Search

Retinal ganglion cells (RGCs) are essential for vision perception. In glaucoma and other optic neuropathies, RGCs and their optic axons undergo degenerative change and cell death; this can result in irreversible vision loss. Here we developed a rapid protocol for directly inducing RGC differentiation from human induced pluripotent stem cells (hiPSCs) by the overexpression of ATOH7, BRN3B, and SOX4. The hiPSC-derived RGC-like cells (iRGCs) show robust expression of various RGC-specific markers by whole transcriptome profiling. A functional assessment was also carried out and this demonstrated that these iRGCs display stimulus-induced neuronal activity, as well as spontaneous neuronal activity. Ethambutol (EMB), an effective first-line anti-tuberculosis agent, is known to cause serious visual impairment and irreversible vision loss due to the RGC degeneration in a significant number of treated patients. Using our iRGCs, EMB was found to induce significant dose-dependent and time-dependent increases in cell death and neurite degeneration. Western blot analysis revealed that the expression levels of p62 and LC3-II were upregulated, and further investigations revealed that EMB caused a blockade of lysosome-autophagosome fusion; this indicates that impairment of autophagic flux is one of the adverse effects of that EMB has on iRGCs. In addition, EMB was found to elevate intracellular reactive oxygen species (ROS) levels increasing apoptotic cell death. This could be partially rescued by the co-treatment with the ROS scavenger NAC. Taken together, our findings suggest that this iRGC model, which achieves both high yield and high purity, is suitable for investigating optic neuropathies, as well as being useful when searching for potential drugs for therapeutic treatment and/or disease prevention., (© 2023. The Author(s).)

Microglia, the immune cells of the central nervous system, play critical roles in brain physiology and pathology. We report a novel approach that produces, within 10 days, the differentiation of human induced pluripotent stem cells (hiPSCs) into microglia (iMG) by forced expression of both SPI1 and CEBPA. High-level expression of the main microglial markers and the purity of the iMG cells were confirmed by RT-qPCR, immunostaining, and flow cytometry analyses. Whole-transcriptome analysis demonstrated that these iMGs resemble human fetal/adult microglia but not human monocytes. Moreover, these iMGs exhibited appropriate physiological functions, including various inflammatory responses, ADP/ATP-evoked migration, and phagocytic ability. When co-cultured with hiPSC-derived neurons, the iMGs respond and migrate toward injured neurons. This study has established a protocol for the rapid conversion of hiPSCs into functional iMGs, which should facilitate functional studies of human microglia using different disease models and also help with drug discovery., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Alzheimer's disease (AD) is the most common type of dementia and also one of the leading causes of death worldwide. However, the underlying mechanisms remain unclear, and currently there is no drug treatment that can prevent or cure AD. Here, we have applied the advantages of using induced pluripotent stem cell (iPSC)-derived neurons (iNs) from AD patients, which are able to offer human-specific drug responsiveness, in order to evaluate therapeutic candidates for AD. Using approach involving an inducible neurogenin-2 transgene, we have established a robust and reproducible protocol for differentiating human iPSCs into glutamatergic neurons. The AD-iN cultures that result have mature phenotypic and physiological properties, together with AD-like biochemical features that include extracellular β-amyloid (Aβ) accumulation and Tau protein phosphorylation. By screening using a gene set enrichment analysis (GSEA) approach, Graptopetalum paraguayense (GP) has been identified as a potential therapeutic agent for AD from among a range of Chinese herbal medicines. We found that administration of a GP extract caused a significantly reduction in the AD-associated phenotypes of the iNs, including decreased levels of extracellular Aβ40 and Aβ42, as well as reduced Tau protein phosphorylation at positions Ser214 and Ser396. Additionally, the effect of GP was more prominent in AD-iNs compared to non-diseased controls. These findings provide valuable information that suggests moving extracts of GP toward drug development, either for treating AD or as a health supplement to prevent AD. Furthermore, our human iN-based platform promises to be a useful strategy when it is used for AD drug discovery.

Based on the protein kinase A (PKA)/GSK3β interaction protein (GSKIP)/glycogen synthase kinase 3β (GSK3β) axis, we hypothesized that these might play a role in Tau phosphorylation. Here, we report that the phosphorylation of Tau Ser409 in SHSY5Y cells was increased by overexpression of GSKIP WT more than by PKA- and GSK3β-binding defective mutants (V41/L45 and L130, respectively). We conducted in vitro assays of various kinase combinations to show that a combination of GSK3β with PKA but not Ca 2+ /calmodulin-dependent protein kinase II (CaMK II) might provide a conformational shelter to harbor Tau Ser409. Cerebrospinal fluid (CSF) was evaluated to extend the clinical significance of Tau phosphorylation status in Alzheimer's disease (AD), neurological disorders (NAD), and mild cognitive impairment (MCI). We found higher levels of different PKA-Tau phosphorylation sites (Ser214, Ser262, and Ser409) in AD than in NAD, MCI, and normal groups. Moreover, we used the CRISPR/Cas9 system to produce amyloid precursor protein ( APP WT/D678H ) isogenic mutants. These results demonstrated an enhanced level of phosphorylation by PKA but not by the control. This study is the first to demonstrate a transient increase in phosphor-Tau caused by PKA, but not GSK3β, in the CSF and induced pluripotent stem cells (iPSCs) of AD, implying that both GSKIP and GSK3β function as anchoring proteins to strengthen the cAMP/PKA/Tau axis signaling during AD pathogenesis., Competing Interests: The authors declare they have no competing interests.

Mutations in RAB18, a member of small G protein, cause Warburg micro syndrome (WARBM), whose clinical features include vision impairment, postnatal microcephaly, and lower limb spasticity. Previously, our Rab18 -/- mice exhibited hind limb weakness and spasticity as well as signs of axonal degeneration in the spinal cord and lumbar spinal nerves. However, the cellular and molecular function of RAB18 and its roles in the pathogenesis of WARBM are still not fully understood. Using immunofluorescence staining and expression of Rab18 and organelle markers, we find that Rab18 associates with lysosomes and actively traffics along neurites in cultured neurons. Interestingly, Rab18 -/- neurons exhibit impaired lysosomal transport. Using autophagosome marker LC3-II, we show that Rab18 dysfunction leads to aberrant autophagy activities in neurons. Electron microscopy further reveals accumulation of lipofuscin-like granules in the dorsal root ganglion of Rab18 -/- mice. Surprisingly, Rab18 colocalizes, cofractionates, and coprecipitates with the lysosomal regulator Rab7, mutations of which cause Charcot-Marie-Tooth (CMT) neuropathy type 2B. Moreover, Rab7 is upregulated in Rab18-deficient neurons, suggesting a compensatory effect. Together, our results suggest that the functions of RAB18 and RAB7 in lysosomal and autophagic activities may constitute an overlapping mechanism underlying WARBM and CMT pathogenesis in the nervous system.

Congenital heart defects, dysmorphic facial features and intellectual developmental disorders (CHDFIDD) syndrome in humans was recently associated with mutation in CDK13 gene. In order to assess the loss of function of Cdk13 during mouse development, we employed gene trap knock-out (KO) allele in Cdk13 gene. Embryonic lethality of Cdk13-deficient animals was observed by the embryonic day (E) 16.5, while live embryos were observed on E15.5. At this stage, improper development of multiple organs has been documented, partly resembling defects observed in patients with mutated CDK13. In particular, overall developmental delay, incomplete secondary palate formation with variability in severity among Cdk13-deficient animals or complete midline deficiency, kidney failure accompanied by congenital heart defects were detected. Based on further analyses, the lethality at this stage is a result of heart failure most likely due to multiple heart defects followed by insufficient blood circulation resulting in multiple organs dysfunctions. Thus, Cdk13 KO mice might be a very useful model for further studies focused on delineating signaling circuits and molecular mechanisms underlying CHDFIDD caused by mutation in CDK13 gene.

DNA damage response (DDR) pathways are critical for ensuring that replication stress and various types of DNA lesion do not perturb production of neural cells during development. Cdk12 maintains genomic stability by regulating expression of DDR genes. Mutant mice in which Cdk12 is conditionally deleted in neural progenitor cells (NPCs) die after birth and exhibit microcephaly with a thinner cortical plate and an aberrant corpus callosum. We show that NPCs of mutant mice accumulate at G2 and M phase, and have lower expression of DDR genes, more DNA double-strand breaks and increased apoptosis. In addition to there being fewer neurons, there is misalignment of layers IV-II neurons and the presence of abnormal axonal tracts of these neurons, suggesting that Cdk12 is also required for the migration of late-arising cortical neurons. Using in utero electroporation, we demonstrate that the migrating mutant cells remain within the intermediate zone and fail to adopt a bipolar morphology. Overexpression of Cdk5 brings about a partially restoration of the neurons reaching layers IV-II in the mutant mice. Thus, Cdk12 is crucial to the repair of DNA damage during the proliferation of NPCs and is also central to the proper migration of late-arising neurons., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The pathogenesis of Parkinson's disease (PD) is not completely understood, Zinc (Zn(2+) ) and dopamine (DA) have been shown to involve in the degeneration of dopaminergic cells. By microarray analysis, we identified Gadd45b as a candidate molecule that mediates Zn(2+) and DA-induced cell death; the mRNA and protein levels of Gadd45b are increased by Zn(2+) treatment and raised to an even higher level by Zn(2+) plus DA treatment. Zn(2+) plus DA treatment-induced PC12 cell death was enhanced when there was over-expression of Gadd45b and was decreased by knock down of Gadd45b. MAPK p38 and JNK signaling was able to cross-talk with Gadd45b during Zn(2+) and DA treatment. The synergistic effects of Zn(2+) and DA on PC12 cell death can be accounted for by an activation of the Gadd45b-induced cell death pathway and an inhibition of p38/JNK survival pathway. Furthermore, the in vivo results show that the levels of Gadd45b protein expression and phosphorylation of p38 were increased in the substantia nigra by the infusion of Zn(2+) /DA in the mouse brain and the level of Gadd45b mRNA is significantly higher in the substantia nigra of male PD patients than normal controls. The novel role of Gadd45b and its interactions with JNK and p38 will help our understanding of the pathogenesis of PD and help the development of future treatments for PD. Zinc and dopamine are implicated in the degeneration of dopaminergic neurons. We previously demonstrated that zinc and dopamine induced synergistic effects on PC12 cell death. Results from this study show that these synergistic effects can be accounted for by activation of the Gadd45b-induced cell death pathway and inhibition of the p38/JNK survival pathway. We provide in vitro and in vivo evidence to support a novel role for Gadd45b in the pathogenesis of Parkinson's disease., (© 2016 International Society for Neurochemistry.)

The maintenance of genomic integrity during early embryonic development is important in order to ensure the proper development of the embryo. Studies from cultured cells have demonstrated that cyclin-dependent kinase 12 (Cdk12) is a multifunctional protein that maintains genomic stability and the pluripotency of embryonic stem cells. Perturbation of its functions is also known to be associated with pathogenesis and drug resistance in human cancers. However, the biological significance of Cdk12 in vivo is unclear. Here we bred mice that are deficient in Cdk12 and demonstrated that Cdk12 depletion leads to embryonic lethality shortly after implantation. We also used an in vitro culture system of blastocysts to examine the molecular mechanisms associated with the embryonic lethality of Cdk12-deficient embryos. Cdk12(-/-) blastocysts fail to undergo outgrowth of the inner cell mass because of an increase in the apoptosis of these cells. Spontaneous DNA damage was revealed by an increase in 53BP1 foci among cells cultured from Cdk12(-/-) embryos. Furthermore, the expression levels of various DNA damage response genes, namely Atr, Brca1, Fanci and Fancd2, are reduced in Cdk12(-/-) embryos. These findings indicate that Cdk12 is important for the correct expression of some DNA damage response genes and indirectly has an influence on the efficiency of DNA repair. Our report also highlights that DNA breaks occurring during DNA replication are frequent in mouse embryonic cells and repair of such damage is critical to the successful development of mouse embryos.

Mutations in the gene of RAB18, a member of Ras superfamily of small G-proteins, cause Warburg Micro Syndrome (WARBM) which is characterized by defective neurodevelopmental and ophthalmological phenotypes. Despite loss of Rab18 had been reported to induce disruption of the endoplasmic reticulum structure and neuronal cytoskeleton organization, parts of the pathogenic mechanism caused by RAB18 mutation remain unclear. From the N-ethyl-N-nitrosourea (ENU)-induced mutagenesis library, we identified a mouse line whose Rab18 was knocked out. This Rab18(-/-) mouse exhibited stomping gait, smaller testis and eyes, mimicking several features of WARBM. Rab18(-/-) mice were obviously less sensitive to pain and touch than WT mice. Histological examinations on Rab18(-/-) mice revealed progressive axonal degeneration in the optic nerves, dorsal column of the spinal cord and sensory roots of the spinal nerves while the motor roots were spared. All the behavioral and pathological changes that resulted from abnormalities in the sensory axons were prevented by introducing an extra copy of Rab18 transgene in Rab18(-/-) mice. Our results reveal that sensory axonal degeneration is the primary cause of stomping gait and progressive weakness of the hind limbs in Rab18(-/-) mice, and optic nerve degeneration should be the major pathology of progressive optic atrophy in children with WARBM. Our results indicate that the sensory nervous system is more vulnerable to Rab18 deficiency and WARBM is not only a neurodevelopmental but also neurodegenerative disease., (Copyright © 2015 Elsevier Inc. All rights reserved.)