Ajai K. Tripathi

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 Alzheimers disease (AD), Parkinsons 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.

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

SIGNIFICANCEnIntracellular 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 Parkinsons disease (PD), a common neurodegenerative condition second only to Alzheimers 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.nnnRECENT ADVANCESnAlthough 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.nnnCRITICAL ISSUESnSince 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.nnnFUTURE DIRECTIONSnImproved 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.

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

Background: Although primarily a neuronal protein, prion protein (PrPC) is expressed in the kidney, suggesting a functional role. Results: PrPC facilitates uptake of iron from the glomerular filtrate across kidney proximal-tubule (PT) epithelial cells. Conclusion: PrPC functions as a ferrireductase on the plasma-membrane of PT cells. Significance: A similar function in neuronal cells is likely to cause brain iron-dyshomeostasis in prion disorders. Brain iron-dyshomeostasis is an important cause of neurotoxicity in prion disorders, a group of neurodegenerative conditions associated with the conversion of prion protein (PrPC) from its normal conformation to an aggregated, PrP-scrapie (PrPSc) isoform. Alteration of iron homeostasis is believed to result from impaired function of PrPC in neuronal iron uptake via its ferrireductase activity. However, unequivocal evidence supporting the ferrireductase activity of PrPC is lacking. Kidney provides a relevant model for this evaluation because PrPC 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 PrPC 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 59Fe 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 59Fe revealed similar results. Expression of exogenous PrPC in immortalized PT cells showed localization on the plasma membrane and intracellular vesicles and increased transepithelial transport of 59Fe-NTBI and to a smaller extent 59Fe-Tf from the apical to the basolateral domain. Notably, the ferrireductase-deficient mutant of PrP (PrPΔ51–89) lacked this activity. Furthermore, excess NTBI and hemin caused aggregation of PrPC to a detergent-insoluble form, limiting iron uptake. Together, these observations suggest that PrPC promotes retrieval of iron from the glomerular filtrate via its ferrireductase activity and modulates kidney iron metabolism.

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 Parkinsons 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.

Transcriptomic dissection of myogenic differentiation signature in caprine by RNA-Seq.

Muscle growth and development from the embryonic to the adult stage of an organism consists of a series of exquisitely regulated and orchestrated changes in expression of genes leading to muscle maturation. In this study, we performed whole transcriptome profiling of adult caprine skeletal muscle derived myoblast and fused myotubes. Using Ion Torrent PGM sequencing platform, a total of 948,776 and 799,976 reads were generated in myoblasts and fused myotubes, respectively. The sequence reads were analyzed on CLC Genomics Workbench using Bos taurus RNA database to study the gene expression in both stages to study different genes responsible for muscle development and regeneration. The up and down-regulated genes were analyzed for gene ontology (GO) and KEGG pathways by Database for Annotation, Visualization and Integrated Discovery (DAVID) database. We found many genes exclusive to multinuclear fused myotubes and contractile nature of skeletal muscle, whereas up-regulated genes in myoblast stage were related to cell division and transcriptional regulation. Out of 27 genes selected for expression validation by RT-qPCR (reverse transcriptase-quantitative polymerase chain reaction), 19 genes showed the expression pattern comparable with CLC Genomics Workbench findings. Further, mRNA originated muscle specific microRNAs (miRNA-1 and miRNA-133b) were also observed in the fused myotubes along with other miRNAs with possible importance in muscle development. This study highlights important genes responsible for muscle development and differentiation in adult skeletal muscle system.

Myostatin knockdown and its effect on myogenic gene expression program in stably transfected goat myoblasts

Myostatin, a negative regulator of skeletal muscle mass, is a proven candidate to modulate skeletal muscle mass through targeted gene knockdown approach. Here, we report myostatin (MSTN) knockdown in goat myoblasts stably expressing small hairpin RNA (shRNAs) against MSTN gene through lentivirus vector-mediated integration. We observed 72% (pu2009=u20090.003) and 54% (pu2009=u20090.022) downregulation of MSTN expression with sh2 shRNA compared to empty vector control and untransduced myoblasts, respectively. The knockdown of MSTN expression was accompanied with concomitant downregulation of myogenic regulatory factor MYOD (77%, pu2009=u20090.001), MYOG (94%, pu2009=u20090.000), and MYF5 (36%, pu2009=u20090.000), cell cycle regulator p21 (62%, pu2009=u20090.000), MSTN receptor ACVR2B (23%, pu2009=u20090.061), MSTN antagonist follistatin (81%, pu2009=u20090.000), and downstream signaling mediators SMAD2 (20%, pu2009=u20090.060) and SMAD3 (49%, pu2009=u20090.006). However, the expression of MYF6 was upregulated by 14% compared to control lentivirus-transduced myoblasts (pu2009=u20090.354) and 79% compared to untransduced myoblasts (pu2009=u20090.018) in sh2 shRNA-transduced goat myoblasts cells. Although, MSTN knockdown led to sustained cell proliferation of myoblasts, the myoblasts fusion was suppressed in both MSTN knocked down and control lentivirus-transduced myoblasts. The expression of interferon response gene OAS1 was significantly upregulated in control lentivirus (10.86-fold; pu2009=u20090.000)- and sh2 (1.71-fold; pu2009=u20090.002)-integrated myoblasts compared to untransduced myoblasts. Our study demonstrates stable knockdown of MSTN in goat myoblasts cells and its potential for use in generation of transgenic goat by somatic cell nuclear transfer.

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

A direct correlation between brain iron and Alzheimers 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 2u200ah, and the brain parenchyma after 24u200ah. 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 2u200ah, 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.

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

ABSTRACT 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 (PrPC), a plasma membrane glycoprotein, from α-helical to a β-sheet rich PrP-scrapie (PrPSc) isoform. Biochemical evidence indicates that PrPC 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 PrPC 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 PrPC with certain members of the ZIP family on the plasma membrane and increased uptake of extracellular iron by cells that co-express PrPC and ZIP14 suggest that PrPC functions as a FR partner for certain members of this family. The connection between PrPC and ZIP proteins therefore extends beyond common ancestry to that of functional cooperation. Here, we summarize evidence supporting the facilitative role of PrPC in cellular iron uptake, and implications of this activity on iron metabolism in sCJD brains.

Goat activin receptor type IIB knockdown by muscle specific promoter driven artificial microRNAs

Activin receptor type IIB (ACVR2B) is a transmembrane receptor which mediates signaling of TGF beta superfamily ligands known to function in regulation of muscle mass, embryonic development and reproduction. ACVR2B antagonism has shown to enhance the muscle growth in several disease and transgenic models. Here, we show ACVR2B knockdown by RNA interference using muscle creatine kinase (MCK) promoter driven artificial microRNAs (amiRNAs). Among the various promoter elements tested, the ∼1.26 kb MCK promoter region showed maximum transcriptional activity in goat myoblasts cells. We observed up to 20% silencing in non-myogenic 293T cells and up to 32% silencing in myogenic goat myoblasts by MCK directed amiRNAs by transient transfection. Goat myoblasts stably integrated with MCK directed amiRNAs showed merely 8% silencing in proliferating myoblasts which was increased to 34% upon induction of differentiation at transcript level whereas up to 57% silencing at protein level. Knockdown of ACVR2B by 5-UTR derived amiRNAs resulted in decreased SMAD2/3 signaling, increased expression of myogenic regulatory factors (MRFs) and enhanced proliferation and differentiation of myoblasts. Unexpectedly, knockdown of ACVR2B by 3-UTR derived amiRNAs resulted in increased SMAD2/3 signaling, reduced expression of MRFs and suppression of myogenesis. Our study offers muscle specific knockdown of ACVR2B as a potential strategy to enhance muscle mass in the farm animal species.