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Effective therapeutics is much needed for amyotrophic lateral sclerosis (ALS), an adult-onset neurodegenerative disease mainly affecting motor neurons. By screening chemical compounds in human patient-derived and aging-relevant motor neurons, we identify a neuroprotective compound and show that MAP4Ks may serve as therapeutic targets for treating ALS. The lead compound broadly improves survival and function of human ALS motor neurons. Mechanistically, it works as an inhibitor of MAP4Ks, regulates the MAP4Ks-HDAC6-TUBA4A-RANGAP1 pathway, and normalizes subcellular distribution of RANGAP1 and TDP-43. Finally, in an ALS mouse model we show that inhibiting MAP4Ks preserves motor neurons and extends animal lifespan.

Abstract: Protein-ligand interactions are necessary for majority protein functions. Adenosine- 5’-triphosphate (ATP) is one such ligand that plays vital role as a coenzyme in providing energy for cellular activities, catalyzing biological reaction and signaling. Knowing ATP binding residues of proteins is helpful for annotation of protein function and drug design. However, due to the huge amounts of protein sequences influx into databases in the post-genome era, experimentally identifying ATP binding residues is costineffective and time-consuming. To address this problem, computational methods have been developed to predict ATP binding residues. In this review, we briefly summarized the application of machine learning methods in detecting ATP binding residues of proteins. We expect this review will be helpful for further research.

High-throughput western blot (WB) analysis of small and precious samples, such as various age-related subtype-specific human induced neurons (hiNs), confers the ability to obtain more consistent, comparable, and informative data from materials with extremely limited availability. In this study, p-toluenesulphonic acid (PTSA), an odorless tissue fixative, was used to inactivate HRP for developing a high-throughput WB method. PTSA-treated blots showed fast and efficient inactivation of HRP without detectable protein loss and epitope damage. With a brief PTSA-treatment before every next probing, 10 proteins of dopaminergic hiNs could be sequentially, sensitively, and specifically detected in a blot. These WB data proved the age-associated and neuron-specific features of hiNs and further revealed a sharp reduction of two Parkinson’s disease-associated proteins, UCHL1 and GAP43, in the normal aging dopaminergic neurons. Together, this study developed a unique and high-efficiency WB analysis and pinpointed its special value for capturing robust useful data from limited, precious samples.GRAPHICAL ABSTRACTHIGHLIGHTS✓P-toluenesulphonic acid (PTSA) quickly and fully deactivated HRP on immunoblots.✓PTSA was an odorless, non-volatile, low cost, and user-friendly HRP inactivator.✓PTSA allowed high-efficiency WB analysis to save small precious samples and time.✓10 proteins were detected in a single blot of age-relevant human induced neurons.✓UCHL1 and GAP43 sharp decline occurred in aging human induced dopaminergic neurons.

CRISPR-Cas, as a tool for gene editing, has received extensive attention in recent years. Anti-CRISPR (Acr) proteins can inactivate the CRISPR-Cas defense system during interference phase, and can be used as a potential tool for the regulation of gene editing. In-depth study of Anti-CRISPR proteins is of great significance for the implementation of gene editing. In this study, we developed a high-accuracy prediction model based on two-step model fusion strategy, called AcrPred, which could produce an AUC of 0.952 with independent dataset validation. To further validate the proposed model, we compared with published tools and correctly identified 9 of 10 new Acr proteins, indicating the strong generalization ability of our model. Finally, for the convenience of related wet-experimental researchers, a user-friendly web-server AcrPred (Anti-CRISPR proteins Prediction) was established at http://lin-group.cn/server/AcrPred, by which users can easily identify potential Anti-CRISPR proteins.

The chloroplast is a type of subcellular organelle of green plants and eukaryotic algae, which plays an important role in the photosynthesis process. Since the function of a protein correlates with its location, knowing its subchloroplast localization is helpful for elucidating its functions. However, due to a large number of chloroplast proteins, it is costly and time-consuming to design biological experiments to recognize subchloroplast localizations of these proteins. To address this problem, during the past ten years, twelve computational prediction methods have been developed to predict protein subchloroplast localization. This review summarizes the research progress in this area. We hope the review could provide important guide for further computational study on protein subchloroplast localization.

Transcription factors play key roles in cell-fate decisions by regulating 3D genome conformation and gene expression. The traditional view is that methylation of DNA hinders transcription factors binding to them, but recent research has shown that many transcription factors prefer to bind to methylated DNA. Therefore, identifying such transcription factors and understanding their functions is a stepping-stone for studying methylation-mediated biological processes. In this paper, a two-step discriminated method was proposed to recognize transcription factors and their preference for methylated DNA based only on sequences information. In the first step, the proposed model was used to discriminate transcription factors from non-transcription factors. The areas under the curve (AUCs) are 0.9183 and 0.9116, respectively, for the 5-fold cross-validation test and independent dataset test. Subsequently, for the classification of transcription factors that prefer methylated DNA and transcription factors that prefer non-methylated DNA, our model could produce the AUCs of 0.7744 and 0.7356, respectively, for the 5-fold cross-validation test and independent dataset test. Based on the proposed model, a user-friendly web server called TFPred was built, which can be freely accessed at http://lin-group.cn/server/TFPred/., Graphical Abstract, Transcription factors binding to methylated DNA perform special and unclear functions. Lin and colleagues developed a machine-learning-based method to predict transcription factors and their preference for methylated DNA, which will help the discovery of methylated DNA-bound transcription factors and the study of their functions.

Transfer RNAs (tRNAs) are products of RNA polymerase III (Pol III) and essential for mRNA translation and ultimately cell growth and proliferation. Whether and how individual tRNA genes are specifically regulated is not clear. Here, we report that SOX4, a well-known Pol II-dependent transcription factor that is critical for neurogenesis and reprogramming of somatic cells, also directly controls, unexpectedly, the expression of a subset of tRNA genes and therefore protein synthesis and proliferation of human glioblastoma cells. Genome-wide location analysis through chromatin immunoprecipitation-sequencing uncovers specific targeting of SOX4 to a subset of tRNA genes, including those for tRNAi Met . Mechanistically, sequence-specific SOX4-binding impedes the recruitment of TATA box binding protein and Pol III to tRNA genes and thereby represses their expression. CRISPR/Cas9-mediated down-regulation of tRNAi Met greatly inhibits growth and proliferation of human glioblastoma cells. Conversely, ectopic tRNAi Met partially rescues SOX4-mediated repression of cell proliferation. Together, these results uncover a regulatory mode of individual tRNA genes to control cell behavior. Such regulation may coordinate codon usage and translation efficiency to meet the demands of diverse tissues and cell types, including cancer cells.

DYT1 dystonia is a hereditary neurologic movement disorder characterized by uncontrollable muscle contractions. It is caused by a heterozygous mutation inTorsin A(TOR1A), a gene encoding a membrane-embedded ATPase. While animal models provide insights into disease mechanisms, significant species-dependent differences exist since animals with the identical heterozygous mutation fail to show pathology. Here, we model DYT1 by using human patient-specific cholinergic motor neurons (MNs) that are generated through either direct conversion of patients' skin fibroblasts or differentiation of induced pluripotent stem cells (iPSCs). These human MNs with the heterozygousTOR1Amutation show reduced neurite length and branches, markedly thickened nuclear lamina, disrupted nuclear morphology, and impaired nucleocytoplasmic transport (NCT) of mRNAs and proteins, whereas they lack the perinuclear “blebs” that are often observed in animal models. Furthermore, we uncover that the nuclear lamina protein LMNB1 is upregulated in DYT1 cells and exhibits abnormal subcellular distribution in a cholinergic MNs-specific manner. Such dysregulation of LMNB1 can be recapitulated by either ectopic expression of the mutantTOR1Agene or shRNA-mediated downregulation of endogenousTOR1Ain healthy control MNs. Interestingly, downregulation of LMNB1 can largely ameliorate all the cellular defects in DYT1 MNs. These results reveal the value of disease modeling with human patient-specific neurons and indicate that dysregulation of LMNB1, a crucial component of the nuclear lamina, may constitute a major molecular mechanism underlying DYT1 pathology.SIGNIFICANCE STATEMENTInaccessibility to patient neurons greatly impedes our understanding of the pathologic mechanisms for dystonia. In this study, we employ reprogrammed human patient-specific motor neurons (MNs) to model DYT1, the most severe hereditary form of dystonia. Our results reveal disease-dependent deficits in nuclear morphology and nucleocytoplasmic transport (NCT). Most importantly, we further identify LMNB1 dysregulation as a major contributor to these deficits, uncovering a new pathologic mechanism for DYT1 dystonia.

Background Alzheimer’s disease (AD) is an adult-onset mental disorder with aging as a major risk factor. Early and progressive degeneration of basal forebrain cholinergic neurons (BFCNs) contributes substantially to cognitive impairments of AD. An aging-relevant cell model of BFCNs will critically help understand AD and identify potential therapeutics. Recent studies demonstrate that induced neurons directly reprogrammed from adult human skin fibroblasts retain aging-associated features. However, human induced BFCNs (hiBFCNs) have yet to be achieved. Methods We examined a reprogramming procedure for the generation of aging-relevant hiBFCNs through virus-mediated expression of fate-determining transcription factors. Skin fibroblasts were obtained from healthy young persons, healthy adults and sporadic AD patients. Properties of the induced neurons were examined by immunocytochemistry, qRT-PCR, western blotting, and electrophysiology. Results We established a protocol for efficient generation of hiBFCNs from adult human skin fibroblasts. They show electrophysiological properties of mature neurons and express BFCN-specific markers, such as CHAT, p75NTR, ISL1, and VACHT. As a proof-of-concept, our preliminary results further reveal that hiBFCNs from sporadic AD patients exhibit time-dependent TAU hyperphosphorylation in the soma and dysfunctional nucleocytoplasmic transport activities. Conclusions Aging-relevant BFCNs can be directly reprogrammed from human skin fibroblasts of healthy adults and sporadic AD patients. They show promises as an aging-relevant cell model for understanding AD pathology and may be employed for therapeutics identification for AD.

Additional file 2: Figure S2. Characterization of the induced neurons, related to Fig. 1 A.

Additional file 4: Figure S4. PhosphoTAU expression in hiBFCNs, related to Fig. 5 A.

Additional file 1: Figure S1. The reprogramming process and optimization, related to Fig. 1 A.

Summary 5hmC, 6mA, and 4mC are three common DNA modifications and are involved in various of biological processes. Accurate genome-wide identification of these sites is invaluable for better understanding their biological functions. Owing to the labor-intensive and expensive nature of experimental methods, it is urgent to develop computational methods for the genome-wide detection of these sites. Keeping this in mind, the current study was devoted to construct a computational method to identify 5hmC, 6mA, and 4mC. We initially used K-tuple nucleotide component, nucleotide chemical property and nucleotide frequency, and mono-nucleotide binary encoding scheme to formulate samples. Subsequently, random forest was utilized to identify 5hmC, 6mA, and 4mC sites. Cross-validated results showed that the proposed method could produce the excellent generalization ability in the identification of the three modification sites. Based on the proposed model, a web-server called iDNA-MS was established and is freely accessible at http://lin-group.cn/server/iDNA-MS., Graphical Abstract, Highlights • A computational tool was developed for identification of 5hmC, 6mA, and 4mC • 6mA and 4mC mark similar regions in the C. equisetifolia and F. vesca genomes • 5hmC enriches in the initial and middle of the DNA loops • A user-friendly webserver was available at http://lin-group.cn/server/iDNA-MS, Genetics; Quantitative Genetics; Bioinformatics

Additional file 6: Table S1. Primary antibodies. Table S2. Primer sequences.

Additional file 5: Figure S5. Effect of LMB on nucleocytoplasmic transport in hiBFCNs, related to Fig. 6 A.

Additional file 3: Figure S3. Characterization of AD-hiBFCNs, related to Fig. 1 A.

The identification of bacteriophage proteins in the host subcellular localization could provide important clues for understanding the interaction between phage and host bacteria as well as antibacterial drug design. To date, computational methods have been reported to identify bacteriophage proteins located in the host cell. However, there is still space for improving the prediction accuracy. The existing methods considering the sequence order correlation and the physicochemical property of protein provide us insights to construct an integrated descriptor based on sequence for phage proteins. Meanwhile, we proposed a feature selection technique to obtain the optimal features. In the jackknife test, the prediction accuracies are 86.7% and 97.9%, respectively for discrimination between PH proteins and non-PH proteins as well as PHM proteins and PHC proteins. Based on our model, we updated the web server PHPred to version 2.0 which can be freely accessed from http://lin-group.cn/server/PHPred2.0 .

Summary Pro-neural transcription factors and small molecules can induce the reprogramming of fibroblasts into functional neurons; however, the immediate-early molecular events that catalyze this conversion have not been well defined. We previously demonstrated that neurogenin 2 (NEUROG2), forskolin (F), and dorsomorphin (D) can reprogram fibroblasts into functional neurons with high efficiency. Here, we used this model to define the genetic and epigenetic events that initiate an acquisition of neuronal identity. We demonstrate that NEUROG2 is a pioneer factor, FD enhances chromatin accessibility and H3K27 acetylation, and synergistic transcription activated by these factors is essential to successful reprogramming. CREB1 promotes neuron survival and acts with NEUROG2 to upregulate SOX4, which co-activates NEUROD1 and NEUROD4. In addition, SOX4 targets SWI/SNF subunits and SOX4 knockdown results in extensive loss of open chromatin and abolishes reprogramming. Applying these insights, adult human glioblastoma cell and skin fibroblast reprogramming can be improved using SOX4 or chromatin-modifying chemicals., Graphical Abstract, Highlights • NEUROG2 acts as a pioneer factor to drive neuronal reprogramming • ATAC-, ChIP-, and RNA-seq profiling reveals genome-wide mechanisms for reprogramming • SOX4 is a critical mediator of chromatin remodeling during reprogramming • SOX4 or FK228 can enhance adult human glioblastoma and skin fibroblast reprogramming, In this article, Zhang and colleagues show that NEUROG2 and two small molecules activate transcription and chromatin remodeling programs that drive fibroblast-to-neuron reprogramming in diverse human cell types. Strikingly, the transcription factor SOX4 mediates genome-wide chromatin remodeling processes critical to efficient conversion, while BCL2L1 and the chemical FK228 can improve reprogramming of adult human cell types.

SummarySubtype-specific neurons obtained from adult humans will be critical to modeling neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS). Here, we show that adult human skin fibroblasts can be directly and efficiently converted into highly pure motor neurons without passing through an induced pluripotent stem cell stage. These adult human induced motor neurons (hiMNs) exhibit the cytological and electrophysiological features of spinal motor neurons and form functional neuromuscular junctions (NMJs) with skeletal muscles. Importantly, hiMNs converted from ALS patient fibroblasts show disease-specific degeneration manifested through poor survival, soma shrinkage, hypoactivity, and an inability to form NMJs. A chemical screen revealed that the degenerative features of ALS hiMNs can be remarkably rescued by the small molecule kenpaullone. Taken together, our results define a direct and efficient strategy to obtain disease-relevant neuronal subtypes from adult human patients and reveal their promising value in disease modeling and drug identification.

The cellular generation of toxic metabolites and subsequent detoxification failure can cause the uncontrolled accumulation of these metabolites in cells, leading to cellular dysfunction. Amyloid-beta protein (A beta), a normal metabolite of neurons, tends to form toxic oligomeric structures that cause neurodegeneration. It is unclear how healthy neurons control the levels of intracellular oligomeric A beta in order to avoid neurodegeneration. Using immunochemical and biochemical studies, we show that the A beta-binding serine protease Omi is a stress-relieving heat-shock protein that protects neurons against neurotoxic oligomeric A beta. Through its PDZ domain, Omi binds preferentially to neurotoxic oligomeric forms of A beta rather than non-toxic monomeric forms to detoxify oligomeric A beta by disaggregation. This specific interaction leads not only to mutual detoxification of the pro-apoptotic activity of Omi and A beta-induced neurotoxicity, but also to a reduction of neurotoxic-A beta accumulation. The neuroprotective role of Omi is further supported by its upregulation during normal neurogenesis and neuronal maturation in mice, which could be in response to the increase in the generation of oligomeric A beta during these processes. These findings provide novel and important insights into the detoxification pathway of intraneuronal oligomeric A beta in mammals and the protective roles of Omi in neurodegeneration, suggesting a novel therapeutic target in neurodegenerative diseases.

Spinal cord injury (SCI) leads to irreversible neuronal loss and glial scar formation, which ultimately result in persistent neurological dysfunction. Cellular regeneration could be an ideal approach to replenish the lost cells and repair the damage. However, the adult spinal cord has limited ability to produce new neurons. Here we show that resident astrocytes can be converted to doublecortin (DCX)-positive neuroblasts by a single transcription factor, SOX2, in the injured adult spinal cord. Importantly, these induced neuroblasts can mature into synapse-forming neurons in vivo. Neuronal maturation is further promoted by treatment with a histone deacetylase inhibitor, valproic acid (VPA). The results of this study indicate that in situ reprogramming of endogenous astrocytes to neurons might be a potential strategy for cellular regeneration after SCI.

Study Design. An in vitro neural hypoxia model and rat spinal cord injury (SCI) model were used to assess the regulation of therapeutic vascular endothelial growth factor (VEGF) gene expression in mouse neural stem cells (mNSCs) by the EPO (erythropoietin) enhancer or RTP801 promoter. Objective. To increase VEGF gene expression in mNSCs under hypoxic conditions in SCI lesions but avoid unwanted overexpression of VEGF in normal sites, we developed a hypoxia-inducible gene expression system consisting of the EPO enhancer and RTP801 promoter fused to VEGF or the luciferase gene, then transfected into mNSCs. Summary of Background Data. On the basis of the ischemic response in the injured area, poor cell survival at the transplantation site is a consistent problem with NSC transplantation after SCI. Although VEGF directly protects neurons and enhances neurite outgrowth, uncontrolled overexpression of VEGF in uninjured tissue may cause serious adverse effects. To effectively improve NSC survival in ischemic sites after transplantation, we evaluated mNSCs modified by a hypoxia-inducible VEGF gene expression system in an SCI model. Methods. Hypoxia-inducible luciferase or VEGF plasmids were constructed using the EPO enhancer or RTP801 promoter. The effect of these systems on targeted gene expression and cell viability was evaluated in mNSCs in both hypoxic in vitro injury and a rat SCI model in vivo. Results. The gene expression system containing the EPO enhancer or RTP801 promoter significantly increased the expression of the luciferase reporter gene and therapeutic VEGF gene under hypoxic conditions. The Epo-SV-VEGF plasmid transfection group had significantly fewer apoptotic cells in vitro. This system also augmented cell viability in the in vivo SCI model. Conclusion. These results strongly suggest the potential utility of mNSCs modified by a hypoxia-inducible VEGF gene expression system in the development of effective stem cell transplantation protocols in SCI.

Background Nonviral ex vivo local gene therapy systems consisting of regulated gene expression vectors and cellular delivery platforms represent a novel strategy for tissue repair and regeneration. We introduced a hypoxia-regulated plasmid-based system into mouse neural stem cells (NSCs) as an efficient gene expression and delivery platform for rapid, robust and persistent hypoxic/ischemic-regulated gene expression in the spinal cord. Methods A synthetic hypoxia-responsive erythropoietin (Epo) enhancer, the SV40 minimal promoter and the luciferase (Luc) reporter gene were incorporated in a DsRed-expressing double-promoter plasmid for cell lipofection and Zeocin-selection to establish a hypoxia-regulated stable NSC line (NSC-Epo-SV-Luc). A nonhypoxia-regulated stable NSC line (NSC-SV-Luc) was also established as a control. Results Under the transcriptional regulation of the Epo enhancer, in vitro luciferase expression in NSC-Epo-SV-Luc, but not in NSC-SV-Luc, was sensitively augmented according to the strength and duration of the hypoxic stimulus and was quickly down-regulated to a low basal level after reoxygenation of the hypoxic cells. Furthermore, deoxygenation of the reoxygenated cells clearly enhanced the luciferase activity again. After transplantation into a rat spinal cord injury (SCI) model, only NSC-Epo-SV-Luc showed ischemic injury-specific luciferase expression Notably, the engineered NSC lines kept the neural differentiation potential and retained the hypoxia-regulated luciferase expression after differentiation. Conclusions We propose that NSCs engineered with the Epo-SV-therapeutic gene will be valuable for developing a controllable stem cell-mediated nonviral gene therapy for SCI or other central nervous system diseases accompanied with chronic or episodic hypoxic/ischemic stresses. Copyright © 2010 John Wiley & Sons, Ltd.

AIM: To identify a method for efficient large-scale purification of functional hepatitis B virus polymerase (HBV-Pol) without addition of cellular factors. METHODS: Full-length HBV-Pol (843 amino acids) tagged with 5’ end Polyhistidine was expressed at a high level in an Escherichia coli (E. coli) system. Sodium dodecyl sulfate lysis buffer was utilized to dissolve insoluble HBVPol, and Ni-NTA resin affinity chromatography was utilized for HBV-Pol purification. Most recombinant HBV-Pol was eluted with 100 mmol/L imidazole in the presence of NP-40, a weak detergent that keeps HBV-Pol in solution. A reducing agent was utilized throughout the purification steps to keep soluble HBV-Pol from redundant disulfide bond formation. RESULTS: The large-scale production of functional intact human HBV-Pol was achieved in an E. coli expression system. Purified HBV-Pol showed stable reverse transcriptase activity and DNA polymerase activity. The purified protein was of high purity and had stable re verse transcriptase activity. CONCLUSION: Large-scale production of HBV-Pol in pure form should facilitate crystallization and detailed analysis of the structure and mechanism of HBV-Pol. Ability of this purification approach to obtain human HBVPol in an enzymatically active form should be helpful for development of drugs for treatment of chronic hepatitis B.

To avoid unwanted adverse effects of higher doses of single treatment of stem cells and gene therapy and increase the therapeutic efficacies, we hypothesized the combined therapy with stem cells and gene therapy. This study assessed the neuroprotective effects of combined gene therapy and stem cell treatment under ischemic hypoxia conditions using hypoxia-inducible vascular endothelial growth factor (VEGF) and bone marrow-derived mesenchymal stem cells (BMSC).Experimental groups included the control which was N2A cells transfected with empty vectors, the transfection only group which was N2A cells treated with pEpo-SV-VEGF alone, the BMSC only group which was N2A cells transfected with empty vectors and cocultured with BMSCs, and the combined treatment group which was N2A cells treated with pEpo-SV-VEGF and cocultured with BMSCs. Each group was transfected for 4 h and cultured at 37 degrees C and 5% CO2 for 24 h. Each group was then cultivated under hypoxic conditions (1% O2) for 12 h. Neuroprotective effects were assessed by reverse transcription polymerase chain reaction, annexin V, and cytotoxicity assay.Neurons exposed to hypoxic conditions exhibited neuronal apoptosis. Compared to single treatments, the combined hypoxia-inducible VEGF and BMSC treatment demonstrated a significant increase in VEGF expression and decreased neuronal apoptosis.These results suggest that combined pEpo-SV-VEGF and BMSC treatment is effective in protecting neurons against hypoxic ischemic injury.

STUDY DESIGN.: An in vitro neural hypoxia model and rat spinal cord injury (SCI) model were used to assess the regulation effect of a reporter or therapeutic gene expression by an oxygen-dependent degradation (ODD) domain in a hypoxia-inducible gene expression system with or without the erythropoietin (EPO) enhancer. OBJECTIVE.: To increase vascular endothelial growth factor (VEGF) gene expression in SCI lesions but avoid unwanted overexpression of VEGF in normal sites, we developed a hypoxia-inducible gene expression system consisting of the EPO enhancer upstream of the SV promoter and an ODD domain C-terminally fused to VEGF. SUMMARY OF BACKGROUND DATA.: ODD domain plays a major role in the degradation of hypoxia-inducible factor 1alpha and has been used in a hypoxia-specific gene expression system as a post-translational regulatory factor. METHODS.: The hypoxia-inducible luciferase or VEGF plasmid was constructed using the EPO enhancer combined with or without the ODD domain. The constructed plasmid was transfected into mouse Neuro 2a (N2a) neuroblastoma cells by Lipofectamine 2000, followed by a 24-hour incubation in hypoxia or normoxia. For in vivo analysis, the naked plasmid DNA was directly injected into the injured rat spinal cord. The gene expression was evaluated by luciferase activity assay, enzyme-linked immunosorbent assay, reverse transcriptase-polymerase chain reaction, and immunofluorescence staining. RESULTS.: The EPO enhancer/ODD domain-combined hypoxia-inducible gene expression system clearly increased the expression of the reporter luciferase gene and therapeutic VEGF gene specifically under hypoxic conditions and SCI, and quickly downregulated protein expression to a very low level after reoxygenation. CONCLUSION.: These results strongly suggest the potential applicability of this EPO enhancer/ODD domain-based hypoxia-inducible gene expression system in the development of a safer and more effective VEGF gene therapy for SCI.

Mammalian serine protease HtrA2/Omi has been known as an apoptosis inducer involved inactivation of caspase-dependent as well as caspase-independent cell death. Recent studies with the HtrA2/Omi mutant and knockout mouse models, however, suggested that HtrA2/Omi might play a protective role in neurons. It is important to establish a transgenic mouse model with neuron-specific overexpression of HtrA2/Omi to clarify the physiological function of mammalian HtrA2/Omi in neurons. In the present study, a transgene containing HtrA2/Omi cDNA downstream of a rat neuron-specific enolase promoter was constructed and microinjected into the pronuclei of fertilized zygotes to establish transgenic mice. Transgenic mice successfully overexpressed HtrA2/Omi in brain tissue. As expected, HtrA2/Omi-overexpressing transgenic mice showed normal development without any sign of apoptotic cell death. Our results suggest that the primary function of neuronal HtrA2/Omi might be to protect neurons against stress in contrast to its role in the somatic system.

HtrA2/Omi is a mammalian mitochondrial serine protease homologous to the E. coli HtrA/DegP gene products. Recently, HtrA2/Omi was found to have a dual role in mammalian cells, acting as an apoptosis-inducing protein and being involved in maintenance of mitochondrial homeostasis. By screening a human brain cDNA library with A beta peptide as bait in a yeast two-hybrid system, we identified HtrA2/Omi as a binding partner of A beta peptide. The interaction between A beta peptide and HtrA2/Omi was confirmed by an immunoblot binding assay. The possible involvement of HtrA2/Omi in A beta peptide metabolism was investigated. In vitro peptide cleavage assays showed that HtrA2/Omi did not directly promote the production of A beta peptide at the beta/gamma-secretase level, or the degradation of A beta peptide. However, overexpression of HtrA2/Omi in K269 cells decreased the production of A beta40 and A beta42 by up to 30%. These results rule out the involvement of HtrA2/Omi in the etiology of Alzheimer's disease. However, the fact that overexpression of HtrA2/Omi reduces the generation of A beta40 and A beta42 suggests that it may play some positive role in mammalian cells.

Malignant gliomas, the most common solid tumors in the central nervous system, are essentially incurable due to their rapid growth and very invasive nature. One potential approach to eradicating glioma cells is to force these cells to undergo terminal differentiation and, in the process, to irreversible postmitotic arrest. Here, we show that neurogenin 2 (NGN2, also known as NEUROG2) synergizes with sex-determining region Y-box 11 (SOX11) to very efficiently convert human glioma cells to terminally differentiated neuron-like cells in both cell culture and adult mouse brains. These cells exhibit neuronal morphology, marker expression, and electrophysiological properties. The conversion process is accompanied by cell cycle exit, which dramatically inhibits glioma cell proliferation and tumor development after orthotopic transplantation. Most importantly, intracranial injection of NGN2- and SOX11-expressing virus into the tumor mass also curtails glioma growth and significantly improves survival of tumor-bearing mice. Taken together, this study shows a simple and highly efficient strategy for reprogramming malignant glioma cells into postmitotic cells, which might be a promising therapeutic approach for brain tumors.