linked to autosomal recessive Parkinson disease (PD). Although more than 50 mutations in PINK1 have been discovered, the role of these mutations in PD pathogenesis remains poorly understood. Here, we characterized 17 representative PINK1 pathogenic mutations in both mammalian cells and Drosophila. These mutations did not affect the typical cleavage patterns and subcellular localization of PINK1 under both normal and damaged mitochondria conditions in mammalian cells. However, PINK1 mutations in the kinase domain failed to translocate Parkin to mitochondria and to induce mitochondrial aggregation. Consistent with the mammalian data, Drosophila PINK1 mutants with mutations in the kinase domain (G426D and L464P) did not genetically interact with Parkin. Furthermore, PINK1-null flies expressing the transgenic G426D mutant displayed defective phenotypes with increasing age, whereas L464P mutant-expressing flies exhibited the phenotypes at an earlier age. Collectively, these results strongly support the hypothesis that the kinase activity of PINK1 is essential for its function and for regulating downstream Parkin functions in mitochondria. We believe that this study provides the basis for understanding the molecular and physiological functions of various PINK1 mutations and provides insights into the pathogenic mechanisms of PINK1-linked PD.

PINK1 controls mitochondrial localization of Parkin through direct phosphorylation.

PTEN-induced putative kinase 1 (PINK1) and Parkin, encoded by their respective genes associated with Parkinson's disease (PD), are linked in a common pathway involved in the protection of mitochondrial integrity and function. However, the mechanism of their interaction at the biochemical level has not been investigated yet. Using both mammalian and Drosophila systems, we here demonstrate that the PINK1 kinase activity is required for its function in mitochondria. PINK1 regulates the localization of Parkin to the mitochondria in its kinase activity-dependent manner. In detail, Parkin phosphorylation by PINK1 on its linker region promotes its mitochondrial translocation, and the RING1 domain of Parkin is critical for this occurrence. These results demonstrate the biochemical relationship between PINK1, Parkin, and the mitochondria and thereby suggest the possible mechanism of PINK-Parkin-associated PD pathogenesis.

Mitochondrial dysfunction in Drosophila PINK1 mutants is complemented by parkin.

Autosomal recessive juvenile parkinsonism (AR-JP) is an early-onset form of Parkinson's disease characterized by motor disturbances and dopaminergic neurodegeneration. To address its underlying molecular pathogenesis, we generated and characterized loss-of-function mutants of Drosophila PTEN-induced putative kinase 1 (PINK1), a novel AR-JP-linked gene. Here, we show that PINK1 mutants exhibit indirect flight muscle and dopaminergic neuronal degeneration accompanied by locomotive defects. Furthermore, transmission electron microscopy analysis and a rescue experiment with Drosophila Bcl-2 demonstrated that mitochondrial dysfunction accounts for the degenerative changes in all phenotypes of PINK1 mutants. Notably, we also found that PINK1 mutants share marked phenotypic similarities with parkin mutants. Transgenic expression of Parkin markedly ameliorated all PINK1 loss-of-function phenotypes, but not vice versa, suggesting that Parkin functions downstream of PINK1. Taken together, our genetic evidence clearly establishes that Parkin and PINK1 act in a common pathway in maintaining mitochondrial integrity and function in both muscles and dopaminergic neurons.