Multiplexed and High-Throughput Label-Free Detection of RNA/Spike Protein/IgG/IgM Biomarkers of SARS-CoV-2 Infection Utilizing Nanoplasmonic Biosensors.

To tackle the COVID-19 outbreak, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there is an unmet need for highly accurate diagnostic tests at all stages of infection with rapid results and high specificity. Here, we present a label-free nanoplasmonic biosensor-based, multiplex screening test for COVID-19 that can quantitatively detect 10 different biomarkers (6 viral nucleic acid genes, 2 spike protein subunits, and 2 antibodies) with a limit of detection in the aM range, all within one biosensor platform. Our newly developed nanoplasmonic biosensors demonstrate high specificity, which is of the upmost importance to avoid false responses. As a proof of concept, we show that our detection approach has the potential to quantify both IgG and IgM antibodies directly from COVID-19-positive patient plasma samples in a single instrument run, demonstrating the high-throughput capability of our detection approach. Most importantly, our assay provides receiving operating characteristics, areas under the curve of 0.997 and 0.999 for IgG and IgM, respectively. The calculated p-value determined through the Mann-Whitney nonparametric test is <0.0001 for both antibodies when the test of COVID-19-positive patients (n = 80) is compared with that of healthy individuals (n = 72). Additionally, the screening test provides a calculated sensitivity (true positive rate) of 100% (80/80), a specificity (true negative rate) >96% (77/80), a positive predictive value of 98% at 5% prevalence, and a negative predictive value of 100% at 5% prevalence. We believe that our very sensitive, multiplex, high-throughput testing approach has potential applications in COVID-19 diagnostics, particularly in determining virus progression and infection severity for clinicians for an appropriate treatment, and will also prove to be a very effective diagnostic test when applied to diseases beyond the COVID-19 pandemic.

Photoswitchable Machine-Engineered Plasmonic Nanosystem with High Optical Response for Ultrasensitive Detection of microRNAs and Proteins Adaptively.

Modulating optoelectronic properties of inorganic nanostructures tethered with light-responsive molecular switches by their conformational change in the solid state is fundamentally important for advanced nanoscale-device fabrication, specifically in biosensing applications. Herein, we present an entirely new solid-state design approach employing the light-induced reversible conformational change of spiropyran (SP)-merocyanine (MC) covalently attached to gold triangular nanoprisms (Au TNPs) via alkylthiolate self-assembled monolayers to produce a large localized surface plasmon resonance response (∼24 nm). This shift is consistent with the increase in thickness of the local dielectric shell-surrounded TNPs and perhaps short-range dipole-dipole (permanent and induced) interactions between TNPs and the zwitterionic MC form. Water contact angle measurement and Raman spectroscopy characterization unequivocally prove the formation of a stable TNP-MC structural motif. Utilizing this form, we fabricated the first adaptable nanoplasmonic biosensor, which uses an identical structural motif for ultrasensitive, highly specific, and programmable detection of microRNAs and proteins at attomolar concentrations in standard human plasma and urine samples, and at femtomolar concentrations from bladder cancer patient plasma (n = 10) and urine (n = 10), respectively. Most importantly, the TNP-MC structural motif displays a strong binding affinity with receptor molecules (i.e., single-stranded DNA and antibody) producing a highly stable biosensor. Taken together, the TNP-MC structural motif represents a multifunctional super biosensor with the potential to expand clinical diagnostics through simplifying biosensor design and providing highly accurate disease diagnosis.

Reversible Tuning of the Plasmoelectric Effect in Noble Metal Nanostructures Through Manipulation of Organic Ligand Energy Levels.

Ligand-controlled tuning of localized surface plasmon resonance (LSPR) properties of noble metal nanostructures is fundamentally important for various optoelectronic applications such as photocatalysis, photovoltaics, and sensing. Here we demonstrate that the free carrier concentration of gold triangular nanoprisms (Au TNPs) can be tuned up to 12% upon functionalization of their surface with different para-substituted thiophenolate (X-Ph-S-) ligands. We achieve this unprecedentedly large optical response (plasmoelectric effect) in TNPs through the selective manipulation of electronic processes at the Au-thiolate interface. Interestingly, thiophenolates with electron withdrawing (donating) groups (X) produce λLSPR blue (red) shifts with broadening (narrowing) of localized surface plasmon resonance peak (λLSPR) line widths. Surprisingly, these experimental results are opposite to a straightforward application of the Drude model. Utilizing density functional theory calculations, we develop here a frontier molecular orbital approach of Au-thiophenolate interactions in the solid-state to delineate the observed spectral response. Importantly, all the spectroscopic properties are fully reversible by exchanging thiophenolates containing electron withdrawing groups with thiophenolates having electron donating groups, and vice versa. On the basis of the experimental data and calculations, we propose that the delocalization of electrons wave function controls the free carrier concentration of Au and thus the LSPR properties rather than simple electronic properties (inductive and/or resonance effects) of thiophenolates. This is further supported by the experimentally determined work functions, which are tunable over 1.9 eV in the X-Ph-S-passivated Au TNPs. We believe that our unexpected finding has great potential to guide in developing unique noble metal nanostructure-organic ligand hybrid nanoconjugates, which could allow us to bypass the complications associated with off-resonance LSPR activation of noble metal-doped semiconductor nanocrystals for various surface plasmon-driven applications.

Ultrathin Plasmonic Tungsten Oxide Quantum Wells with Controllable Free Carrier Densities.

Localized surface plasmon resonances (LSPR) of nanostructures can be tuned by controlling their morphology, local dielectric environment, and free carrier concentration. We report the colloidal synthesis of an ∼3 tungsten-oxygen (W-O) layer thick (∼1 nm), two-dimensional (2D) WO3-x nanoplatelets (NPLs) (x ≈ 0.55-1.03), which display tunable near-infrared LSPR properties and additionally high free electron density (Ne) that arises predominantly from the large shape factor of 2D NPLs. Importantly, the W to O composition ratios inferred from their LSPR measurements show much higher percentage of oxygen vacancies than those determined by X-ray diffraction analysis, suggesting that the aspect ratio of ultrathin WO3-x NPLs is the key to producing an unprecedentedly large Ne, although synthesis temperature is also an independent factor. We find that NPL formation is kinetically controlled, whereas thermodynamic parameter manipulation leads to Ne values as high as 4.13 × 1022 cm-3, which is close to that of plasmonic noble metals, and thus our oxide-based nanostructures can be considered as quasi-metallic. The unique structural properties of 2D nanomaterials along with the high Ne of WO3-x NPLs provide an attractive alternative to plasmonic noble metal nanostructures for various plasmon-driven energy conversions and design of photochromic nanodevices.

Dual Role of Electron-Accepting Metal-Carboxylate Ligands: Reversible Expansion of Exciton Delocalization and Passivation of Nonradiative Trap-States in Molecule-like CdSe Nanocrystals.

This paper reports large bathochromic shifts of up to 260 meV in both the excitonic absorption and emission peaks of oleylamine (OLA)-passivated molecule-like (CdSe)34 nanocrystals caused by postsynthetic treatment with the electron accepting Cd(O2CPh)2 complex at room temperature. These shifts are found to be reversible upon removal of Cd(O2CPh)2 by N,N,N',N'-tetramethylethylene-1,2-diamine. 1H NMR and FTIR characterizations of the nanocrystals demonstrate that the OLA remained attached to the surface of the nanocrystals during the reversible removal of Cd(O2CPh)2. On the basis of surface ligand characterization, X-ray powder diffraction measurements, and additional control experiments, we propose that these peak red shifts are a consequence of the delocalization of confined exciton wave functions into the interfacial electronic states that are formed from interaction of the LUMO of the nanocrystals and the LUMO of Cd(O2CPh)2, as opposed to originating from a change in size or reorganization of the inorganic core. Furthermore, attachment of Cd(O2CPh)2 to the OLA-passivated (CdSe)34 nanocrystal surface increases the photoluminescence quantum yield from 5% to an unprecedentedly high 70% and causes a 3-fold increase of the photoluminescence lifetime, which are attributed to a combination of passivation of nonradiative surface trap states and relaxation of exciton confinement. Taken together, our work demonstrates the unique aspects of surface ligand chemistry in controlling the excitonic absorption and emission properties of ultrasmall (CdSe)34 nanocrystals, which could expedite their potential applications in solid-state device fabrication.

Ultrasensitive photoreversible molecular sensors of azobenzene-functionalized plasmonic nanoantennas.

This Letter describes an unprecedentedly large and photoreversible localized surface plasmon resonance (LSPR) wavelength shift caused by photoisomerization of azobenzenes attached to gold nanoprisms that act as nanoantennas. The blue light-induced cis to trans azobenzene conformational change occurs in the solid state and controls the optical properties of the nanoprisms shifting their LSPR peak up to 21 nm toward longer wavelengths. This shift is consistent with the increase in thickness of the local dielectric environment (0.6 nm) surrounding the nanoprism and perhaps a contribution from plasmonic energy transfer between the nanoprism and azobenzenes. The effects of the azobenzene conformational change and its photoreversibility were also probed through surface-enhanced Raman spectroscopy (SERS) showing that the electronic interaction between the nanoprisms and bound azobenzenes in their cis conformation significantly enhances the intensity of the Raman bands of the azobenzenes. The SERS data suggests that the isomerization is controlled by first-order kinetics with a rate constant of 1.0 × 10(-4) s(-1). Our demonstration of light-induced photoreversibility of this type of molecular machine is the first-step toward removing present limitations on detection of molecular motion in solid-state devices using LSPR spectroscopy with nanoprisms. Modulating the LSPR peak position and controlling energy transfer across the nanostructure-organic molecule interface are very important for the fabrication of plasmonic-based nanoscale devices.

Hybrid Metal-Ligand Interfacial Dipole Engineering of Functional Plasmonic Nanostructures for Extraordinary Responses of Optoelectronic Properties.

Programmable manipulation of inorganic-organic interfacial electronic properties of ligand-functionalized plasmonic nanoparticles (NPs) is the key parameter dictating their applications such as catalysis, photovoltaics, and biosensing. Here we report the localized surface plasmon resonance (LSPR) properties of gold triangular nanoprisms (Au TNPs) in solid state that are functionalized with dipolar, conjugated ligands. A library of thiocinnamate ligands with varying surface dipole moments were used to functionalize TNPs, which results in ∼150 nm reversible tunability of LSPR peak wavelength with significant peak broadening (∼230 meV). The highly adjustable chemical system of thiocinnamate ligands is capable of shifting the Au work function down to 2.4 eV versus vacuum, i.e., ∼2.9 eV lower than a clean Au (111) surface, and this work function can be modulated up to 3.3 eV, the largest value reported to date through the formation of organothiolate SAMs on Au. Interestingly, the magnitude of plasmonic responses and work function modulation is NP shape dependent. By combining first-principles calculations and experiments, we have established the mechanism of direct wave function delocalization of electrons residing near the Fermi level into hybrid electronic states that are mostly dictated by the inorganic-organic interfacial dipole moments. We determine that both interfacial dipole and hybrid electronic states, and vinyl conjugation together are the key to achieving such extraordinary changes in the optoelectronic properties of ligand-functionalized, plasmonic NPs. The present study provides a quantitative relationship describing how specifically constructed organic ligands can be used to control the interfacial properties of NPs and thus the plasmonic and electronic responses of these functional plasmonics for a wide range of plasmon-driven applications.

Iron loading-induced aggregation and reduction of iron incorporation in heteropolymeric ferritin containing a mutant light chain that causes neurodegeneration.

Hereditary ferritinopathy (HF) is a neurodegenerative disease characterized by intracellular ferritin inclusion bodies (IBs) and iron accumulation throughout the central nervous system. Ferritin IBs are composed of mutant ferritin light chain as well as wild-type light (Wt-FTL) and heavy chain (FTH1) polypeptides. In vitro studies have shown that the mutant light chain polypeptide p.Phe167SerfsX26 (Mt-FTL) forms soluble ferritin 24-mer homopolymers having a specific structural disruption that explains its functional problems of reduced ability to incorporate iron and aggregation during iron loading. However, because ferritins are usually 24-mer heteropolymers and all three polypeptides are found in IBs, we investigated the properties of Mt-FTL/FTH1 and Mt-FTL/Wt-FTL heteropolymeric ferritins. We show here the facile assembly of Mt-FTL and FTH1 subunits into soluble ferritin heteropolymers, but their ability to incorporate iron was significantly reduced relative to Wt-FTL/FTH1 heteropolymers. In addition, Mt-FTL/FTH1 heteropolymers formed aggregates during iron loading, contrasting Wt-FTL/FTH1 heteropolymers and similar to what was seen for Mt-FTL homopolymers. The resulting precipitate contained both Mt-FTL and FTH1 polypeptides as do ferritin IBs in patients with HF. The presence of Mt-FTL subunits in Mt-FTL/Wt-FTL heteropolymers also caused iron loading-induced aggregation relative to Wt-FTL homopolymers, with the precipitate containing Mt- and Wt-FTL polypeptides again paralleling HF. Our data demonstrate that co-assembly with wild-type subunits does not circumvent the functional problems caused by mutant subunits. Furthermore, the functional problems characterized here in heteropolymers that contain mutant subunits parallel those problems previously reported in homopolymers composed exclusively of mutant subunits, which strongly suggests that the structural disruption characterized previously in Mt-FTL homopolymers occurs in a similar manner and to a significant extent in both Mt-FTL/FTH1 and Mt-FTL/Wt-FTL heteropolymers.

Using substantial reductant concentration with chelation therapy to enhance small aggregate dispersal, iron mobilization, and its clearance in neurodegenerative diseases.

Connections between altered iron homeostasis and certain neurodegenerative diseases are highlighted by numerous studies suggesting iron neurotoxicity. Iron causes aggregation in neurodegenerative disease-linked proteins as well as others and additionally facilitates oxidative damage. Iron and oxidative damage can cause cell death including by ferroptosis. As treatment for neurodegeneration, chelation therapy alone is sometimes used with modest, varying efficacy and has not in general proven to reverse or halt the damage long term. Questions often focus on optimal chelator partitioning and fine-tuning binding strength; however iron oxidation state chemistry implies a different approach. More specifically, my perspective is that applying a redox-based component to iron mobilization and handling is crucial because ferrous iron is in general a more soluble, weaker biological binder than ferric. Once cellular iron becomes oxidized to ferric, it binds tenaciously, exchanges ligands more slowly, and enhances protein aggregation, which importantly can be reversed by iron reduction. This situation escalates with age as brain reducing ability decreases, iron concentration increases, autophagic clearance decreases, and cell stress diminishes iron handling capacity. Taken together, treatment employing chelation therapy together with a strong biological reductant may effectively remove inappropriately bound cellular iron or at least inhibit accumulation. This approach would likely require high concentration ascorbate or glutathione by IV along with chelation to enhance iron mobilization and elimination, thus reducing cumulative cellular damage and perhaps restoring partial function. Potential treatment-induced oxidative damage may be attenuated by high reductant concentration, appropriate choice of chelator, and/or treatment sequence. Comprehensive study is urged.

Unraveling of the E-helices and disruption of 4-fold pores are associated with iron mishandling in a mutant ferritin causing neurodegeneration.

Mutations in the coding sequence of the ferritin light chain (FTL) gene cause a neurodegenerative disease known as neuroferritinopathy or hereditary ferritinopathy, which is characterized by the presence of intracellular inclusion bodies containing the mutant FTL polypeptide and by abnormal accumulation of iron in the brain. Here, we describe the x-ray crystallographic structure and report functional studies of ferritin homopolymers formed from the mutant FTL polypeptide p.Phe167SerfsX26, which has a C terminus that is altered in amino acid sequence and length. The structure was determined and refined to 2.85 A resolution and was very similar to the wild type between residues Ile-5 and Arg-154. However, instead of the E-helices normally present in wild type ferritin, the C-terminal sequences of all 24 mutant subunits showed substantial amounts of disorder, leading to multiple C-terminal polypeptide conformations and a large disruption of the normally tiny 4-fold axis pores. Functional studies underscored the importance of the mutant C-terminal sequence in iron-induced precipitation and revealed iron mishandling by soluble mutant FTL homopolymers in that only wild type incorporated iron when in direct competition in solution with mutant ferritin. Even without competition, the amount of iron incorporation over the first few minutes differed severalfold. Our data suggest that disruption at the 4-fold pores may lead to direct iron mishandling through attenuated iron incorporation by the soluble form of mutant ferritin and that the disordered C-terminal polypeptides may play a major role in iron-induced precipitation and formation of ferritin inclusion bodies in hereditary ferritinopathy.

What Can Cellular Redox, Iron, and Reactive Oxygen Species Suggest About the Mechanisms and Potential Therapy of COVID-19?

Accumulating evidence suggests that there are important contributions to coronavirus disease (COVID-19) from redox imbalance and improperly coordinated iron, which cause cellular oxidative damage and stress. Cells have developed elaborate redox-dependent processes to handle and store iron, and their disfunction leads to several serious diseases. Cellular reductants are important as reactive oxygen species (ROS) scavengers and to power enzymatic repair mechanisms, but they also may help generate toxic ROS. These complicated interrelationships are presented in terms of a cellular redox/iron/ROS triad, including ROS generation both at improperly coordinated iron and enzymatically, ROS interconvertibility, cellular signaling and damage, and reductant and iron chelator concentration-dependent effects. This perspective provides the rational necessary to strongly suggest that COVID-19 disrupts this interdependent triad, producing a substantial contribution to the ROS load, which causes direct ROS-induced protein and phospholipid damage, taxes cellular resources and repair mechanisms, and alters cellular signaling, especially in the more critical acute respiratory distress syndrome (ARDS) phase of the infection. Specific suggestions for therapeutic interventions using reductants and chelators that may help treat COVID-19 are discussed.

Cryo-EM structures and functional characterization of homo- and heteropolymers of human ferritin variants.

The role of abnormal brain iron metabolism in neurodegenerative diseases is still insufficiently understood. Here, we investigate the molecular basis of the neurodegenerative disease hereditary ferritinopathy (HF), in which dysregulation of brain iron homeostasis is the primary cause of neurodegeneration. We mutagenized ferritin's three-fold pores (3FPs), i.e. the main entry route for iron, to investigate ferritin's iron management when iron must traverse the protein shell through the disrupted four-fold pores (4FPs) generated by mutations in the ferritin light chain (FtL) gene in HF. We assessed the structure and properties of ferritins using cryo-electron microscopy and a range of functional analyses in vitro. Loss of 3FP function did not alter ferritin structure but led to a decrease in protein solubility and iron storage. Abnormal 4FPs acted as alternate routes for iron entry and exit in the absence of functional 3FPs, further reducing ferritin iron-storage capacity. Importantly, even a small number of MtFtL subunits significantly compromises ferritin solubility and function, providing a rationale for the presence of ferritin aggregates in cell types expressing different levels of FtLs in patients with HF. These findings led us to discuss whether modifying pores could be used as a pharmacological target in HF.

Iron, Ferritin, Hereditary Ferritinopathy, and Neurodegeneration.

Cellular growth, function, and protection require proper iron management, and ferritin plays a crucial role as the major iron sequestration and storage protein. Ferritin is a 24 subunit spherical shell protein composed of both light (FTL) and heavy chain (FTH1) subunits, possessing complimentary iron-handling functions and forming three-fold and four-fold pores. Iron uptake through the three-fold pores is well-defined, but the unloading process somewhat less and generally focuses on lysosomal ferritin degradation although it may have an additional, energetically efficient pore mechanism. Hereditary Ferritinopathy (HF) or neuroferritinopathy is an autosomal dominant neurodegenerative disease caused by mutations in the FTL C-terminal sequence, which in turn cause disorder and unraveling at the four-fold pores allowing iron leakage and enhanced formation of toxic, improperly coordinated iron (ICI). Histopathologically, HF is characterized by iron deposition and formation of ferritin inclusion bodies (IBs) as the cells overexpress ferritin in an attempt to address iron accumulation while lacking the ability to clear ferritin and its aggregates. Overexpression and IB formation tax cells materially and energetically, i.e., their synthesis and disposal systems, and may hinder cellular transport and other spatially dependent functions. ICI causes cellular damage to proteins and lipids through reactive oxygen species (ROS) formation because of high levels of brain oxygen, reductants and metabolism, taxing cellular repair. Iron can cause protein aggregation both indirectly by ROS-induced protein modification and destabilization, and directly as with mutant ferritin through C-terminal bridging. Iron release and ferritin degradation are also linked to cellular misfunction through ferritinophagy, which can release sufficient iron to initiate the unique programmed cell death process ferroptosis causing ROS formation and lipid peroxidation. But IB buildup suggests suppressed ferritinophagy, with elevated iron from four-fold pore leakage together with ROS damage and stress leading to a long-term ferroptotic-like state in HF. Several of these processes have parallels in cell line and mouse models. This review addresses the roles of ferritin structure and function within the above-mentioned framework, as they relate to HF and associated disorders characterized by abnormal iron accumulation, protein aggregation, oxidative damage, and the resulting contributions to cumulative cellular stress and death.

Elucidating the role of surface passivating ligand structural parameters in hole wave function delocalization in semiconductor cluster molecules.

This article describes the mechanisms underlying electronic interactions between surface passivating ligands and (CdSe)34 semiconductor cluster molecules (SCMs) that facilitate band-gap engineering through the delocalization of hole wave functions without altering their inorganic core. We show here both experimentally and through density functional theory calculations that the expansion of the hole wave function beyond the SCM boundary into the ligand monolayer depends not only on the pre-binding energetic alignment of interfacial orbitals between the SCM and surface passivating ligands but is also strongly influenced by definable ligand structural parameters such as the extent of their π-conjugation [π-delocalization energy; pyrene (Py), anthracene (Anth), naphthalene (Naph), and phenyl (Ph)], binding mode [dithiocarbamate (DTC, -NH-CS2-), carboxylate (-COO-), and amine (-NH2)], and binding head group [-SH, -SeH, and -TeH]. We observe an unprecedentedly large ∼650 meV red-shift in the lowest energy optical absorption band of (CdSe)34 SCMs upon passivating their surface with Py-DTC ligands and the trend is found to be Ph- < Naph- < Anth- < Py-DTC. This shift is reversible upon removal of Py-DTC by triethylphosphine gold(i) chloride treatment at room temperature. Furthermore, we performed temperature-dependent (80-300 K) photoluminescence lifetime measurements, which show longer lifetime at lower temperature, suggesting a strong influence of hole wave function delocalization rather than carrier trapping and/or phonon-mediated relaxation. Taken together, knowledge of how ligands electronically interact with the SCM surface is crucial to semiconductor nanomaterial research in general because it allows the tuning of electronic properties of nanomaterials for better charge separation and enhanced charge transfer, which in turn will increase optoelectronic device and photocatalytic efficiencies.

Effect of Systemic Iron Overload and a Chelation Therapy in a Mouse Model of the Neurodegenerative Disease Hereditary Ferritinopathy.

Mutations in the ferritin light chain (FTL) gene cause the neurodegenerative disease neuroferritinopathy or hereditary ferritinopathy (HF). HF is characterized by a severe movement disorder and by the presence of nuclear and cytoplasmic iron-containing ferritin inclusion bodies (IBs) in glia and neurons throughout the central nervous system (CNS) and in tissues of multiple organ systems. Herein, using primary mouse embryonic fibroblasts from a mouse model of HF, we show significant intracellular accumulation of ferritin and an increase in susceptibility to oxidative damage when cells are exposed to iron. Treatment of the cells with the iron chelator deferiprone (DFP) led to a significant improvement in cell viability and a decrease in iron content. In vivo, iron overload and DFP treatment of the mouse model had remarkable effects on systemic iron homeostasis and ferritin deposition, without significantly affecting CNS pathology. Our study highlights the role of iron in modulating ferritin aggregation in vivo in the disease HF. It also puts emphasis on the potential usefulness of a therapy based on chelators that can target the CNS to remove and redistribute iron and to resolubilize or prevent ferritin aggregation while maintaining normal systemic iron stores.

Systemic and cerebral iron homeostasis in ferritin knock-out mice.

Ferritin, a 24-mer heteropolymer of heavy (H) and light (L) subunits, is the main cellular iron storage protein and plays a pivotal role in iron homeostasis by modulating free iron levels thus reducing radical-mediated damage. The H subunit has ferroxidase activity (converting Fe(II) to Fe(III)), while the L subunit promotes iron nucleation and increases ferritin stability. Previous studies on the H gene (Fth) in mice have shown that complete inactivation of Fth is lethal during embryonic development, without ability to compensate by the L subunit. In humans, homozygous loss of the L gene (FTL) is associated with generalized seizure and atypical restless leg syndrome, while mutations in FTL cause a form of neurodegeneration with brain iron accumulation. Here we generated mice with genetic ablation of the Fth and Ftl genes. As previously reported, homozygous loss of the Fth allele on a wild-type Ftl background was embryonic lethal, whereas knock-out of the Ftl allele (Ftl-/-) led to a significant decrease in the percentage of Ftl-/- newborn mice. Analysis of Ftl-/- mice revealed systemic and brain iron dyshomeostasis, without any noticeable signs of neurodegeneration. Our findings indicate that expression of the H subunit can rescue the loss of the L subunit and that H ferritin homopolymers have the capacity to sequester iron in vivo. We also observed that a single allele expressing the H subunit is not sufficient for survival when both alleles encoding the L subunit are absent, suggesting the need of some degree of complementation between the subunits as well as a dosage effect.

Abnormal iron homeostasis and neurodegeneration.

Abnormal iron metabolism is observed in many neurodegenerative diseases, however, only two have shown dysregulation of brain iron homeostasis as the primary cause of neurodegeneration. Herein, we review one of these - hereditary ferritinopathy (HF) or neuroferritinopathy, which is an autosomal dominant, adult onset degenerative disease caused by mutations in the ferritin light chain (FTL) gene. HF has a clinical phenotype characterized by a progressive movement disorder, behavioral disturbances, and cognitive impairment. The main pathologic findings are cystic cavitation of the basal ganglia, the presence of ferritin inclusion bodies (IBs), and substantial iron deposition. Mutant FTL subunits have altered sequence and length but assemble into soluble 24-mers that are ultrastructurally indistinguishable from those of the wild type. Crystallography shows substantial localized disruption of the normally tiny 4-fold pores between the ferritin subunits because of unraveling of the C-termini into multiple polypeptide conformations. This structural alteration causes attenuated net iron incorporation leading to cellular iron mishandling, ferritin aggregation, and oxidative damage at physiological concentrations of iron and ascorbate. A transgenic murine model parallels several features of HF, including a progressive neurological phenotype, ferritin IB formation, and misregulation of iron metabolism. These studies provide a working hypothesis for the pathogenesis of HF by implicating (1) a loss of normal ferritin function that triggers iron accumulation and overproduction of ferritin polypeptides, and (2) a gain of toxic function through radical production, ferritin aggregation, and oxidative stress. Importantly, the finding that ferritin aggregation can be reversed by iron chelators and oxidative damage can be inhibited by radical trapping may be used for clinical investigation. This work provides new insights into the role of abnormal iron metabolism in neurodegeneration.

A mutant light-chain ferritin that causes neurodegeneration has enhanced propensity toward oxidative damage.

Intracellular inclusion bodies (IBs) containing ferritin and iron are hallmarks of hereditary ferritinopathy (HF). This neurodegenerative disease is caused by mutations in the coding sequence of the ferritin light chain (FTL) gene that generate FTL polypeptides with a C-terminus that is altered in amino acid sequence and length. Previous studies of ferritin formed with p.Phe167SerfsX26 mutant FTL (Mt-FTL) subunits found disordered 4-fold pores, iron mishandling, and proaggregative behavior, as well as a general increase in cellular oxidative stress when expressed in vivo. Herein, we demonstrate that Mt-FTL is also a target of iron-catalyzed oxidative damage in vitro and in vivo. Incubation of recombinant Mt-FTL ferritin with physiological concentrations of iron and ascorbate resulted in shell structural disruption and polypeptide cleavage not seen with the wild type, as well as a 2.5-fold increase in carbonyl group formation. However, Mt-FTL shell disruption and polypeptide cleavage were completely inhibited by the addition of the radical trap 5,5-dimethyl-1-pyrroline N-oxide. These results indicate an enhanced propensity of Mt-FTL toward free radical-induced oxidative damage in vitro. We also found evidence of extensive carbonylation in IBs from a patient with HF together with isolation of a C-terminal Mt-FTL fragment, which are both indicative of oxidative ferritin damage in vivo. Our data demonstrate an enhanced propensity of mutant ferritin to undergo iron-catalyzed oxidative damage and support this as a mechanism causing disruption of ferritin structure and iron mishandling that contribute to the pathology of HF.

Iron-mediated aggregation and a localized structural change characterize ferritin from a mutant light chain polypeptide that causes neurodegeneration.

Nucleotide insertions in the ferritin light chain (FTL) polypeptide gene cause hereditary ferritinopathy, a neurodegenerative disease characterized by abnormal accumulation of ferritin and iron in the central nervous system. Here we describe for the first time the protein structure and iron storage function of the FTL mutant p.Phe167SerfsX26 (MT-FTL), which has a C terminus altered in sequence and extended in length. MT-FTL polypeptides assembled spontaneously into soluble, spherical 24-mers that were ultrastructurally indistinguishable from those of the wild type. Far-UV CD showed a decrease in alpha-helical content, and 8-anilino-1-naphthalenesulfonate fluorescence revealed the appearance of hydrophobic binding sites. Near-UV CD and proteolysis studies suggested little or no structural alteration outside of the C-terminal region. In contrast to wild type, MT-FTL homopolymers precipitated at much lower iron loading, had a diminished capacity to incorporate iron, and were less thermostable. However, precipitation was significantly reversed by addition of iron chelators both in vitro and in vivo. Our results reveal substantial protein conformational changes localized at the 4-fold pore of MT-FTL homopolymers and imply that the C terminus of the MT-FTL polypeptide plays an important role in ferritin solubility, stability, and iron management. We propose that the protrusion of some portion of the C terminus above the spherical shell allows it to cross-link with other mutant polypeptides through iron bridging, leading to enhanced mutant precipitation by iron. Our data suggest that hereditary ferritinopathy pathogenesis is likely to result from a combination of reduction in iron storage function and enhanced toxicity associated with iron-induced ferritin aggregates.