Developmental excitation-inhibition imbalance underlying psychoses revealed by single-cell analyses of discordant twins-derived cerebral organoids.

Despite extensive genetic and neuroimaging studies, detailed cellular mechanisms underlying schizophrenia and bipolar disorder remain poorly understood. Recent progress in single-cell RNA sequencing (scRNA-seq) technologies enables identification of cell-type-specific pathophysiology. However, its application to psychiatric disorders is challenging because of methodological difficulties in analyzing human brains and the confounds due to a lifetime of illness. Brain organoids derived from induced pluripotent stem cells (iPSCs) of the patients are a powerful avenue to investigate the pathophysiological processes. Here, we generated iPSC-derived cerebral organoids from monozygotic twins discordant for psychosis. scRNA-seq analysis of the organoids revealed enhanced GABAergic specification and reduced cell proliferation following diminished Wnt signaling in the patient, which was confirmed in iPSC-derived forebrain neuronal cells. Two additional monozygotic twin pairs discordant for schizophrenia also confirmed the excess GABAergic specification of the patients' neural progenitor cells. With a well-controlled genetic background, our data suggest that unbalanced specification of excitatory and inhibitory neurons during cortical development underlies psychoses.

Tricornered/NDR kinase signaling mediates PINK1-directed mitochondrial quality control and tissue maintenance.

Eukaryotes employ elaborate mitochondrial quality control (MQC) to maintain the function of the power-generating organelle. Parkinson's disease-associated PINK1 and Parkin actively participate in MQC. However, the signaling events involved are largely unknown. Here we show that mechanistic target of rapamycin 2 (mTORC2) and Tricornered (Trc) kinases act downstream from PINK1 to regulate MQC. Trc is phosphorylated in mTORC2-dependent and mTORC2-independent manners and is specifically localized to mitochondria in response to PINK1, which regulates mTORC2 through mitochondrial complex-I activity. Genetically, mTORC2 and Trc act upstream of Parkin. Thus, multiplex kinase signaling is acting between PINK1 and Parkin to regulate MQC, a process highly conserved in mammals.

Parkinson's disease-associated kinase PINK1 regulates Miro protein level and axonal transport of mitochondria.

Mutations in Pten-induced kinase 1 (PINK1) are linked to early-onset familial Parkinson's disease (FPD). PINK1 has previously been implicated in mitochondrial fission/fusion dynamics, quality control, and electron transport chain function. However, it is not clear how these processes are interconnected and whether they are sufficient to explain all aspects of PINK1 pathogenesis. Here we show that PINK1 also controls mitochondrial motility. In Drosophila, downregulation of dMiro or other components of the mitochondrial transport machinery rescued dPINK1 mutant phenotypes in the muscle and dopaminergic (DA) neurons, whereas dMiro overexpression alone caused DA neuron loss. dMiro protein level was increased in dPINK1 mutant but decreased in dPINK1 or dParkin overexpression conditions. In Drosophila larval motor neurons, overexpression of dPINK1 inhibited axonal mitochondria transport in both anterograde and retrograde directions, whereas dPINK1 knockdown promoted anterograde transport. In HeLa cells, overexpressed hPINK1 worked together with hParkin, another FPD gene, to regulate the ubiquitination and degradation of hMiro1 and hMiro2, apparently in a Ser-156 phosphorylation-independent manner. Also in HeLa cells, loss of hMiro promoted the perinuclear clustering of mitochondria and facilitated autophagy of damaged mitochondria, effects previously associated with activation of the PINK1/Parkin pathway. These newly identified functions of PINK1/Parkin and Miro in mitochondrial transport and mitophagy contribute to our understanding of the complex interplays in mitochondrial quality control that are critically involved in PD pathogenesis, and they may explain the peripheral neuropathy symptoms seen in some PD patients carrying particular PINK1 or Parkin mutations. Moreover, the different effects of loss of PINK1 function on Miro protein level in Drosophila and mouse cells may offer one explanation of the distinct phenotypic manifestations of PINK1 mutants in these two species.

Parkin interacts with Klokin1 for mitochondrial import and maintenance of membrane potential.

Parkin is a multifunctional protein, including maintaining mitochondrial homeostasis. Recent evidence suggests that Parkin is recruited from the cytoplasm to damaged mitochondria with low membrane potential. We found that intracellular localization of Parkin changed with cellular growth phase. Parkin was preferentially localized in the mitochondria of cultured cells. The mitochondria with large amounts of Parkin showed preserved membrane potentials even during treatment with carbonyl cyanide m-chlorophenylhydrazone. Here we report a novel protein named Klokin 1 that transports Parkin to the mitochondria. Klokin 1 was localized to the mitochondria and enhanced mitochondrial expression of Parkin. Klokin 1 enhanced cell viability in Parkin-silenced cells. Klokin 1 expression was enhanced in the brains of Parkin-deficient mice but not in an autopsied PARK2 brain. Our findings indicate that mitochondrial Parkin prevents mitochondrial depolarization and that Klokin 1 may compensate for Parkin deficiency.

Human archetypal pluripotent stem cells differentiate into trophoblast stem cells via endogenous BMP5/7 induction without transitioning through naive state.

Primary human trophoblast stem cells (TSCs) and TSCs derived from human pluripotent stem cells (hPSCs) can potentially model placental processes in vitro. Yet, the pluripotent states and factors involved in the differentiation of hPSCs to TSCs remain poorly understood. In this study, we demonstrate that the primed pluripotent state can generate TSCs by activating pathways such as Epidermal Growth Factor (EGF) and Wingless-related integration site (WNT), and by suppressing tumor growth factor beta (TGFß), histone deacetylases (HDAC), and Rho-associated protein kinase (ROCK) signaling pathways, all without the addition of exogenous Bone morphogenetic protein 4 (BMP4)-a condition we refer to as the TS condition. We characterized this process using temporal single-cell RNA sequencing to compare TS conditions with differentiation protocols involving BMP4 activation alone or BMP4 activation in conjunction with WNT inhibition. The TS condition consistently produced a stable, proliferative cell type that closely mimics first-trimester placental cytotrophoblasts, marked by the activation of endogenous retroviral genes and the absence of amnion expression. This was observed across multiple cell lines, including various primed induced pluripotent stem cell (iPSC) and embryonic stem cell (ESC) lines. Primed-derived TSCs can proliferate for over 30 passages and further specify into multinucleated syncytiotrophoblasts and extravillous trophoblast cells. Our research establishes that the differentiation of primed hPSCs to TSC under TS conditions triggers the induction of TMSB4X, BMP5/7, GATA3, and TFAP2A without progressing through a naive state. These findings propose that the primed hPSC state is part of a continuum of potency with the capacity to differentiate into TSCs through multiple routes.

The loss of PGAM5 suppresses the mitochondrial degeneration caused by inactivation of PINK1 in Drosophila.

PTEN-induced kinase 1 (PINK1), which is required for mitochondrial homeostasis, is a gene product responsible for early-onset Parkinson's disease (PD). Another early onset PD gene product, Parkin, has been suggested to function downstream of the PINK1 signalling pathway based on genetic studies in Drosophila. PINK1 is a serine/threonine kinase with a predicted mitochondrial target sequence and a probable transmembrane domain at the N-terminus, while Parkin is a RING-finger protein with ubiquitin-ligase (E3) activity. However, how PINK1 and Parkin regulate mitochondrial activity is largely unknown. To explore the molecular mechanism underlying the interaction between PINK1 and Parkin, we biochemically purified PINK1-binding proteins from human cultured cells and screened the genes encoding these binding proteins using Drosophila PINK1 (dPINK1) models to isolate a molecule(s) involved in the PINK1 pathology. Here we report that a PINK1-binding mitochondrial protein, PGAM5, modulates the PINK1 pathway. Loss of Drosophila PGAM5 (dPGAM5) can suppress the muscle degeneration, motor defects, and shorter lifespan that result from dPINK1 inactivation and that can be attributed to mitochondrial degeneration. However, dPGAM5 inactivation fails to modulate the phenotypes of parkin mutant flies. Conversely, ectopic expression of dPGAM5 exacerbated the dPINK1 and Drosophila parkin (dParkin) phenotypes. These results suggest that PGAM5 negatively regulates the PINK1 pathway related to maintenance of the mitochondria and, furthermore, that PGAM5 acts between PINK1 and Parkin, or functions independently of Parkin downstream of PINK1.

Recapitulation of Perturbed Striatal Gene Expression Dynamics of Donors' Brains With Ventral Forebrain Organoids Derived From the Same Individuals With Schizophrenia.

OBJECTIVE: Schizophrenia is a brain disorder that originates during neurodevelopment and has complex genetic and environmental etiologies. Despite decades of clinical evidence of altered striatal function in affected patients, studies examining its cellular and molecular mechanisms in humans are limited. To explore neurodevelopmental alterations in the striatum associated with schizophrenia, the authors established a method for the differentiation of induced pluripotent stem cells (iPSCs) into ventral forebrain organoids (VFOs). METHODS: VFOs were generated from postmortem dural fibroblast-derived iPSCs of four individuals with schizophrenia and four neurotypical control individuals for whom postmortem caudate genotypes and transcriptomic data were profiled in the BrainSeq neurogenomics consortium. Individuals were selected such that the two groups had nonoverlapping schizophrenia polygenic risk scores (PRSs). RESULTS: Single-cell RNA sequencing analyses of VFOs revealed differences in developmental trajectory between schizophrenia and control individuals in which inhibitory neuronal cells from the patients exhibited accelerated maturation. Furthermore, upregulated genes in inhibitory neurons in schizophrenia VFOs showed a significant overlap with upregulated genes in postmortem caudate tissue of individuals with schizophrenia compared with control individuals, including the donors of the iPSC cohort. CONCLUSIONS: The findings suggest that striatal neurons derived from high-PRS individuals with schizophrenia carry abnormalities that originated during early brain development and that the VFO model can recapitulate disease-relevant cell type-specific neurodevelopmental phenotypes in a dish.

Variation in TAF1 expression in female carrier induced pluripotent stem cells and human brain ontogeny has implications for adult neostriatum vulnerability in X-linked Dystonia Parkinsonism.

X-linked Dystonia-Parkinsonism (XDP) is an inherited, X-linked, adult-onset movement disorder characterized by degeneration in the neostriatum. No therapeutics alter disease progression. The mechanisms underlying regional differences in degeneration and adult onset are unknown. Developing therapeutics requires a deeper understanding of how XDP-relevant features vary in health and disease. XDP is possibly due, in part, to a partial loss of TAF1 function. A disease-specific SINE-VNTR-Alu (SVA) retrotransposon insertion occurs within intron 32 of TAF1, a subunit of TFIID involved in transcription initiation. While all XDP males are usually clinically affected, females are heterozygous carriers generally not manifesting the full syndrome. As a resource for disease modeling, we characterized eight iPSC lines from three XDP female carrier individuals for X chromosome inactivation status and identified clonal lines that express either the wild-type X or XDP haplotype. Furthermore, we characterized XDP-relevant transcript expression in neurotypical humans, and found that SVA-F expression decreases after 30 years of age in the brain and that TAF1 is decreased in most female samples. Uniquely in the caudate nucleus, TAF1 expression is not sexually dimorphic and decreased after adolescence. These findings indicate that regional-, age- and sex-specific mechanisms regulate TAF1, highlighting the importance of disease-relevant models and postmortem tissue. We propose that the decreased TAF1 expression in the adult caudate may synergize with the XDP-specific partial loss of TAF1 function in patients, thereby passing a minimum threshold of TAF1 function, and triggering degeneration in the neostriatum.Significance StatementXDP is an inherited, X-linked, adult-onset movement disorder characterized by degeneration in the neostriatum. No therapeutics alter disease progression. Developing therapeutics requires a deeper understanding of how XDP-relevant features vary in health and disease. XDP is possibly due to a partial loss of TAF1 function. While all XDP males are usually affected, females are heterozygous carriers generally not manifesting the full syndrome. As a resource for disease modeling, we characterized eight stem cell lines from XDP female carrier individuals. Furthermore, we found that, uniquely in the caudate nucleus, TAF1 expression decreases after adolescence in healthy humans. We hypothesize that the decrease of TAF1 after adolescence in human caudate, in general, may underlie the vulnerability of the adult neostriatum in XDP.

A case of anti-BP230 antibody-positive bullous pemphigoid receiving DPP-4 inhibitor.

Bullous pemphigoid (BP) is a cutaneous autoimmune blistering disorder. Recently, it has been reported that dipeptidyl peptidase-4 inhibitors (DPP4i) is associated with the development of BP (DPP4i-BP). Patients with DPP4i-BP have autoantibodies (autoAbs) preferentially targeting full-length BP180, but not the BP180NC16a domain. In this report, we described a case of anti-BP230 antibody (Ab)-positive BP receiving DPP4i. A 78-year-old male with a medical history of type 2 diabetes receiving vildagliptin at 100 mg daily for 38 months was referred to our hospital with itching blisters on his body and extremities. Skin biopsy showed subepidermal bulla with eosinophil infiltration. Direct immunofluorescence staining revealed a linear staining pattern with complement C3 and IgG at the subepidermal basement membrane zone. By laboratory testing, autoAbs against BP180NC16a and full-length BP180 were negative by enzyme-linked immunosorbent assay (ELISA); however, anti-BP230 Abs were positive by ELISA (index: 123.91). His HLA genotype was DQB1*04:01 and 05:01. Based on these results, we diagnosed the patient with anti-BP230 Abs-positive BP associated with DPP4i. To the best of our knowledge, this is the first case of DPP4i-BP with only anti-BP230 Abs. Further accumulation of DPP4i-BP cases is needed to clarify the relationship between overall features of DPP4i-BP and anti-BP230 Abs.

Generation of four postmortem dura-derived iPS cell lines from four control individuals with genotypic and brain-region-specific transcriptomic data available through the BrainSEQ consortium.

In this study, we established induced pluripotent stem (iPS) cell lines from postmortem dura-derived fibroblasts of four control individuals with low polygenic risk score for psychiatric disorders including schizophrenia and bipolar disorder. The fibroblasts were reprogrammed into iPS cells using episomal vectors carrying OCT3/4, SOX2, KLF4, L-Myc, LIN28 and shRNA-p53. All iPS cell lines showed the same genotype with parental postmortem brain tissues, expressed pluripotency markers, and exhibited the differentiation potency into three embryonic germ layers.

Relation of the Glu298Asp polymorphism of the nitric oxide synthase gene to hypertension and serum cholesterol in Japanese workers.

OBJECTIVE: To assess whether the Glu298Asp polymorphism of the endothelial nitric oxide synthase (eNOS) gene possibly mediates the relation of blood pressure and serum cholesterol. METHOD: Regular health examination in 2003 of 1,694 Japanese workers from the Shimane Prefecture, Japan. RESULTS: The frequencies of the Glu/Glu, Glu/Asp, and Asp/Asp genotypes were 85.9%, 13.4%, and 0.7%, respectively. After adjustment for age, sex, BMI, and lifestyle (drinking, smoking, exercise and stress), the odds ratio (OR) of hypertension associated with high (> or = 220 mg/dl or under treatment) total cholesterol was 2.08 (95% Confidence Interval (CI) 1.02-4.24) among carriers of the eNOS 298Asp allele versus 1.18 (95% CI 0.89-1.55, p for interaction=0.50) among non-carriers. Similarly, the ORs of hypertension associated with counseling-need (120-139 mg/dl) and high (> or = 140 mg/dl) levels of LDL cholesterol among carriers of the eNOS 298Asp allele were significantly higher than those among non-carriers, at 2.65 (95% CI 1.16-6.01) versus 1.03 (95% CI 0.77-1.39, p for interaction=0.01), and 2.80 (95% CI 1.33-5.89) versus 0.95 (95% CI 0.71-1.26, p for interaction=0.04), respectively. CONCLUSION: These results indicate that the eNOS 298Asp allele, which is weakly associated with hypertension, may increase the risk of hypertension when associated with high serum lipid levels.

BAFF inhibition attenuates fibrosis in scleroderma by modulating the regulatory and effector B cell balance.

Systemic sclerosis (SSc) is an autoimmune disease characterized by skin and lung fibrosis. More than 90% of patients with SSc are positive for autoantibodies. In addition, serum B cell activating factor (BAFF) level is correlated with SSc severity and activity. Thus, B cells are considered to play a pathogenic role in SSc. However, there are two opposing subsets: regulatory B cells (Bregs) and effector B cells (Beffs). Interleukin-10 (IL-10)-producing Bregs negatively regulate the immune response, while IL-6-producing Beffs positively regulate it. Therefore, a protocol that selectively depletes Beffs would represent a potent therapy for SSc. The aims of this study were to investigate the roles of Bregs and Beffs in SSc and to provide a scientific basis for developing a new treatment strategy targeting B cells. A bleomycin-induced scleroderma model was induced in mice with a B cell-specific deficiency in IL-6 or IL-10. We also examined whether BAFF regulates cytokine-producing B cells and its effects on the scleroderma model. IL-6-producing Beffs increased in number and infiltrated the inflamed skin in the scleroderma model. The skin and lung fibrosis was attenuated in B cell-specific IL-6-deficient mice, whereas B cell-specific IL-10-deficient mice showed more severe fibrosis. In addition, BAFF increased Beffs but suppressed Bregs. Furthermore, BAFF antagonist attenuated skin and lung fibrosis in the scleroderma model with reduction of Beffs but not of Bregs. The current study indicates that Beffs play a pathogenic role in the scleroderma model, while Bregs play a protective role. BAFF inhibition is a potential therapeutic strategy for SSc via alteration of B cell balance.

S-nitrosylation regulates mitochondrial quality control via activation of parkin.

Parkin, a ubiquitin E3 ligase of the ring between ring fingers family, has been implicated in mitochondrial quality control. A series of recent reports have suggested that the recruitment of parkin is regulated by phosphorylation. However, the molecular mechanism that activates parkin to induce mitochondrial degradation is not well understood. Here, and in contrast to previous reports that S-nitrosylation of parkin is exclusively inhibitory, we identify a previously unrecognized site of S-nitrosylation in parkin (Cys323) that induces mitochondrial degradation. We demonstrate that endogenous S-nitrosylation of parkin is in fact responsible for activation of its E3 ligase activity to induce aggregation and degradation. We further demonstrate that mitochondrial uncoupling agents result in denitrosylation of parkin, and that prevention of denitrosylation restores mitochondrial degradation. Our data indicates that NO both positive effects on mitochondrial quality control, and suggest that targeted S-nitrosylation could provide a novel therapeutic strategy against Parkinson's disease.

The nitric oxide-cyclic GMP pathway regulates FoxO and alters dopaminergic neuron survival in Drosophila.

Activation of the forkhead box transcription factor FoxO is suggested to be involved in dopaminergic (DA) neurodegeneration in a Drosophila model of Parkinson's disease (PD), in which a PD gene product LRRK2 activates FoxO through phosphorylation. In the current study that combines Drosophila genetics and biochemical analysis, we show that cyclic guanosine monophosphate (cGMP)-dependent kinase II (cGKII) also phosphorylates FoxO at the same residue as LRRK2, and Drosophila orthologues of cGKII and LRRK2, DG2/For and dLRRK, respectively, enhance the neurotoxic activity of FoxO in an additive manner. Biochemical assays using mammalian cGKII and FoxO1 reveal that cGKII enhances the transcriptional activity of FoxO1 through phosphorylation of the FoxO1 S319 site in the same manner as LRRK2. A Drosophila FoxO mutant resistant to phosphorylation by DG2 and dLRRK (dFoxO S259A corresponding to human FoxO1 S319A) suppressed the neurotoxicity and improved motor dysfunction caused by co-expression of FoxO and DG2. Nitric oxide synthase (NOS) and soluble guanylyl cyclase (sGC) also increased FoxO's activity, whereas the administration of a NOS inhibitor L-NAME suppressed the loss of DA neurons in aged flies co-expressing FoxO and DG2. These results strongly suggest that the NO-FoxO axis contributes to DA neurodegeneration in LRRK2-linked PD.