Interspecies chimerism with human embryonic stem cells generates functional human dopamine neurons at low efficiency.

Interspecies chimeras offer great potential for regenerative medicine and the creation of human disease models. Whether human pluripotent stem cell-derived neurons in an interspecies chimera can differentiate into functional neurons and integrate into host neural circuity is not known. Here, we show, using Engrailed 1 (En1) as a development niche, that human naive-like embryonic stem cells (ESCs) can incorporate into embryonic and adult mouse brains. Human-derived neurons including tyrosine hydroxylase (TH)+ neurons integrate into the mouse brain at low efficiency. These TH+ neurons have electrophysiologic properties consistent with their human origin. In addition, these human-derived neurons in the mouse brain accumulate pathologic phosphorylated α-synuclein in response to α-synuclein preformed fibrils. Optimization of human/mouse chimeras could be used to study human neuronal differentiation and human brain disorders.

Enhanced mTORC1 signaling and protein synthesis in pathologic α-synuclein cellular and animal models of Parkinson's disease.

Pathologic α-synuclein plays an important role in the pathogenesis of α-synucleinopathies such as Parkinson's disease (PD). Disruption of proteostasis is thought to be central to pathologic α-synuclein toxicity; however, the molecular mechanism of this deregulation is poorly understood. Complementary proteomic approaches in cellular and animal models of PD were used to identify and characterize the pathologic α-synuclein interactome. We report that the highest biological processes that interacted with pathologic α-synuclein in mice included RNA processing and translation initiation. Regulation of catabolic processes that include autophagy were also identified. Pathologic α-synuclein was found to bind with the tuberous sclerosis protein 2 (TSC2) and to trigger the activation of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1), which augmented mRNA translation and protein synthesis, leading to neurodegeneration. Genetic and pharmacologic inhibition of mTOR and protein synthesis rescued the dopamine neuron loss, behavioral deficits, and aberrant biochemical signaling in the α-synuclein preformed fibril mouse model and Drosophila transgenic models of pathologic α-synuclein-induced degeneration. Pathologic α-synuclein furthermore led to a destabilization of the TSC1-TSC2 complex, which plays an important role in mTORC1 activity. Constitutive overexpression of TSC2 rescued motor deficits and neuropathology in α-synuclein flies. Biochemical examination of PD postmortem brain tissues also suggested deregulated mTORC1 signaling. These findings establish a connection between mRNA translation deregulation and mTORC1 pathway activation that is induced by pathologic α-synuclein in cellular and animal models of PD.

Study on PI3k gene expression in breast cancer samples and its association with clinical factors and patient survival.

Breast cancer is the most common cancer among women in the world. The phosphatidylinositol 3-Kinase (PI3k), which regulates various cellular signaling pathways, is often elevated in human cancers. This study aimed to evaluate the expression of the PI3k gene in breast cancer. In this case-control study, 40 paraffin-embedded tissues of breast cancer and 40 adjacent non-tumor tissues were examined. After total RNA extraction and cDNA synthesis, the relative expression of the gene was obtained using the real-time-PCR method and evaluated by the 2-ΔΔCT method. Also, the association of gene expression with clinical factors and survival rate was investigated. Data analysis was performed by SPSS statistical software (version 22), t-test, and ANOVA. A p-value of less than 0.05 was considered significant. The results showed that PI3k expression was significantly increased in breast tumor tissues compared to non-tumor tissues (p = 0001). Consistent with these results, PI3k expression was associated with metastasis (p = 0.008) and high tumor grade (p = 0.01). In addition, increasing PI3k expression decreased overall survival compared to its low expression (p = 0.03). In general, PI3k plays a tumor-enhancing role in the progression of breast cancer. In addition, increased PI3k expression is associated with metastasis and poor prognosis of cancer, so that PI3k may be useful in the diagnosis, treatment, and prognosis of people with the disease. However, further investigation is needed to substantiate this claim.

PAAN/MIF nuclease inhibition prevents neurodegeneration in Parkinson's disease.

Parthanatos-associated apoptosis-inducing factor (AIF) nuclease (PAAN), also known as macrophage migration inhibitor factor (MIF), is a member of the PD-D/E(X)K nucleases that acts as a final executioner in parthanatos. PAAN's role in Parkinson's disease (PD) and whether it is amenable to chemical inhibition is not known. Here, we show that neurodegeneration induced by pathologic α-synuclein (α-syn) occurs via PAAN/MIF nuclease activity. Genetic depletion of PAAN/MIF and a mutant lacking nuclease activity prevent the loss of dopaminergic neurons and behavioral deficits in the α-syn preformed fibril (PFF) mouse model of sporadic PD. Compound screening led to the identification of PAANIB-1, a brain-penetrant PAAN/MIF nuclease inhibitor that prevents neurodegeneration induced by α-syn PFF, AAV-α-syn overexpression, or MPTP intoxication in vivo. Our findings could have broad relevance in human pathologies where parthanatos plays a role in the development of cell death inhibitors targeting the druggable PAAN/MIF nuclease.

Dysregulated mRNA Translation in the G2019S LRRK2 and LRRK2 Knock-Out Mouse Brains.

The G2019S mutation in leucine-rich repeat kinase 2 (LRRK2) causes familial Parkinson's disease (PD) and is also found in a subset of idiopathic cases. Prior studies in Drosophila and human induced pluripotent stem cell (iPSC)-derived dopamine neurons uncovered a pronounced effect of G2019S LRRK2 on mRNA translation. It was previously reported that G2019S LRRK2 promotes translation of mRNAs with complex 5' untranslated region (UTR) secondary structure, resulting in increased expression of calcium channels and dysregulated calcium homeostasis in human dopamine neurons. Here, we show that dysregulated translation occurs in the brains of mammalian LRRK2 models in vivo Through ribosome profiling studies of global translation, we observe that mRNAs with complex 5'UTR structure are also preferentially translated in the G2019S LRRK2-expressing mouse brain. Reporter assays suggest that this 5'UTR preference is independent of translation initiation factors. Conversely, translation of mRNAs with complex 5'UTR secondary structure is downregulated in LRRK2 knock-out (KO) mouse brain, indicating a robust link between LRRK2 kinase activity and translation of mRNA with complex 5'UTR structure. Further, substantia nigra pars compacta (SNpc) dopamine neurons in the G2019S LRRK2-expressing brain exhibit increased calcium influx, which is consistent with the previous report from human dopamine neurons. These results collectively suggest that LRRK2 plays a mechanistic role in translational regulation, and the G2019S mutation in LRRK2 causes translational defects leading to calcium dysregulation in the mammalian brain.

Analysis of the Curative Effect of Posterior Approach on Lumbar Brucellar Spondylitis with Abscess through Magnetic Resonance Imaging under Improved Watershed Algorithm.

To explore the performance of improved watershed algorithm in processing magnetic resonance imaging (MRI) images and the effect of the processed images on the treatment of lumbar brucellar spondylitis (BS) with abscess by the posterior approach, the watershed algorithm was improved by adding constraints such as noise reduction and regional area attribute. 50 patients with abscessed lumbar disc herniation admitted to the hospital from January 2018 to January 2019 were selected, and all of them were examined by MRI. They were rolled into two groups in random. The treatment group (n = 25) accepted surgery with the aid of MRI images processed by the improved watershed algorithm, and the control group (Ctrl group) (n = 25) accepted surgery with the aid of unprocessed MRI images. The improved watershed algorithm can accurately segment the spine, and the segmentation results were relatively excellent. In contrast with the unprocessed MRI image, that processed by the improved watershed algorithm had a positive effect on the operation. In contrast with the Ctrl group, the visual analogue scale pain score (VAS), oxygen desaturation index (ODI), erythrocyte sedimentation rate (ESR), and high sensitivity C-reactive protein (CRP) were obviously lower (p < 0.05). The improved watershed algorithm proposed performs better in MRI image processing and can effectively enhance the resolution of MRI images. At the same time, the posterior approach has a good effect in the treatment of lumbar BS with abscess and is worthy of clinical promotion.

Protocol for measurement of calcium dysregulation in human induced pluripotent stem cell-derived dopaminergic neurons.

Calcium regulation is a critical process in neurons, and Ca2+ signaling is a major contributor to neurological disorders including Parkinson's disease (PD). Here, combining calcium imaging with whole-cell Ca2+ current recording, we provide a detailed protocol for measuring Ca2+ homeostasis in dopaminergic (DA) neurons derived from human induced pluripotent stem cells (hiPSCs). This approach can be applied to investigate the role of Ca2+ homeostasis in neuronal functionality as well as in disease processes. For complete details on the use and execution of this protocol, please refer to Kim et al. (2020).

The injury mechanisms and injury pyramids among children and adolescents in Zhuhai City, China.

BACKGROUND: The unclear mechanisms and severity of injuries in the injury pyramids for Chinese children and adolescents prevent the prioritization of interventions. This study aimed to describe the injury mechanisms and injury pyramids in this population to provide a priority for injury prevention strategies. METHODS: Death, hospitalization, and outpatient/emergency department visit data from patients aged 0 ~ 17 years with injuries were obtained from January 1, 2013, to December 31, 2017, in Zhuhai City, China. The injury mechanism ratios were calculated, and the injury pyramid ratios were drawn in proportion using injury mortality and the incidence of both injury hospitalizations and outpatient/emergency department injury visits. RESULTS: The top three mechanisms for injuries in children and adolescents treated in outpatient/emergency departments were falls (52.02%), animal bites (14.57%), and blunt injuries (10.60%). The top three mechanisms for injury hospitalizations were falls (37.33%), road traffic injuries (17.87%), and fire/burns (14.29%), while the top three mechanisms for injury deaths were drowning (32.91%), road traffic injuries (20.25%) and falls (13.92%). The incidence rate of outpatient/emergency department injury visits for children and adolescents was 11,210.87/100,000; the incidence rate of injury hospitalization was 627.09/100,000, and the injury death rate was 10.70/100,000. For each injury death, there were 59 injury hospitalizations and 1048 outpatient/emergency injury visits. CONCLUSIONS: The injury mechanisms were different for injury-related outpatient/emergency department visits, hospitalizations, and deaths among children and adolescents. The injury mechanisms by sex at different stages of child development, and interventions should be formulated based on this finding. The ratios of the injury pyramids varied by age, sex, region, and injury mechanisms; minor nonfatal injuries were more common in children and adolescents. The differences in the severity and extent of the injuries suggested that injury interventions in children and adolescents still have a long way to go.

Identifying intentional injuries among children and adolescents based on Machine Learning.

BACKGROUND: Compared to other studies, the injury monitoring of Chinese children and adolescents has captured a low level of intentional injuries on account of self-harm/suicide and violent attacks. Intentional injuries in children and adolescents have not been apparent from the data. It is possible that there has been a misclassification of existing intentional injuries, and there is a lack of research literature on the misclassification of intentional injuries. This study aimed to discuss the feasibility of discriminating the intention of injury based on Machine Learning (ML) modelling and provided ideas for understanding whether there was a misclassification of intentional injuries. METHODS: Information entropy was used to determine the correlation between variables and the intention of injury, and Naive Bayes (NB), Decision Tree (DT), Random Forest (RF), Adaboost algorithms and Deep Neural Networks (DNN) were used to create an intention of injury discrimination model. The models were compared by comprehensively testing the discrimination effect to determine stability and consistency. RESULTS: For the area under the ROC curve with different intentions of injuries, the NB model was 0.891, 0.880, and 0.897, respectively; the DT model was 0.870, 0.803, and 0.871, respectively; the RF model was 0.850, 0.809, and 0.845, respectively; the Adaboost model was 0.914, 0.846, and 0.914, respectively; the DNN model was 0.927, 0.835, and 0.934, respectively. In a comprehensive comparison of the five models, DNN and Adaboost models had higher values for the determination of the intention of injury. A discrimination of cases with unclear intentions of injury showed that on average, unintentional injuries, violent attacks, and self-harm/suicides accounted for 86.57%, 6.81%, and 6.62%, respectively. CONCLUSION: It was feasible to use the ML algorithm to determine the injury intention of children and adolescents. The research suggested that the DNN and Adaboost models had higher values for the determination of the intention of injury. This study could build a foundation for transforming the model into a tool for rapid diagnosis and excavating potential intentional injuries of children and adolescents by widely collecting the influencing factors, extracting the influence variables characteristically, reducing the complexity and improving the performance of the models in the future.

AMPA Receptor Surface Expression Is Regulated by S-Nitrosylation of Thorase and Transnitrosylation of NSF.

The regulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) trafficking affects multiple brain functions, such as learning and memory. We have previously shown that Thorase plays an important role in the internalization of AMPARs from the synaptic membrane. Here, we show that N-methyl-d-aspartate receptor (NMDAR) activation leads to increased S-nitrosylation of Thorase and N-ethylmaleimide-sensitive factor (NSF). S-nitrosylation of Thorase stabilizes Thorase-AMPAR complexes and enhances the internalization of AMPAR and interaction with protein-interacting C kinase 1 (PICK1). S-nitrosylated NSF is dependent on the S-nitrosylation of Thorase via trans-nitrosylation, which modulates the surface insertion of AMPARs. In the presence of the S-nitrosylation-deficient C137L Thorase mutant, AMPAR trafficking, long-term potentiation, and long-term depression are impaired. Overall, our data suggest that both S-nitrosylation and interactions of Thorase and NSF/PICK1 are required to modulate AMPAR-mediated synaptic plasticity. This study provides critical information that elucidates the mechanism underlying Thorase and NSF-mediated trafficking of AMPAR complexes.

Defects in mRNA Translation in LRRK2-Mutant hiPSC-Derived Dopaminergic Neurons Lead to Dysregulated Calcium Homeostasis.

The G2019S mutation in leucine-rich repeat kinase 2 (LRRK2) is a common cause of familial Parkinson's disease (PD). This mutation results in dopaminergic neurodegeneration via dysregulated protein translation, although how alterations in protein synthesis contribute to neurodegeneration in human neurons is not known. Here we define the translational landscape in LRRK2-mutant dopaminergic neurons derived from human induced pluripotent stem cells (hiPSCs) via ribosome profiling. We found that mRNAs that have complex secondary structure in the 5' untranslated region (UTR) are translated more efficiently in G2019S LRRK2 neurons. This leads to the enhanced translation of multiple genes involved in Ca2+ regulation and to increased Ca2+ influx and elevated intracellular Ca2+ levels, a major contributor to PD pathogenesis. This study reveals a link between dysregulated translation control and Ca2+ homeostasis in G2019S LRRK2 human dopamine neurons, which potentially contributes to the progressive and selective dopaminergic neurotoxicity in PD.

Integration of Human Induced Pluripotent Stem Cell (hiPSC)-Derived Neurons into Rat Brain Circuits.

Human neuron transplantation offers novel opportunities for modeling human neurologic diseases and potentially replacement therapies. However, the complex structure of the human cerebral cortex, which is organized in six layers with tightly interconnected excitatory and inhibitory neuronal networks, presents significant challenges for in vivo transplantation techniques to obtain a balanced, functional and homeostatically stable neuronal network. Here, we present a protocol to introduce human induced pluripotent stem cell (hiPSC)-derived neural progenitors to rat brains. Using this approach, hiPSC-derived neurons structurally integrate into the rat forebrain, exhibit electrophysiological characteristics, including firing, excitatory and inhibitory synaptic activity, and establish neuronal connectivity with the host circuitry.

Comparison of Intentional and Unintentional Injuries Among Chinese Children and Adolescents.

BACKGROUND: The patterns and risk factors of intentional injuries compared to unintentional injuries among Chinese children and adolescents have not been examined in depth. This work comprehensively describes patterns of intentional injuries in China, for which little information has been previously published. METHODS: All cases involving individuals 0-17 years old registered at emergency rooms and outpatient clinics were examined using data submitted to the National Injury Surveillance System from 2006 through 2017. A logistic regression model was performed to explore the risk factors related to intentional injuries compared to unintentional injuries. RESULTS: A total of 81,459 (95.1%) unintentional injuries, 4,218 (4.9%) intentional injuries (4,013 violent attacks and 205 self-mutilation/suicide) cases were identified. Blunt injuries accounted for 59.4% of violent attacks, while cuts and poisoning accounted for 37.1% and 23.4% of injuries involving self-mutilation/suicide, respectively. For unintentional injuries, falls (50.4%) ranked first. Additional risk factors for intentional injuries included being male (odds ratio [OR] 1.6), coming from rural areas (OR 1.9), being staff or workers (OR 2.2), and being a student (OR 1.8). As the age of the patients increased, so did the risk of intentional injuries (OR 5.0 in the 15-17 age group). Intentional injuries were more likely to occur at 00:00-03:00 am (OR 2.0). CONCLUSIONS: Intentional injuries affected more males, rural and older children, school students, and staff or workers. The mechanisms and occurrence times differed according to age group. Preventive measures should be taken to reduce the dropout of rural students, strengthen the school's violence prevention plan, and reduce self-harm.

Neurons Derived from Human Induced Pluripotent Stem Cells Integrate into Rat Brain Circuits and Maintain Both Excitatory and Inhibitory Synaptic Activities.

The human cerebral cortex is a complex structure with tightly interconnected excitatory and inhibitory neuronal networks. In order to study human cortical function, we recently developed a method to generate cortical neurons from human induced pluripotent stem cells (hiPSCs) that form both excitatory and inhibitory neuronal networks resembling the composition of the human cortex. These cultures and organoids recapitulate neuronal populations representative of the six cortical layers and a balanced excitatory and inhibitory network that is functional and homeostatically stable. To determine whether hiPSC-derived neurons can integrate and retain physiologic activities in vivo, we labeled hiPSCs with red fluorescent protein (RFP) and introduced hiPSC-derived neural progenitors to rat brains. Efficient neural induction, followed by differentiation resulted in a RFP+ neural population with traits of forebrain identity and a balanced synaptic activity composed of both excitatory neurons and inhibitory interneurons. Ten weeks after transplantation, grafted cells structurally integrated into the rat forebrain. Remarkably, these hiPSC-derived neurons were able to fire, exhibiting both excitatory and inhibitory postsynaptic currents, which culminates in the establishment of neuronal connectivity with the host circuitry. This study demonstrates that neural progenitors derived from hiPSCs can differentiate into functional cortical neurons and can participate in neural network activity through functional synaptic integration in vivo, thereby contributing to information processing.

GBA1 deficiency negatively affects physiological α-synuclein tetramers and related multimers.

Accumulating evidence suggests that α-synuclein (α-syn) occurs physiologically as a helically folded tetramer that resists aggregation. However, the mechanisms underlying the regulation of formation of α-syn tetramers are still mostly unknown. Cellular membrane lipids are thought to play an important role in the regulation of α-syn tetramer formation. Since glucocerebrosidase 1 (GBA1) deficiency contributes to the aggregation of α-syn and leads to changes in neuronal glycosphingolipids (GSLs) including gangliosides, we hypothesized that GBA1 deficiency may affect the formation of α-syn tetramers. Here, we show that accumulation of GSLs due to GBA1 deficiency decreases α-syn tetramers and related multimers and increases α-syn monomers in CRISPR-GBA1 knockout (KO) SH-SY5Y cells. Moreover, α-syn tetramers and related multimers are decreased in N370S GBA1 Parkinson's disease (PD) induced pluripotent stem cell (iPSC)-derived human dopaminergic (hDA) neurons and murine neurons carrying the heterozygous L444P GBA1 mutation. Treatment with miglustat to reduce GSL accumulation and overexpression of GBA1 to augment GBA1 activity reverse the destabilization of α-syn tetramers and protect against α-syn preformed fibril-induced toxicity in hDA neurons. Taken together, these studies provide mechanistic insights into how GBA1 regulates the transition from monomeric α-syn to α-syn tetramers and multimers and suggest unique therapeutic opportunities for PD and dementia with Lewy bodies.

Thorase variants are associated with defects in glutamatergic neurotransmission that can be rescued by Perampanel.

The AAA+ adenosine triphosphatase (ATPase) Thorase plays a critical role in controlling synaptic plasticity by regulating the expression of surface α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). Bidirectional sequencing of exons of ATAD1, the gene encoding Thorase, in a cohort of patients with schizophrenia and healthy controls revealed rare Thorase variants. These variants caused defects in glutamatergic signaling by impairing AMPAR internalization and recycling in mouse primary cortical neurons. This contributed to increased surface expression of the AMPAR subunit GluA2 and enhanced synaptic transmission. Heterozygous Thorase-deficient mice engineered to express these Thorase variants showed altered synaptic transmission and several behavioral deficits compared to heterozygous Thorase-deficient mice expressing wild-type Thorase. These behavioral impairments were rescued by the competitive AMPAR antagonist Perampanel, a U.S. Food and Drug Administration-approved drug. These findings suggest that Perampanel may be useful for treating disorders involving compromised AMPAR-mediated glutamatergic neurotransmission.

Pathological α-synuclein transmission initiated by binding lymphocyte-activation gene 3.

Emerging evidence indicates that the pathogenesis of Parkinson's disease (PD) may be due to cell-to-cell transmission of misfolded preformed fibrils (PFF) of α-synuclein (α-syn). The mechanism by which α-syn PFF spreads from neuron to neuron is not known. Here, we show that LAG3 (lymphocyte-activation gene 3) binds α-syn PFF with high affinity (dissociation constant = 77 nanomolar), whereas the α-syn monomer exhibited minimal binding. α-Syn-biotin PFF binding to LAG3 initiated α-syn PFF endocytosis, transmission, and toxicity. Lack of LAG3 substantially delayed α-syn PFF-induced loss of dopamine neurons, as well as biochemical and behavioral deficits in vivo. The identification of LAG3 as a receptor that binds α-syn PFF provides a target for developing therapeutics designed to slow the progression of PD and related α-synucleinopathies.

Cultured networks of excitatory projection neurons and inhibitory interneurons for studying human cortical neurotoxicity.

Translating neuroprotective treatments from discovery in cell and animal models to the clinic has proven challenging. To reduce the gap between basic studies of neurotoxicity and neuroprotection and clinically relevant therapies, we developed a human cortical neuron culture system from human embryonic stem cells or human inducible pluripotent stem cells that generated both excitatory and inhibitory neuronal networks resembling the composition of the human cortex. This methodology used timed administration of retinoic acid to FOXG1(+) neural precursor cells leading to differentiation of neuronal populations representative of the six cortical layers with both excitatory and inhibitory neuronal networks that were functional and homeostatically stable. In human cortical neuronal cultures, excitotoxicity or ischemia due to oxygen and glucose deprivation led to cell death that was dependent on N-methyl-D-aspartate (NMDA) receptors, nitric oxide (NO), and poly(ADP-ribose) polymerase (PARP) (a cell death pathway called parthanatos that is distinct from apoptosis, necroptosis, and other forms of cell death). Neuronal cell death was attenuated by PARP inhibitors that are currently in clinical trials for cancer treatment. This culture system provides a new platform for the study of human cortical neurotoxicity and suggests that PARP inhibitors may be useful for ameliorating excitotoxic and ischemic cell death in human neurons.