RNA Modifications and RNA Metabolism in Neurological Disease Pathogenesis.

The intrinsic cellular heterogeneity and molecular complexity of the mammalian nervous system relies substantially on the dynamic nature and spatiotemporal patterning of gene expression. These features of gene expression are achieved in part through mechanisms involving various epigenetic processes such as DNA methylation, post-translational histone modifications, and non-coding RNA activity, amongst others. In concert, another regulatory layer by which RNA bases and sugar residues are chemically modified enhances neuronal transcriptome complexity. Similar RNA modifications in other systems collectively constitute the cellular epitranscriptome that integrates and impacts various physiological processes. The epitranscriptome is dynamic and is reshaped constantly to regulate vital processes such as development, differentiation and stress responses. Perturbations of the epitranscriptome can lead to various pathogenic conditions, including cancer, cardiovascular abnormalities and neurological diseases. Recent advances in next-generation sequencing technologies have enabled us to identify and locate modified bases/sugars on different RNA species. These RNA modifications modulate the stability, transport and, most importantly, translation of RNA. In this review, we discuss the formation and functions of some frequently observed RNA modifications-including methylations of adenine and cytosine bases, and isomerization of uridine to pseudouridine-at various layers of RNA metabolism, together with their contributions to abnormal physiological conditions that can lead to various neurodevelopmental and neurological disorders.

A Positive Regulatory Feedback Loop between EKLF/KLF1 and TAL1/SCL Sustaining the Erythropoiesis.

The erythroid Krüppel-like factor EKLF/KLF1 is a hematopoietic transcription factor binding to the CACCC DNA motif and participating in the regulation of erythroid differentiation. With combined use of microarray-based gene expression profiling and the promoter-based ChIP-chip assay of E14.5 fetal liver cells from wild type (WT) and EKLF-knockout (Eklf-/-) mouse embryos, we identified the pathways and direct target genes activated or repressed by EKLF. This genome-wide study together with the molecular/cellular analysis of the mouse erythroleukemic cells (MEL) indicate that among the downstream direct target genes of EKLF is Tal1/Scl. Tal1/Scl encodes another DNA-binding hematopoietic transcription factor TAL1/SCL, known to be an Eklf activator and essential for definitive erythroid differentiation. Further identification of the authentic Tal gene promoter in combination with the in vivo genomic footprinting approach and DNA reporter assay demonstrate that EKLF activates the Tal gene through binding to a specific CACCC motif located in its promoter. These data establish the existence of a previously unknow positive regulatory feedback loop between two DNA-binding hematopoietic transcription factors, which sustains mammalian erythropoiesis.

Negative Regulation of the Differentiation of Flk2- CD34- LSK Hematopoietic Stem Cells by EKLF/KLF1.

Erythroid Krüppel-like factor (EKLF/KLF1) was identified initially as a critical erythroid-specific transcription factor and was later found to be also expressed in other types of hematopoietic cells, including megakaryocytes and several progenitors. In this study, we have examined the regulatory effects of EKLF on hematopoiesis by comparative analysis of E14.5 fetal livers from wild-type and Eklf gene knockout (KO) mouse embryos. Depletion of EKLF expression greatly changes the populations of different types of hematopoietic cells, including, unexpectedly, the long-term hematopoietic stem cells Flk2- CD34- Lin- Sca1+ c-Kit+ (LSK)-HSC. In an interesting correlation, Eklf is expressed at a relatively high level in multipotent progenitor (MPP). Furthermore, EKLF appears to repress the expression of the colony-stimulating factor 2 receptor ß subunit (CSF2RB). As a result, Flk2- CD34- LSK-HSC gains increased differentiation capability upon depletion of EKLF, as demonstrated by the methylcellulose colony formation assay and by serial transplantation experiments in vivo. Together, these data demonstrate the regulation of hematopoiesis in vertebrates by EKLF through its negative regulatory effects on the differentiation of the hematopoietic stem and progenitor cells, including Flk2- CD34- LSK-HSCs.

Znf179 E3 ligase-mediated TDP-43 polyubiquitination is involved in TDP-43- ubiquitinated inclusions (UBI) (+)-related neurodegenerative pathology.

BACKGROUND: The brain predominantly expressed RING finger protein, Znf179, is known to be important for embryonic neuronal differentiation during brain development. Downregulation of Znf179 has been observed in motor neurons of adult mouse models for amyotrophic lateral sclerosis (ALS), yet the molecular function of Znf179 in neurodegeneration has never been previously described. Znf179 contains the classical C3HC4 RING finger domain, and numerous proteins containing C3HC4 RING finger domain act as E3 ubiquitin ligases. Hence, we are interested to identify whether Znf179 possesses E3 ligase activity and its role in ALS neuropathy. METHODS: We used in vivo and in vitro ubiquitination assay to examine the E3 ligase autoubiquitination activity of Znf179 and its effect on 26S proteasome activity. To search for the candidate substrates of Znf179, we immunoprecipitated Znf179 and subjected to mass spectrometry (MS) analysis to identify its interacting proteins. We found that ALS/ FTLD-U (frontotemporal lobar degeneration (FTLD) with ubiquitin inclusions)-related neurodegenerative TDP-43 protein is the E3 ligase substrate of Znf179. To further clarify the role of E3 ubiquitin ligase Znf179 in neurodegenerative TDP-43-UBI (ubiquitinated inclusions) (+) proteinopathy, the effect of Znf179-mediated TDP-43 polyubiquitination on TDP-43 protein stability, aggregate formation and nucleus/cytoplasm mislocalization were evaluated in vitro cell culture system and in vivo animal model. RESULTS: Here we report that Znf179 is a RING E3 ubiquitin ligase which possesses autoubiquitination feature and regulates 26S proteasome activity through modulating the protein expression levels of 19S/20S proteasome subunits. Our immunoprecipitation assay and MS analysis results revealed that the neuropathological TDP-43 protein is one of its E3 ligase substrate. Znf179 interactes with TDP-43 protein and mediates polyubiquitination of TDP-43 in vitro and in vivo. In neurodegenerative TDP-43 proteinopathy, we found that Znf179-mediated polyubiquitination of TDP-43 accelerates its protein turnover rate and attenuates insoluble pathologic TDP-43 aggregates, while knockout of Znf179 in mouse brain results in accumulation of insoluble TDP-43 and cytosolic TDP-43 inclusions in cortex, hippocampus and midbrain regions. CONCLUSIONS: Here we unveil the important role for the novel E3 ligase Znf179 in TDP-43-mediated neuropathy, and provide a potential therapeutic strategy for combating ALS/ FTLD-U neurodegenerative pathologies.

Metabolism and mis-metabolism of the neuropathological signature protein TDP-43.

TDP-43 (also known as TARDBP) is a pathological signature protein of neurodegenerative diseases, with TDP-43 proteinopathies including frontotemporal lobar degeneration (FTLD)-TDP and amyotrophic lateral sclerosis (ALS)-TDP. These TDP-43 proteinopathies are characterized by cytoplasmic insoluble TDP-43-positive aggregates in the diseased cells, the formation of which requires the seeding of TDP-25 fragment generated by caspase cleavage of TDP-43. We have investigated the metabolism and mis-metabolism of TDP-43 in cultured cells and found that endogenous and exogenously overexpressed TDP-43 is degraded not only by the ubiquitin proteasome system (UPS) and macroautophagy, but also by the chaperone-mediated autophagy (CMA) mediated through an interaction between Hsc70 (also known as HSPA8) and ubiquitylated TDP-43. Furthermore, proteolytic cleavage of TDP-43 by caspase(s) is a necessary intermediate step for degradation of the majority of the TDP-43 protein, with the TDP-25 and TDP-35 fragments being the main substrates. Finally, we have determined the threshold level of the TDP-25 fragment that is necessary for formation of the cytosolic TDP-43-positive aggregates in cells containing the full-length TDP-43 at an elevated level close to that found in patients with TDP-43 proteinopathies. A comprehensive model of the metabolism and mis-metabolism of TDP-43 in relation to these findings is presented.

Co-regulation of mRNA translation by TDP-43 and Fragile X Syndrome protein FMRP.

For proper mammalian brain development and functioning, the translation of many neuronal mRNAs needs to be repressed without neuronal activity stimulations. We have discovered that the expression of a subclass of neuronal proteins essential for neurodevelopment and neuron plasticity is co-regulated at the translational level by TDP-43 and the Fragile X Syndrome protein FMRP. Using molecular, cellular and imaging approaches, we show that these two RNA-binding proteins (RBP) co-repress the translation initiation of Rac1, Map1b and GluR1 mRNAs, and consequently the hippocampal spinogenesis. The co-repression occurs through binding of TDP-43 to mRNA(s) at specific UG/GU sequences and recruitment of the inhibitory CYFIP1-FMRP complex by its glycine-rich domain. This novel regulatory scenario could be utilized to silence a significant portion of around 160 common target mRNAs of the two RBPs. The study establishes a functional/physical partnership between FMRP and TDP-43 that mechanistically links several neurodevelopmental disorders and neurodegenerative diseases.

Therapeutic effect of berberine on TDP-43-related pathogenesis in FTLD and ALS.

BACKGROUND: In the central nervous system regions of the sporadic and familial FTLD and ALS patients, TDP-43 has been identified as the major component of UBIs inclusions which is abnormally hyperphosphorylated, ubiquitinated, and cleaved into C-terminal fragments to form detergent-insoluble aggregates. So far, the effective drugs for FTLD and ALS neurodegenerative diseases are yet to be developed. Autophagy has been demonstrated as the major metabolism route of the pathological TDP-43 inclusions, hence activation of autophagy is a potential therapeutic strategy for TDP-43 pathogenesis in FTLD and ALS. Berberine, a traditional herbal medicine, is an inhibitor of mTOR signal and an activator for autophagy. Berberine has been implicated in several kinds of diseases, including the neuronal-related pathogenesis, such as Parkinson's, Huntington's and Alzheimer's diseases. However, the therapeutic effect of berberine on FTLD or ALS pathology has never been investigated. RESULTS: Here we studied the molecular mechanism of berberine in cell culture model with TDP-43 proteinopathies, and found that berberine is able to reverse the processing of insoluble TDP-43 aggregates formation through deregulation of mTOR/p70S6K signal and activation of autophagic degradation pathway. And inhibition of autophagy by specific autophagosome inhibitor, 3-MA, reverses the effect of berberine on reducing the accumulation of insoluble TDP-43 and aggregates formation. These results gave us the notion that inhibition of autophagy by 3-MA reverses the effect of berberine on TDP-43 pathogenesis, and activation of mTOR-regulated autophagy plays an important role in berberine-mediated therapeutic effect on TDP-43 proteinopathies. CONCLUSION: We supported an important notion that the traditional herb berberine is a potential alternative therapy for TDP-43-related neuropathology. Here we demonstrated that berberine is able to reverse the processing of insoluble TDP-43 aggregates formation through deregulation of mTOR/p70S6K signal and activation of autophagic degradation pathway. mTOR-autophagy signals plays an important role in berberine-mediated autophagic clearance of TDP-43 aggregates. Exploring the detailed mechanism of berberine on TDP-43 proteinopathy provides a better understanding for the therapeutic development in FTLD and ALS.

Rapamycin alleviates pathogenesis of a new Drosophila model of ALS-TDP.

TDP-43 is a multi-functional RNA/DNA-binding protein, well-conserved among many species including mammals and Drosophila. However, it is also a major component of the pathological inclusions associated with degenerating motor neurons of amyotrophic lateral sclerosis (ALS). Further, TDP-43 is a signature protein in one subtype of frontotemporal degeneration, FTLD-U. Currently, there are no effective drugs for these neurodegenerative diseases. We describe the generation and characterization of a new fly model of ALS-TDP with transgenic expression of the Drosophila ortholog of TDP-43, dTDP, in adult flies under the control of a temperature-sensitive motor neuron-specific GAL4, thus bypassing the deleterious effect of dTDP during development. Diminished lifespan as well as impaired locomotor activities of the flies following induction of dTDP overexpression have been observed. Dissection of the T1/T2 region of the thoracic ganglia has revealed loss of these neurons. To counter the defects in this fly model of ALS-TDP, we have examined the therapeutic effects of the autophagy activator, rapamycin. Although harmful to the control flies, administration of 400 µM rapamycin before the induction of dTDP overexpression can significantly reduce the number of neurons bearing dTDP (+) aggregates, as well as partially rescue the diminished lifespan and locomotive defects of the ALS-TDP flies. Furthermore, we identify S6K, a downstream mediator of the TOR pathway, as one genetic modifier of dTDP. In sum, this Drosophila model of ALS-TDP under temporal and spatial control presents a useful new genetic tool for the screening and validation of therapeutic drugs for ALS. Furthermore, the data support our previous finding that autophagy activators including rapamycin are potential therapeutic drugs for the progression of neurodegenerative diseases with TDP-43 proteinopathies.

Autophagy activators rescue and alleviate pathogenesis of a mouse model with proteinopathies of the TAR DNA-binding protein 43.

TDP-43 is a multifunctional DNA/RNA-binding protein that has been identified as the major component of the cytoplasmic ubiquitin (+) inclusions (UBIs) in diseased cells of frontotemporal lobar dementia (FTLD-U) and amyotrophic lateral sclerosis (ALS). Unfortunately, effective drugs for these neurodegenerative diseases are yet to be developed. We have tested the therapeutic potential of rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR) and three other autophagy activators (spermidine, carbamazepine, and tamoxifen) in a FTLD-U mouse model with TDP-43 proteinopathies. Rapamycin treatment has been reported to be beneficial in some animal models of neurodegenerative diseases but not others. Furthermore, the effects of rapamycin treatment in FTLD-U have not been investigated. We show that rapamycin treatment effectively rescues the learning/memory impairment of these mice at 3 mo of age, and it significantly slows down the age-dependent loss of their motor function. These behavioral improvements upon rapamycin treatment are accompanied by a decreased level of caspase-3 and a reduction of neuron loss in the forebrain of FTLD-U mice. Furthermore, the number of cells with cytosolic TDP-43 (+) inclusions and the amounts of full-length TDP-43 as well as its cleavage products (35 kDa and 25 kDa) in the urea-soluble fraction of the cellular extract are significantly decreased upon rapamycin treatment. These changes in TDP-43 metabolism are accompanied by rapamycin-induced decreases in mTOR-regulated phospho-p70 S6 kinase (P-p70) and the p62 protein, as well as increases in the autophagic marker LC3. Finally, rapamycin as well as spermidine, carbamazepine, and tamoxifen could also rescue the motor dysfunction of 7-mo-old FTLD-U mice. These data suggest that autophagy activation is a potentially useful route for the therapy of neurodegenerative diseases with TDP-43 proteinopathies.

Modulation of mGluR-dependent MAP1B translation and AMPA receptor endocytosis by microRNA miR-146a-5p.

The translation of dendritic microtubule-associated protein 1B (MAP1B) is exaggerated upon group I mGluR activation leading to AMPA receptor (AMPAR) endocytosis and consequent long-term depression. However, the mechanisms of regulation of MAP1B protein synthesis in the mature dendrites remain unclear. Here we have identified miR-146a-5p that targets the 3' UTR of MAP1B mRNA and represses its translation. Inhibition of the endogenous miR-146a-5p in mouse cultured hippocampal neurons triggers an increase of the dendritic MAP1B protein as well as the internalization of AMPARs, resulting in a decline in synaptic transmission. Conversely, enforced expression of miR-146a-5p inhibits MAP1B translation and attenuates group I mGluR-induced AMPAR endocytosis. Moreover, siRNA-mediated knockdown of MAP1B recovers the impairment of synaptic transmission caused by inhibition of miR-146a-5p. These results reveal that miR-146a-5p modulates the synaptic plasticity via repression of MAP1B protein synthesis.

DNA 5-methylcytosine demethylation activities of the mammalian DNA methyltransferases.

Methylation at the 5-position of DNA cytosine on the vertebrate genomes is accomplished by the combined catalytic actions of three DNA methyltransferases (DNMTs), the de novo enzymes DNMT3A and DNMT3B and the maintenance enzyme DNMT1. Although several metabolic routes have been suggested for demethylation of the vertebrate DNA, whether active DNA demethylase(s) exist has remained elusive. Surprisingly, we have found that the mammalian DNMTs, and likely the vertebrates DNMTs in general, can also act as Ca(2+) ion- and redox state-dependent active DNA demethylases. This finding suggests new directions for reinvestigation of the structures and functions of these DNMTs, in particular their roles in Ca(2+) ion-dependent biological processes, including the genome-wide/local DNA demethylation during early embryogenesis, cell differentiation, neuronal activity-regulated gene expression, and carcinogenesis.

The mammalian de novo DNA methyltransferases DNMT3A and DNMT3B are also DNA 5-hydroxymethylcytosine dehydroxymethylases.

For cytosine (C) demethylation of vertebrate DNA, it is known that the TET proteins could convert 5-methyl C (5-mC) to 5-hydroxymethyl C (5-hmC). However, DNA dehydroxymethylase(s), or enzymes able to directly convert 5-hmC to C, have been elusive. We present in vitro evidence that the mammalian de novo DNA methyltransferases DNMT3A and DNMT3B, but not the maintenance enzyme DNMT1, are also redox-dependent DNA dehydroxymethylases. Significantly, intactness of the C methylation catalytic sites of these de novo enzymes is also required for their 5-hmC dehydroxymethylation activity. That DNMT3A and DNMT3B function bidirectionally both as DNA methyltransferases and as dehydroxymethylases raises intriguing and new questions regarding the structural and functional aspects of these enzymes and their regulatory roles in the dynamic modifications of the vertebrate genomes during development, carcinogenesis, and gene regulation.

G-CSF rescues the memory impairment of animal models of Alzheimer's disease.

Most of the current clinical treatments for Alzheimer's disease (AD) are largely symptomatic and can have serious side effects. We have tested the feasibility of using the granulocyte colony-stimulating factor (G-CSF), which is known to mobilize hematopoietic stem cells (HSCs) from the bone marrow into the peripheral blood, as a therapeutic agent for AD. Subcutaneous administration of G-CSF into two different beta-amyloid (Abeta)-induced AD mouse models substantially rescued their cognitive/memory functions. The rescue was accompanied by the accumulation of 5-bromo-2'deoxyuridine-positive HSCs, as well as local neurogenesis surrounding the Abeta aggregates. Furthermore, the level of acetylcholine in the brains of Tg2576 mice was considerably enhanced upon G-CSF treatment. We suggest that G-CSF, a drug already extensively used for treating chemotherapy-induced neutropenia, should be pursued as a novel, noninvasive therapeutic agent for the treatment of AD.

Misregulated progesterone secretion and impaired pregnancy in Cyp11a1 transgenic mice.

Normal pregnancy is supported by increased levels of progesterone (P4), which is secreted from ovarian luteal cells via enzymatic steps catalyzed by P450scc (CYP11A1) and HSD3B. The development and maintenance of corpora lutea during pregnancy, however, are less well understood. Here we used Cyp11a1 transgenic mice to delineate the steps of luteal cell differentiation during pregnancy. Cyp11a1 in a bacterial artificial chromosome was injected into mouse embryos to generate transgenic mice with transgene expression that recapitulated endogenous Cyp11a1 expression. Cyp11a1 transgenic females displayed reduced pregnancy rate, impaired implantation and placentation, and decreased litter size in utero, although they produced comparable numbers of blastocysts. The differentiation of transgenic luteal cells was delayed during early pregnancy as shown by the delayed activation of genes involved in steroidogenesis and cholesterol availability. Luteal cell mitochondria were elongated, and their numbers were reduced, with morphology and numbers similar to those observed in granulosa cells. Transgenic luteal cells accumulated lipid droplets and secreted less progesterone during early pregnancy. The progesterone level returned to normal on gestation day 9 but was not properly withdrawn at term, leading to delayed stillbirth. P4 supplementation rescued the implantation rates but not the ovarian defects. Thus, overexpression of Cyp11a1 disrupts normal development of the corpus luteum, leading to progesterone insufficiency during early pregnancy. Misregulation of the progesterone production in Cyp11a1 transgenic mice during pregnancy resulted in aberrant implantation, anomalous placentation, and delayed parturition.

Similar dose-dependence of motor neuron cell death caused by wild type human TDP-43 and mutants with ALS-associated amino acid substitutions.

BACKGROUND: TDP-43, a multi-functional DNA/ RNA-binding protein encoded by the TARDBP gene, has emerged as a major patho-signature factor of the ubiquitinated intracellular inclusions (UBIs) in the diseased cells of a range of neurodegenerative diseases. Mutations in at least 9 different genes including TARDBP have been identified in ALS with TDP-43 (+)-UBIs. Thus far, the pathogenic role(s) of the more than 30 ALS-associated mutations in the TARDBP gene has not been well defined. RESULTS: By transient DNA transfection studies, we show that exogenously expressed human TDP-43 (hTDP-43), either wild type (WT) or 2 different ALS mutant (MT) forms, could cause significantly higher apoptotic death rate of a mouse spinal motor neuron-like cell line (NSC34) than other types of cells, e.g. mouse neuronal Neuro2a and human fibroblast HEK293T cells. Furthermore, at the same plasmid DNA dose(s) used for transfection, the percentages of NSC34 cell death caused by the 2 exogenously expressed hTDP-43 mutants are all higher than that caused by the WT hTDP-43. Significantly, the above observations are correlated with higher steady-state levels of the mutant hTDP-43 proteins as well as their stabilities than the WT. CONCLUSIONS: Based on these data and previous transgenic TDP-43 studies in animals or cell cultures, we suggest that one major common consequence of the different ALS-associated TDP-43 mutations is the stabilization of the hTDP-43 polypeptide. The resulting elevation of the steady state level of hTDP-43 in combination with the relatively low tolerance of the spinal motor neurons to the increased amount of hTDP-43 lead to the neurodegeneration and pathogenesis of ALS, and of diseases with TDP-43 proteinopathies in general.

JNK-mediated turnover and stabilization of the transcription factor p45/NF-E2 during differentiation of murine erythroleukemia cells.

Regulation of the homeostatic concentrations of specific sets of transcription factors is essential for correct programming of cell proliferation and differentiation. We have characterized the signal transduction pathways regulating the catabolisis of p45/NF-E2, a bZIP factor activating the erythroid and megakaryocytic gene transcription. Through use of different approaches including nano-scale proteomics, we show that activated-JNK, or Phospho-JNK (P-JNK), physically interacts with p45/NF-E2 and phosphorylates its Ser157 residue. This reaction leads to the poly-ubiquitination of p45/NF-E2 at one or more of six Lys residues, one of which being also a sumoylation site, and its degradation through the proteasome pathway. Significantly, this regulatory pathway of p45/NF-E2 by P-JNK exists only in uninduced murine erythroleukemia (MEL) cells but not in differentiated MEL cells in which JNK is inactivated on DMSO induction. Based on the above data and analysis of the chromatin-binding kinetics of p45/NF-E2 and the erythroid gene repressor Bach1 during the early phase of MEL differentiation, we suggest a model for the regulation of erythroid maturation. In the model, the posttranslational modifications and turnover of p45/NF-E2, as mediated by P-JNK, contribute to the control of its homeostatic concentration and consequently, its regulatory functions in the progression of erythroid differentiation and erythroid gene expression.

TDP-43 regulates the mammalian spinogenesis through translational repression of Rac1.

Impairment of learning and memory is a significant pathological feature of many neurodegenerative diseases including FTLD-TDP. Appropriate regulation and fine tuning of spinogenesis of the dendrites, which is an integral part of the learning/memory program of the mammalian brain, are essential for the normal function of the hippocampal neurons. TDP-43 is a nucleic acid-binding protein implicated in multi-cellular functions and in the pathogenesis of a range of neurodegenerative diseases including FTLD-TDP and ALS. We have combined the use of single-cell dye injection, shRNA knockdown, plasmid rescue, immunofluorescence staining, Western blot analysis and patch clamp electrophysiological measurement of primary mouse hippocampal neurons in culture to study the functional role of TDP-43 in mammalian spinogenesis. We found that depletion of TDP-43 leads to an increase in the number of protrusions/spines as well as the percentage of matured spines among the protrusions. Significantly, the knockdown of TDP-43 also increases the level of Rac1 and its activated form GTP-Rac1, a known positive regulator of spinogenesis. Clustering of the AMPA receptors on the dendritic surface and neuronal firing are also induced by depletion of TDP-43. Furthermore, use of an inhibitor of Rac1 activation negatively regulated spinogenesis of control hippocampal neurons as well as TDP-43-depleted hippocampal neurons. Mechanistically, RT-PCR assay and cycloheximide chase experiments have indicated that increases in Rac1 protein upon TDP-43 depletion is regulated at the translational level. These data together establish that TDP-43 is an upstream regulator of spinogenesis in part through its action on the Rac1 â†’ GTP-Rac1 â†’ AMPAR pathway. This study provides the first evidence connecting TDP-43 with the GTP-Rac1 â†’ AMPAR regulatory pathway of spinogenesis. It establishes that mis-metabolism of TDP-43, as occurs in neurodegenerative diseases with TDP-43 proteinopathies, e.g., FTLD-TDP, would alter its homeostatic cellular concentration, thus leading to impairment of hippocampal plasticity.

Cabozantinib promotes erythroid differentiation in K562 erythroleukemia cells through global changes in gene expression and JNK activation.

Cabozantinib is a potent tyrosine kinase inhibitor with multiple targets including MET, VEGFR2, RET, KIT, and FLT3. Cabozantinib is widely used for the treatment of medullary thyroid cancer and renal cell carcinoma. We recently suggested cabozantinib as a potential therapeutic alternative for acute myeloid leukemia (AML) patients with FLT3-internal tandem duplication (FLT3-ITD). Here, we report that cabozantinib can promote differentiation in erythroid leukemia cells. We found that K562 erythroid leukemia cells treated with 1 µM cabozantinib for 72 h underwent erythroid lineage differentiation. Transcriptomic analysis revealed that various pathways associated with heme biosynthesis, hemoglobin production, and GATA1 targets were upregulated, whereas cell survival pathways were downregulated. Further examination revealed that cabozantinib-induced erythroid differentiation is at least in part regulated by JNK activation and phosphorylation. Levels of phosphorylated BCR-ABL, AKT, STAT5, ERK, and p38 also decreased following cabozantinib treatment. Therefore, we indicate that cabozantinib has dual functions. First, it induces K562 cell differentiation toward the erythroid lineage by upregulating heme biosynthesis, globin synthesis, and erythroid-associated reactions. Second, cabozantinib inhibits K562 cell proliferation by inhibiting the phosphorylation of BCR-ABL and the downstream MAPK, PI3K-AKT, and JAK-STAT signaling pathways.

Genetic Disruption of KLF1 K74 SUMOylation in Hematopoietic System Promotes Healthy Longevity in Mice.

The quest for rejuvenation and prolonged lifespan through transfusion of young blood has been studied for decades with the hope of unlocking the mystery of the key substance(s) that exists in the circulating blood of juvenile organisms. However, a pivotal mediator has yet been identified. Here, atypical findings are presented that are observed in a knockin mouse model carrying a lysine to arginine substitution at residue 74 of Krüppel-like factor 1 (KLF1/EKLF), the SUMOylation-deficient Klf1K74R/K74R mouse, that displayed significant improvement in geriatric disorders and lifespan extension. Klf1K74R/K74R mice exhibit a marked delay in age-related physical performance decline and disease progression as evidenced by physiological and pathological examinations. Furthermore, the KLF1(K74R) knockin affects a subset of lymphoid lineage cells; the abundance of tumor infiltrating effector CD8+ T cells and NKT cells is increased resulting in antitumor immune enhancement in response to tumor cell administration. Significantly, infusion of hematopoietic stem cells (HSCs) from Klf1K74R/K74R mice extends the lifespan of the wild-type mice. The Klf1K74R/K74R mice appear to be an ideal animal model system for further understanding of the molecular/cellular basis of aging and development of new strategies for antiaging and prevention/treatment of age-related diseases thus extending the healthspan as well as lifespan.

Structural insights into TDP-43 in nucleic-acid binding and domain interactions.

TDP-43 is a pathogenic protein: its normal function in binding to UG-rich RNA is related to cystic fibrosis, and inclusion of its C-terminal fragments in brain cells is directly linked to frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Here we report the 1.65 A crystal structure of the C-terminal RRM2 domain of TDP-43 in complex with a single-stranded DNA. We show that TDP-43 is a dimeric protein with two RRM domains, both involved in DNA and RNA binding. The crystal structure reveals the basis of TDP-43's TG/UG preference in nucleic acids binding. It also reveals that RRM2 domain has an atypical RRM-fold with an additional beta-strand involved in making protein-protein interactions. This self association of RRM2 domains produced thermal-stable RRM2 assemblies with a melting point greater than 85 degrees C as monitored by circular dichroism at physiological conditions. These studies thus characterize the recognition between TDP-43 and nucleic acids and the mode of RRM2 self association, and provide molecular models for understanding the role of TDP-43 in cystic fibrosis and the neurodegenerative diseases related to TDP-43 proteinopathy.