Identification of miRNAs That Mediate Protective Functions of Anti-Cancer Drugs During White Matter Ischemic Injury.

We have previously shown that two anti-cancer drugs, CX-4945 and MS-275, protect and preserve white matter (WM) architecture and improve functional recovery in a model of WM ischemic injury. While both compounds promote recovery, CX-4945 is a selective Casein kinase 2 (CK2) inhibitor and MS-275 is a selective Class I histone deacetylase (HDAC) inhibitor. Alterations in microRNAs (miRNAs) mediate some of the protective actions of these drugs. In this study, we aimed to (1) identify miRNAs expressed in mouse optic nerves (MONs); (2) determine which miRNAs are regulated by oxygen glucose deprivation (OGD); and (3) determine the effects of CX-4945 and MS-275 treatment on miRNA expression. RNA isolated from MONs from control and OGD-treated animals with and without CX-4945 or MS-275 treatment were quantified using NanoString nCounter® miRNA expression profiling. Comparative analysis of experimental groups revealed that 12 miRNAs were expressed at high levels in MONs. OGD upregulated five miRNAs (miR-1959, miR-501-3p, miR-146b, miR-201, and miR-335-3p) and downregulated two miRNAs (miR-1937a and miR-1937b) compared to controls. OGD with CX-4945 upregulated miR-1937a and miR-1937b, and downregulated miR-501-3p, miR-200a, miR-1959, and miR-654-3p compared to OGD alone. OGD with MS-275 upregulated miR-2134, miR-2141, miR-2133, miR-34b-5p, miR-153, miR-487b, miR-376b, and downregulated miR-717, miR-190, miR-27a, miR-1959, miR-200a, miR-501-3p, and miR-200c compared to OGD alone. Interestingly, miR-501-3p and miR-1959 were the only miRNAs upregulated by OGD, and downregulated by OGD plus CX-4945 and MS-275. Therefore, we suggest that protective functions of CX-4945 or MS-275 against WM injury maybe mediated, in part, through miRNA expression.

The BHMT-betaine methylation pathway epigenetically modulates oligodendrocyte maturation.

Research into the epigenome is of growing importance as a loss of epigenetic control has been implicated in the development of neurodegenerative diseases. Previous studies have implicated aberrant DNA and histone methylation in multiple sclerosis (MS) disease pathogenesis. We have previously reported that the methyl donor betaine is depleted in MS and is linked to changes in histone H3 trimethylation (H3K4me3) in neurons. We have also shown that betaine increases histone methyltransferase activity by activating chromatin bound betaine homocysteine S-methyltransferase (BHMT). Here, we investigated the role of the BHMT-betaine methylation pathway in oligodendrocytes. Immunocytochemistry in the human MO3.13 cell line, primary rat oligodendrocytes, and tissue from MS postmortem brain confirmed the presence of the BHMT enzyme in the nucleus in oligodendrocytes. BHMT expression is increased 2-fold following oxidative insult, and qRT-PCR demonstrated that betaine can promote an increase in expression of oligodendrocyte maturation genes SOX10 and NKX-2.2 under oxidative conditions. Chromatin fractionation provided evidence of a direct interaction of BHMT on chromatin and co-IP analysis indicates an interaction between BHMT and DNMT3a. Our data show that both histone and DNA methyltransferase activity are increased following betaine administration. Betaine effects were shown to be dependent on BHMT expression following siRNA knockdown of BHMT. This is the first report of BHMT expression in oligodendrocytes and suggests that betaine acts through BHMT to modulate histone and DNA methyltransferase activity on chromatin. These data suggest that methyl donor availability can impact epigenetic changes and maturation in oligodendrocytes.

CK2 inhibition confers functional protection to young and aging axons against ischemia by differentially regulating the CDK5 and AKT signaling pathways.

White matter (WM) is injured in most strokes, which contributes to functional deficits during recovery. Casein kinase 2 (CK2) is a protein kinase that is expressed in brain, including WM. To assess the impact of CK2 inhibition on axon recovery following oxygen glucose deprivation (OGD), mouse optic nerves (MONs), which are pure WM tracts, were subjected to OGD with or without the selective CK2 inhibitor CX-4945. CX-4945 application preserved axon function during OGD and promoted axon function recovery when applied before or after OGD. This protective effect of CK2 inhibition correlated with preservation of oligodendrocytes and conservation of axon structure and axonal mitochondria. To investigate the pertinent downstream signaling pathways, siRNA targeting the CK2α subunit identified CDK5 and AKT as downstream molecules. Consequently, MK-2206 and roscovitine, which are selective AKT and CDK5 inhibitors, respectively, protected young and aging WM function only when applied before OGD. However, a novel pan-AKT allosteric inhibitor, ARQ-092, which targets both the inactive and active conformations of AKT, conferred protection to young and aging axons when applied before or after OGD. These results suggest that AKT and CDK5 signaling contribute to the WM functional protection conferred by CK2 inhibition during ischemia, while inhibition of activated AKT signaling plays the primary role in post-ischemic protection conferred by CK2 inhibition in WM independent of age. CK2 inhibitors are currently being used in clinical trials for cancer patients; therefore, our results will provide rationale for repurposing these drugs as therapeutic options for stroke patients by adding novel targets.

Transport of Non-Transferrin Bound Iron to the Brain: Implications for Alzheimer's Disease.

A direct correlation between brain iron and Alzheimer's disease (AD) raises questions regarding the transport of non-transferrin-bound iron (NTBI), a toxic but less researched pool of circulating iron that is likely to increase due to pathological and/or iatrogenic systemic iron overload. Here, we compared the distribution of radiolabeled-NTBI (59Fe-NTBI) and transferrin-bound iron (59Fe-Tf) in mouse models of iron overload in the absence or presence of inflammation. Following a short pulse, most of the 59Fe-NTBI was taken up by the liver, followed by the kidney, pancreas, and heart. Notably, a strong signal of 59Fe-NTBI was detected in the brain ventricular system after 2 h, and the brain parenchyma after 24 h. 59Fe-Tf accumulated mainly in the femur and spleen, and was transported to the brain at a much slower rate than 59Fe-NTBI. In the kidney, 59Fe-NTBI was detected in the cortex after 2 h, and outer medulla after 24 hours. Most of the 59Fe-NTBI and 59Fe-Tf from the kidney was reabsorbed; negligible amount was excreted in the urine. Acute inflammation increased the uptake of 59Fe-NTBI by the kidney and brain from 2-24 hours. Chronic inflammation, on the other hand, resulted in sequestration of iron in the liver and kidney, reducing its transport to the brain. These observations provide direct evidence for the transport of NTBI to the brain, and reveal a complex interplay between inflammation and brain iron homeostasis. Further studies are necessary to determine whether transient increase in NTBI due to systemic iron overload is a risk factor for AD.

Alpha-synuclein modulates retinal iron homeostasis by facilitating the uptake of transferrin-bound iron: Implications for visual manifestations of Parkinson's disease.

Aggregation of α-synuclein (α-syn) in neurons of the substantia nigra is diagnostic of Parkinson's disease (PD), a neuro-motor disorder with prominent visual symptoms. Here, we demonstrate that α-syn, the principal protein involved in the pathogenesis of PD, is expressed widely in the neuroretina, and facilitates the uptake of transferrin-bound iron (Tf-Fe) by retinal pigment epithelial (RPE) cells that form the outer blood-retinal barrier. Absence of α-syn in knock-out mice (α-syn(-/-)) resulted in down-regulation of ferritin in the neuroretina, indicating depletion of cellular iron stores. A similar phenotype of iron deficiency was observed in the spleen, femur, and brain tissue of α-syn(-)(/-) mice, organs that utilize mainly Tf-Fe for their metabolic needs. The liver and kidney, organs that take up significant amounts of non-Tf-bound iron (NTBI), showed minimal change. Evaluation of the underlying mechanism in the human RPE47 cell line suggested a prominent role of α-syn in the uptake of Tf-Fe by modulating the endocytosis and recycling of transferrin (Tf)/transferrin-receptor (TfR) complex. Down-regulation of α-syn in RPE cells by RNAi resulted in the accumulation of Tf/TfR complex in common recycling endosomes (CREs), indicating disruption of recycling to the plasma membrane. Over-expression of exogenous α-syn in RPE cells, on the other hand, up-regulated ferritin and TfR expression. Interestingly, exposure to exogenous iron increased membrane association and co-localization of α-syn with TfR, supporting its role in iron uptake by the Tf/TfR complex. Together with our observations indicating basolateral expression of α-syn and TfR on RPE cells in vivo, this study reveals a novel function of α-syn in the uptake of Tf-Fe by the neuroretina. It is likely that retinal iron dyshomeostasis due to impaired or altered function of α-syn contributes to the visual symptoms associated with PD.

Prion Protein-Hemin Interaction Upregulates Hemoglobin Synthesis: Implications for Cerebral Hemorrhage and Sporadic Creutzfeldt-Jakob Disease.

Hemin is known to induce endocytosis of prion-protein (PrP(C)) from the neuronal plasma membrane, potentially limiting propagation of the disease causing PrP-scrapie (PrP(Sc)) isoform. Hemin is therefore an attractive disease-modifying option for sporadic Creutzfeldt-Jakob disease (sCJD), a human prion disorder with no effective treatment. The hemin-PrP(C) interaction is also of interest in cerebral-hemorrhage (CH), a condition where potentially toxic hemin molecules come in contact with neuronal PrP(C). Interestingly, PrP(C) is upregulated in penumbric neurons surrounding CH and is known to confer neuroprotection in a dose-dependent manner. The underlying mechanism, however, is not clear. Here, we report that hemin binds PrP(C) on diverse cell lines, resulting in its aggregation or degradation in a cell-type specific manner. Surprisingly, the hemin-PrP(C) interaction upregulates Hb synthesis in hematopoietic cells, a response reversed by deleting the hemin-binding octa-peptide repeat region of PrP(C). A similar response is noted in brain organotypic cultures where exposure to hemin induces significantly more α-globin in wild-type (PrP(+/+)) relative to PrP-knock-out (PrP(-/-)) samples. Furthermore, red blood cells and brain tissue from PrP(-/-) mice show significantly less α-globin relative to PrP(+/+) controls, indicating a positive effect of PrP(C) on Hb synthesis under physiological conditions as well. Surprisingly, levels of α-globin are significantly higher in sCJD brain tissue relative to controls, suggesting compensatory upregulation of Hb synthesis by surviving neurons or misregulation in diseased brains. These observations reveal a unique function of PrP(C) that is likely to impact the therapeutic management of CH and sCJD.

The prion-ZIP connection: From cousins to partners in iron uptake.

Converging observations from disparate lines of inquiry are beginning to clarify the cause of brain iron dyshomeostasis in sporadic Creutzfeldt-Jakob disease (sCJD), a neurodegenerative condition associated with the conversion of prion protein (PrP(C)), a plasma membrane glycoprotein, from α-helical to a ß-sheet rich PrP-scrapie (PrP(Sc)) isoform. Biochemical evidence indicates that PrP(C) facilitates cellular iron uptake by functioning as a membrane-bound ferrireductase (FR), an activity necessary for the transport of iron across biological membranes through metal transporters. An entirely different experimental approach reveals an evolutionary link between PrP(C) and the Zrt, Irt-like protein (ZIP) family, a group of proteins involved in the transport of zinc, iron, and manganese across the plasma membrane. Close physical proximity of PrP(C) with certain members of the ZIP family on the plasma membrane and increased uptake of extracellular iron by cells that co-express PrP(C) and ZIP14 suggest that PrP(C) functions as a FR partner for certain members of this family. The connection between PrP(C) and ZIP proteins therefore extends beyond common ancestry to that of functional cooperation. Here, we summarize evidence supporting the facilitative role of PrP(C) in cellular iron uptake, and implications of this activity on iron metabolism in sCJD brains.

Prion protein functions as a ferrireductase partner for ZIP14 and DMT1.

Excess circulating iron is stored in the liver, and requires reduction of non-Tf-bound iron (NTBI) and transferrin (Tf) iron at the plasma membrane and endosomes, respectively, by ferrireductase (FR) proteins for transport across biological membranes through divalent metal transporters. Here, we report that prion protein (PrP(C)), a ubiquitously expressed glycoprotein most abundant on neuronal cells, functions as a FR partner for divalent-metal transporter-1 (DMT1) and ZIP14. Thus, absence of PrP(C) in PrP-knock-out (PrP(-/-)) mice resulted in markedly reduced liver iron stores, a deficiency that was not corrected by chronic or acute administration of iron by the oral or intraperitoneal routes. Likewise, preferential radiolabeling of circulating NTBI with (59)Fe revealed significantly reduced uptake and storage of NTBI by the liver of PrP(-/-) mice relative to matched PrP(+/+) controls. However, uptake, storage, and utilization of ferritin-bound iron that does not require reduction for uptake were increased in PrP(-/-) mice, indicating a compensatory response to the iron deficiency. Expression of exogenous PrP(C) in HepG2 cells increased uptake and storage of ferric iron (Fe(3+)), not ferrous iron (Fe(2+)), from the medium, supporting the function of PrP(C) as a plasma membrane FR. Coexpression of PrP(C) with ZIP14 and DMT1 in HepG2 cells increased uptake of Fe(3+) significantly, and surprisingly, increased the ratio of N-terminally truncated PrP(C) forms lacking the FR domain relative to full-length PrP(C). Together, these observations indicate that PrP(C) promotes, and possibly regulates, the uptake of NTBI through DMT1 and Zip14 via its FR activity. Implications of these observations for neuronal iron homeostasis under physiological and pathological conditions are discussed.

Prion protein promotes kidney iron uptake via its ferrireductase activity.

Brain iron-dyshomeostasis is an important cause of neurotoxicity in prion disorders, a group of neurodegenerative conditions associated with the conversion of prion protein (PrP(C)) from its normal conformation to an aggregated, PrP-scrapie (PrP(Sc)) isoform. Alteration of iron homeostasis is believed to result from impaired function of PrP(C) in neuronal iron uptake via its ferrireductase activity. However, unequivocal evidence supporting the ferrireductase activity of PrP(C) is lacking. Kidney provides a relevant model for this evaluation because PrP(C) is expressed in the kidney, and ∼370 µg of iron are reabsorbed daily from the glomerular filtrate by kidney proximal tubule cells (PT), requiring ferrireductase activity. Here, we report that PrP(C) promotes the uptake of transferrin (Tf) and non-Tf-bound iron (NTBI) by the kidney in vivo and mainly NTBI by PT cells in vitro. Thus, uptake of (59)Fe administered by gastric gavage, intravenously, or intraperitoneally was significantly lower in PrP-knock-out (PrP(-/-)) mouse kidney relative to PrP(+/+) controls. Selective in vivo radiolabeling of plasma NTBI with (59)Fe revealed similar results. Expression of exogenous PrP(C) in immortalized PT cells showed localization on the plasma membrane and intracellular vesicles and increased transepithelial transport of (59)Fe-NTBI and to a smaller extent (59)Fe-Tf from the apical to the basolateral domain. Notably, the ferrireductase-deficient mutant of PrP (PrP(Δ51-89)) lacked this activity. Furthermore, excess NTBI and hemin caused aggregation of PrP(C) to a detergent-insoluble form, limiting iron uptake. Together, these observations suggest that PrP(C) promotes retrieval of iron from the glomerular filtrate via its ferrireductase activity and modulates kidney iron metabolism.

Iron in neurodegenerative disorders of protein misfolding: a case of prion disorders and Parkinson's disease.

SIGNIFICANCE: Intracellular and extracellular aggregation of a specific protein or protein fragments is the principal pathological event in several neurodegenerative conditions. We describe two such conditions: sporadic Creutzfeldt-Jakob disease (sCJD), a rare but potentially infectious and invariably fatal human prion disorder, and Parkinson's disease (PD), a common neurodegenerative condition second only to Alzheimer's disease in prevalence. In sCJD, a cell surface glycoprotein known as the prion protein (PrP(C)) undergoes a conformational change to PrP-scrapie, a pathogenic and infectious isoform that accumulates in the brain parenchyma as insoluble aggregates. In PD, α-synuclein, a cytosolic protein, forms insoluble aggregates that accumulate in neurons of the substantia nigra and cause neurotoxicity. RECENT ADVANCES: Although distinct processes are involved in the pathogenesis of sCJD and PD, both share brain iron dyshomeostasis as a common associated feature that is reflected in the cerebrospinal fluid in a disease-specific manner. CRITICAL ISSUES: Since PrP(C) and α-synuclein play a significant role in maintaining cellular iron homeostasis, it is important to understand whether the aggregation of these proteins and iron dyshomeostasis are causally related. Here, we discuss recent information on the normal function of PrP(C) and α-synuclein in cellular iron metabolism and the cellular and biochemical processes that contribute to iron imbalance in sCJD and PD. FUTURE DIRECTIONS: Improved understanding of the relationship between brain iron imbalance and protein aggregation is likely to help in the development of therapeutic strategies that can restore brain iron homeostasis and mitigate neurotoxicity.

Brain iron homeostasis: from molecular mechanisms to clinical significance and therapeutic opportunities.

Iron has emerged as a significant cause of neurotoxicity in several neurodegenerative conditions, including Alzheimer's disease (AD), Parkinson's disease (PD), sporadic Creutzfeldt-Jakob disease (sCJD), and others. In some cases, the underlying cause of iron mis-metabolism is known, while in others, our understanding is, at best, incomplete. Recent evidence implicating key proteins involved in the pathogenesis of AD, PD, and sCJD in cellular iron metabolism suggests that imbalance of brain iron homeostasis associated with these disorders is a direct consequence of disease pathogenesis. A complete understanding of the molecular events leading to this phenotype is lacking partly because of the complex regulation of iron homeostasis within the brain. Since systemic organs and the brain share several iron regulatory mechanisms and iron-modulating proteins, dysfunction of a specific pathway or selective absence of iron-modulating protein(s) in systemic organs has provided important insights into the maintenance of iron homeostasis within the brain. Here, we review recent information on the regulation of iron uptake and utilization in systemic organs and within the complex environment of the brain, with particular emphasis on the underlying mechanisms leading to brain iron mis-metabolism in specific neurodegenerative conditions. Mouse models that have been instrumental in understanding systemic and brain disorders associated with iron mis-metabolism are also described, followed by current therapeutic strategies which are aimed at restoring brain iron homeostasis in different neurodegenerative conditions. We conclude by highlighting important gaps in our understanding of brain iron metabolism and mis-metabolism, particularly in the context of neurodegenerative disorders.

The landscape of alternative splicing in buccal mucosa squamous cell carcinoma.

OBJECTIVES: Alternative splicing (AS) is a key regulatory mechanism in the process of protein synthesis generating transcriptome and proteome diversity. In this study, we attempted to identify alternative splicing in a pair of BMSCC cancer and adjacent normal tissue using RNAseq datasets and also assessed the potential of these datasets to provide quantitative measurements for alternative splicing levels. MATERIALS AND METHODS: We performed high-throughput sequencing of buccal mucosal cancer and healthy tissue cDNA library which resulted in a transcriptome map of BMSCC cancer. RNAseq analysis was performed to assess alternative splicing complexity in cancer tissue and to search splice junction sequences that represent candidate 'new' splicing events. The splice junctions were predicted by SpliceMap software and putative assembled transcripts validated using the RT-PCR. We also analyzed the coding potential of alternative spliced candidate by HMMER. RESULTS: We detected a total of 11 novel splice junctions derived mostly from alternate 5' splice site; including two of them which contained new translation initiation sites (TISs). We have identified novel IgG pseudogene and a fusion transcript of MEMO1 and RPS9, which were further confirmed by PCR from genomic DNA. We also found novel putative long non-coding RNA (lncRNA), which is antisense to SPINK5 gene. The coding potential of these AS variants revealed that alternative splicing caused premature termination, insertion/deletion of amino acid (s) or formation of novel N-terminus. CONCLUSIONS: Differential splicing of these novel AS variants between cancer and adjacent normal tissue suggests their involvement in BMSCC cancer development and progression.

Identification of novel splice variants in horn cancer by RNA-Seq analysis in Zebu cattle.

Horn cancer accounts for nearly 83% of total tumors found in Indian Zebu cattle, which results in chronic suffering and causes heavy economic losses. Alternative splicing has been frequently implicated in the various types of cancer progression. Utilizing the transcriptome sequence generated by next generation sequencing, we analyzed the transcript data for the presence of alternative splicing using BLAT program and identified 27 alternatively spliced genes, of which 12 spliced variants appeared to be the novel spliced candidates. Protein prediction of these novel spliced variants revealed that splice variation has caused either truncation of protein, insertion/deletion of stretch of amino acids or formation of unique carboxy terminus. The RT-PCR analysis confirmed the expression of 8 of the 12 novel spliced variants observed by transcriptome sequencing. Additionally, altered splicing/expression of these novel candidates between cancer and normal tissues revealed by qPCR suggests their potential involvement in the development of horn cancer.

Identification of novel transcripts deregulated in buccal cancer by RNA-seq.

The differential transcriptome analysis provides better understanding of molecular pathways leading to cancer, which in turn allows designing the effective strategies for diagnosis, therapeutic intervention and prediction of therapeutic outcome. This study describes the transcriptome analysis of buccal cancer and normal tissue by CLC Genomics Workbench from the data generated by Roche's 454 sequencing platform, which identified total of 1797 and 2655 genes uniquely expressed in normal and cancer tissues, respectively with 2466 genes expressed in both tissues. Among the genes expressed in both tissues, 1842 were up-regulated whereas 624 were down-regulated in cancer tissue. Besides transcripts known to be involved in cancer, this study led to the identification of novel transcripts, with significantly altered expression in buccal cancer tissue, providing potential targets for diagnosis and cancer therapeutics. The functional categorization by the KEGG pathway and gene ontology analysis revealed enrichment of differentially expressed transcripts to various pathways leading to cancer, including the p53 signaling pathway. Moreover, the gene ontology analysis unfolded suppression of transcripts involved in actin mediated cell contraction process. The down-regulation of four of these transcripts MYL1, ACTA1, TCAP and DESMIN in buccal cancer were further supported by quantitative PCR signifying its possible implication in the cancer progression.

A preliminary sketch of horn cancer transcriptome in Indian zebu cattle.

Horn cancer, a type of squamous cell carcinoma, in zebu cattle is an expensive affair in Indian agriculture sector, which accounts for 83.34% of total tumors found. In general, cancer tissue confirms considerably different expression patterns when compared to a normal stage. This includes not only up/down regulation, but also, the aberrant gene expression, the presence of different non-coding RNAs (ncRNAs), pseudogenes expression and genes involved in unusual pathways. We employed Roche 454 next generation sequencing platform to sequence Bos indicus cancerous and normal horn tissue transcripts. This resulted into a total of 909,345 high-confidence deep sequencing reads and detected a range of unusual transcriptional events including tumor associated genes. We also validated expression of two of the four tested genes in five other similar tissue samples by RT-qPCR. Further, seven cancer specific non-coding transcripts were accessed and a few of them have been suggested as cancer specific markers. This study for the first time provides primary transcriptome sketch of Bos indicus horn cancer tissue, and also demonstrates the suitability of the 454 sequencer for transcriptome analysis, which supports the concept of varied gene expression in cancerous condition.