TAF15 amyloid filaments in frontotemporal lobar degeneration.

Frontotemporal lobar degeneration (FTLD) causes frontotemporal dementia (FTD), the most common form of dementia after Alzheimer's disease, and is often also associated with motor disorders1. The pathological hallmarks of FTLD are neuronal inclusions of specific, abnormally assembled proteins2. In the majority of cases the inclusions contain amyloid filament assemblies of TAR DNA-binding protein 43 (TDP-43) or tau, with distinct filament structures characterizing different FTLD subtypes3,4. The presence of amyloid filaments and their identities and structures in the remaining approximately 10% of FTLD cases are unknown but are widely believed to be composed of the protein fused in sarcoma (FUS, also known as translocated in liposarcoma). As such, these cases are commonly referred to as FTLD-FUS. Here we used cryogenic electron microscopy (cryo-EM) to determine the structures of amyloid filaments extracted from the prefrontal and temporal cortices of four individuals with FTLD-FUS. Surprisingly, we found abundant amyloid filaments of the FUS homologue TATA-binding protein-associated factor 15 (TAF15, also known as TATA-binding protein-associated factor 2N) rather than of FUS itself. The filament fold is formed from residues 7-99 in the low-complexity domain (LCD) of TAF15 and was identical between individuals. Furthermore, we found TAF15 filaments with the same fold in the motor cortex and brainstem of two of the individuals, both showing upper and lower motor neuron pathology. The formation of TAF15 amyloid filaments with a characteristic fold in FTLD establishes TAF15 proteinopathy in neurodegenerative disease. The structure of TAF15 amyloid filaments provides a basis for the development of model systems of neurodegenerative disease, as well as for the design of diagnostic and therapeutic tools targeting TAF15 proteinopathy.

ß-Bracelets: Macrocyclic Cross-ß Epitope Mimics Based on a Tau Conformational Strain.

The aggregation of misfolded tau into neurotoxic fibrils is linked to the progression of Alzheimer's disease (AD) and related tauopathies. Disease-associated conformations of filamentous tau are characterized by hydrophobic interactions between side chains on unique and distant ß-strand modules within each protomer. Here, we report the design and diversity-oriented synthesis of ß-arch peptide macrocycles composed of the aggregation-prone PHF6 hexapeptide of tau and the cross-ß module specific to the AD tau fold. Termed "ß-bracelets", these proteomimetics assemble in a sequence- and macrocycle-dependent fashion, resulting in amyloid-like fibrils that feature in-register parallel ß-sheet structure. Backbone N-amination of a selected ß-bracelet affords soluble inhibitors of tau aggregation. We further demonstrate that the N-aminated macrocycles block the prion-like cellular seeding activity of recombinant tau as well as mature fibrils from AD patient extracts. These studies establish ß-bracelets as a new class of cross-ß epitope mimics and demonstrate their utility in the rational design of molecules targeting amyloid propagation and seeding.

Lack of ApoE inhibits ADan amyloidosis in a mouse model of familial Danish dementia.

The Apolipoprotein E-ε4 allele (APOE-ε4) is the strongest genetic risk factor for late onset Alzheimer disease (AD). ApoE plays a critical role in amyloid-ß (Aß) accumulation in AD, and genetic deletion of the murine ApoE gene in mouse models results in a decrease or inhibition of Aß deposition. The association between the presence of ApoE and amyloid in amyloidoses suggests a more general role for ApoE in the fibrillogenesis process. However, whether decreasing levels of ApoE would attenuate amyloid pathology in different amyloidoses has not been directly addressed. Familial Danish dementia (FDD) is an autosomal dominant neurodegenerative disease characterized by the presence of widespread parenchymal and vascular Danish amyloid (ADan) deposition and neurofibrillary tangles. A transgenic mouse model for FDD (Tg-FDD) is characterized by parenchymal and vascular ADan deposition. To determine the effect of decreasing ApoE levels on ADan accumulation in vivo, we generated a mouse model by crossing Tg-FDD mice with ApoE KO mice (Tg-FDD+/-/ApoE-/-). Lack of ApoE results in inhibition of ADan deposition up to 18 months of age. Additionally, our results from a genetic screen of Tg-FDD+/-/ApoE-/- mice emphasize the significant role for ApoE in neurodegeneration in FDD via glial-mediated mechanisms. Taken together, our findings suggest that the interaction between ApoE and ADan plays a key role in FDD pathogenesis, in addition to the known role for ApoE in amyloid plaque formation in AD.

Cryo-EM structures of prion protein filaments from Gerstmann-Sträussler-Scheinker disease.

Prion protein (PrP) aggregation and formation of PrP amyloid (APrP) are central events in the pathogenesis of prion diseases. In the dominantly inherited prion protein amyloidosis known as Gerstmann-Sträussler-Scheinker (GSS) disease, plaques made of PrP amyloid are present throughout the brain. The c.593t > c mutation in the prion protein gene (PRNP) results in a phenylalanine to serine amino acid substitution at PrP residue 198 (F198S) and causes the most severe amyloidosis among GSS variants. It has been shown that neurodegeneration in this disease is associated with the presence of extracellular APrP plaques and neuronal intracytoplasmic Tau inclusions, that have been shown to contain paired helical filaments identical to those found in Alzheimer disease. Using cryogenic electron microscopy (cryo-EM), we determined for the first time the structures of filaments of human APrP, isolated post-mortem from the brain of two symptomatic PRNP F198S mutation carriers. We report that in GSS (F198S) APrP filaments are composed of dimeric, trimeric and tetrameric left-handed protofilaments with their protomers sharing a common protein fold. The protomers in the cross-ß spines consist of 62 amino acids and span from glycine 80 to phenylalanine 141, adopting a previously unseen spiral fold with a thicker outer layer and a thinner inner layer. Each protomer comprises nine short ß-strands, with the ß1 and ß8 strands, as well as the ß4 and ß9 strands, forming a steric zipper. The data obtained by cryo-EM provide insights into the structural complexity of the PrP filament in a dominantly inherited human PrP amyloidosis. The novel findings highlight the urgency of extending our knowledge of the filaments' structures that may underlie distinct clinical and pathologic phenotypes of human neurodegenerative diseases.

Age-dependent formation of TMEM106B amyloid filaments in human brains.

Many age-dependent neurodegenerative diseases, such as Alzheimer's and Parkinson's, are characterized by abundant inclusions of amyloid filaments. Filamentous inclusions of the proteins tau, amyloid-ß, α-synuclein and transactive response DNA-binding protein (TARDBP; also known as TDP-43) are the most common1,2. Here we used structure determination by cryogenic electron microscopy to show that residues 120-254 of the lysosomal type II transmembrane protein 106B (TMEM106B) also form amyloid filaments in human brains. We determined the structures of TMEM106B filaments from a number of brain regions of 22 individuals with abundant amyloid deposits, including those resulting from sporadic and inherited tauopathies, amyloid-ß amyloidoses, synucleinopathies and TDP-43 proteinopathies, as well as from the frontal cortex of 3 individuals with normal neurology and no or only a few amyloid deposits. We observed three TMEM106B folds, with no clear relationships between folds and diseases. TMEM106B filaments correlated with the presence of a 29-kDa sarkosyl-insoluble fragment and globular cytoplasmic inclusions, as detected by an antibody specific to the carboxy-terminal region of TMEM106B. The identification of TMEM106B filaments in the brains of older, but not younger, individuals with normal neurology indicates that they form in an age-dependent manner.

Structure of Tau filaments in Prion protein amyloidoses.

In human neurodegenerative diseases associated with the intracellular aggregation of Tau protein, the ordered cores of Tau filaments adopt distinct folds. Here, we analyze Tau filaments isolated from the brain of individuals affected by Prion-Protein cerebral amyloid angiopathy (PrP-CAA) with a nonsense mutation in the PRNP gene that leads to early termination of translation of PrP (Q160Ter or Q160X), and Gerstmann-Sträussler-Scheinker (GSS) disease, with a missense mutation in the PRNP gene that leads to an amino acid substitution at residue 198 (F198S) of PrP. The clinical and neuropathologic phenotypes associated with these two mutations in PRNP are different; however, the neuropathologic analyses of these two genetic variants have consistently shown the presence of numerous neurofibrillary tangles (NFTs) made of filamentous Tau aggregates in neurons. We report that Tau filaments in PrP-CAA (Q160X) and GSS (F198S) are composed of 3-repeat and 4-repeat Tau isoforms, having a striking similarity to NFTs in Alzheimer disease (AD). In PrP-CAA (Q160X), Tau filaments are made of both paired helical filaments (PHFs) and straight filaments (SFs), while in GSS (F198S), only PHFs were found. Mass spectrometry analyses of Tau filaments extracted from PrP-CAA (Q160X) and GSS (F198S) brains show the presence of post-translational modifications that are comparable to those seen in Tau aggregates from AD. Cryo-EM analysis reveals that the atomic models of the Tau filaments obtained from PrP-CAA (Q160X) and GSS (F198S) are identical to those of the Tau filaments from AD, and are therefore distinct from those of Pick disease, chronic traumatic encephalopathy, and corticobasal degeneration. Our data support the hypothesis that in the presence of extracellular amyloid deposits and regardless of the primary amino acid sequence of the amyloid protein, similar molecular mechanisms are at play in the formation of identical Tau filaments.

Tau as a mediator of neurotoxicity associated to cerebral amyloid angiopathy.

Cerebral amyloid angiopathy (CAA) is typified by the cerebrovascular deposition of amyloid. Currently, there is no clear understanding of the mechanisms underlying the contribution of CAA to neurodegeneration. Despite the fact that CAA is highly associated with accumulation of Aß, other types of amyloids have been shown to associate with the vasculature. Interestingly, in many cases, vascular amyloidosis is accompanied by significant tau pathology. However, the contribution of tau to neurodegeneration associated to CAA remains to be determined. We used a mouse model of Familial Danish Dementia (FDD), a neurodegenerative disease characterized by the accumulation of Danish amyloid (ADan) in the vasculature, to characterize the contribution of tau to neurodegeneration associated to CAA. We performed histological and biochemical assays to establish tau modifications associated with CAA in conjunction with cell-based and electrophysiological assays to determine the role of tau in the synaptic dysfunction associated with ADan. We demonstrated that ADan aggregates induced hyperphosphorylation and misfolding of tau. Moreover, in a mouse model for CAA, we observed tau oligomers closely associated to astrocytes in the vicinity of vascular amyloid deposits. We finally determined that the absence of tau prevents synaptic dysfunction induced by ADan oligomers. In addition to demonstrating the effect of ADan amyloid on tau misfolding, our results provide compelling evidence of the role of tau in neurodegeneration associated with ADan-CAA and suggest that decreasing tau levels could be a feasible approach for the treatment of CAA.

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.

Amyloid and intracellular accumulation of BRI2.

Familial British dementia (FBD) and familial Danish dementia (FDD) are caused by mutations in the BRI2 gene. These diseases are characterized clinically by progressive dementia and ataxia and neuropathologically by amyloid deposits and neurofibrillary tangles. Herein, we investigate BRI2 protein accumulation in FBD, FDD, Alzheimer disease and Gerstmann-Sträussler-Scheinker disease. In FBD and FDD, we observed reduced processing of the mutant BRI2 pro-protein, which was found accumulating intracellularly in the Golgi of neurons and glial cells. In addition, we observed an accumulation of a mature form of BRI2 protein in dystrophic neurites, surrounding amyloid cores. Accumulation of BRI2 was also observed in dystrophic neurites of Alzheimer disease and Gerstmann-Sträussler-Scheinker disease cases. Although it remains to be determined whether intracellular accumulation of BRI2 may lead to cell damage in these degenerative diseases, our study provides new insights into the role of mutant BRI2 in the pathogenesis of FBD and FDD and implicates BRI2 as a potential indicator of neuritic damage in diseases characterized by cerebral amyloid deposition.

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.

A human monoclonal IgG that binds aß assemblies and diverse amyloids exhibits anti-amyloid activities in vitro and in vivo.

Alzheimer's disease (AD) and familial Danish dementia (FDD) are degenerative neurological diseases characterized by amyloid pathology. Normal human sera contain IgG antibodies that specifically bind diverse preamyloid and amyloid proteins and have shown therapeutic potential in vitro and in vivo. We cloned one of these antibodies, 3H3, from memory B cells of a healthy individual using a hybridoma method. 3H3 is an affinity-matured IgG that binds a pan-amyloid epitope, recognizing both Aß and λ Ig light chain (LC) amyloids, which are associated with AD and primary amyloidosis, respectively. The pan-amyloid-binding properties of 3H3 were demonstrated using ELISA, immunohistochemical studies, and competition binding assays. Functional studies showed that 3H3 inhibits both Aß and LC amyloid formation in vitro and abrogates disruption of hippocampal synaptic plasticity by AD-patient-derived soluble Aß in vivo. A 3H3 single-chain variable fragment (scFv) retained the binding specificity of the 3H3 IgG and, when expressed in the brains of transgenic mice using an adeno-associated virus (AAV) vector, decreased parenchymal Aß amyloid deposition in TgCRND8 mice and ADan (Danish Amyloid) cerebral amyloid angiopathy in the mouse model of FDD. These data indicate that naturally occurring human IgGs can recognize a conformational, amyloid-specific epitope and have potent anti-amyloid activities, providing a rationale to test their potential as antibody therapeutics for diverse neurological and other amyloid diseases.

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.

Amyloid peptides ABri and ADan show differential neurotoxicity in transgenic Drosophila models of familial British and Danish dementia.

BACKGROUND: Familial British and Familial Danish dementias (FBD and FDD, respectively) are associated with mutations in the BRI2 gene. Processing of the mutated BRI2 protein leads to the accumulation in the brain of the 34-mer amyloid Bri (ABri) and amyloid Dan (ADan) peptides, accompanied by neurofibrillary tangles. Recently, transgenic mice successfully reproduced different aspects of FDD, while modeling of FBD in vivo has been more difficult. In this work we have modeled FBD and FDD in Drosophila and tested the hypothesis that ABri and ADan are differentially neurotoxic. RESULTS: By using site-directed insertion, we generated transgenic lines carrying ABri, ADan, Bri2-23 (the normal product of wild-type BRI2 processing) and amyloid-ß (Aß) 1-42 as a well-characterized neurotoxic peptide, alone or with a His-tag. Therefore, we avoided random insertion effects and were able to compare levels of accumulation accurately. Peptides were expressed with the GAL4-Upstream Activating Sequence (UAS) system using specific drivers. Despite low levels of expression, toxicity in the eye was characterized by mild disorganization of ommatidia and amyloid peptides accumulation. The highest toxicity was seen for ADan, followed by Aß42 and ABri. Pan-neuronal expression in the CNS revealed an age-dependent toxicity of amyloid peptides as determined by the ability of flies to climb in a geotaxis paradigm when compared to Bri2-23. This effect was stronger for ADan, detected at 7 days post-eclosion, and followed by ABri and Aß42, whose toxicity became evident after 15 and 21 days, respectively. Histological analysis showed mild vacuolization and thioflavine-S-negative deposits of amyloid peptides. In contrast, the over-expression of amyloid peptides in the specific subset of lateral neurons that control circadian locomotor activity showed no toxicity. CONCLUSIONS: Our results support the differential neurotoxicity of ADan and ABri in the Drosophila eye and CNS at low expression levels. Such differences may be partially attributed to rates of aggregation and accumulation. In the CNS, both peptides appear to be more neurotoxic than wild-type Aß42. These Drosophila models will allow a systematic and unambiguous comparison of differences and similarities in the mechanisms of toxicity of diverse amyloid peptides associated with dementia.

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.

Neurodegeneración causada por mutaciones en el gen de la cadena liviana de ferritina / Neurodegeneration caused by mutations in the ferritin light chain gene / Neurodegeneração causada por mutações no gene da cadeia leve de ferritina

La duplicación de nucleótidos en el cuarto exón del gen de la cadena liviana de ferritina (FTL) causa la enfermedad neurodegenerativa autosómica dominante neuroferritinopatía o ferritinopatía hereditaria (hereditary ferritinopathy, HF). La presentación clínica de la HF se caracteriza por la presencia de trastornos del movimiento, alteraciones del comportamiento y deterioro cognitivo. El estudio de resonancia magnética nuclear muestra señales anormales en ambos globos pálidos y el putamen, así como también la cavitación del putamen. También puede observarse una atrofia moderada en el cerebro y el cerebelo. Desde el punto de vista neuropatológico la enfermedad de HF se caracteriza por una pérdida neuronal severa en los ganglios de la base, atrofia del cerebelo y de la corteza cerebral, acumulación de hierro, y por la presencia de cuerpos de inclusión (inclusion bodies, IB) de ferritina en neuronas y células de la glía. Los IBs de ferritina también pueden hallarse en células de otros órganos, incluyendo piel, riñones, hígado y músculo. Los niveles séricos de ferritina pueden hallarse dentro del rango normal o encontrarse anormalmente disminuidos en presencia de niveles séricos normales de hierro. Todas las mutaciones reportadas en el gen de la FTL generan polipéptidos con un extremo C-terminal anormal que altera la incorporación de hierro y favorece la agregación de ferritina mediada por la presencia de hierro. La expresión de una mutante de FTL en ratones ha recapitulado diversas facetas de la enfermedad, incluyendo la formación de IBs de ferritina en neuronas y células de la glía, desregulación de la homeostasis de hierro y daño oxidativo de proteínas en el cerebro, similarmente a lo que ha sido observado en individuos con la enfermedad de HF.

The Psen1-L166P-knock-in mutation leads to amyloid deposition in human wild-type amyloid precursor protein YAC transgenic mice.

Genetically engineered mice have been generated to model cerebral ß-amyloidosis, one of the hallmarks of Alzheimer disease (AD) pathology, based on the overexpression of a mutated cDNA of the amyloid-ß precursor protein (AßPP) or by knock-in of the murine Aßpp gene alone or with presenilin1 mutations. Here we describe the generation and initial characterization of a new mouse line based on the presence of 2 copies of the human genomic region encoding the wild-type AßPP and the L166P presenilin 1 mutation. At ∼6 mo of age, double-mutant mice develop amyloid pathology, with signs of neuritic dystrophy, intracellular Aß accumulation, and glial inflammation, an increase in AßPP C-terminal fragments, and an 8 times increase in Aß42 levels with a 40% decrease in Aß40 levels, leading to a significant increase (14 times) of Aß42/Aß40 ratios, with minimal effects on presenilin or the Notch1 pathway in the brain. We conclude that in mice, neither mutations in AßPP nor overexpression of an AßPP isoform are a prerequisite for Aß pathology. This model will allow the study of AD pathogenesis and testing of therapeutic strategies in a more relevant environment without experimental artifacts due to the overexpression of a single-mutant AßPP isoform using exogenous promoters.

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 deficiency drives an autosomal dominant hypophosphatemic rickets (ADHR) phenotype in fibroblast growth factor-23 (Fgf23) knock-in mice.

Autosomal dominant hypophosphatemic rickets (ADHR) is unique among the disorders involving Fibroblast growth factor 23 (FGF23) because individuals with R176Q/W and R179Q/W mutations in the FGF23 (176)RXXR(179)/S(180) proteolytic cleavage motif can cycle from unaffected status to delayed onset of disease. This onset may occur in physiological states associated with iron deficiency, including puberty and pregnancy. To test the role of iron status in development of the ADHR phenotype, WT and R176Q-Fgf23 knock-in (ADHR) mice were placed on control or low-iron diets. Both the WT and ADHR mice receiving low-iron diet had significantly elevated bone Fgf23 mRNA. WT mice on a low-iron diet maintained normal serum intact Fgf23 and phosphate metabolism, with elevated serum C-terminal Fgf23 fragments. In contrast, the ADHR mice on the low-iron diet had elevated intact and C-terminal Fgf23 with hypophosphatemic osteomalacia. We used in vitro iron chelation to isolate the effects of iron deficiency on Fgf23 expression. We found that iron chelation in vitro resulted in a significant increase in Fgf23 mRNA that was dependent upon Mapk. Thus, unlike other syndromes of elevated FGF23, our findings support the concept that late-onset ADHR is the product of gene-environment interactions whereby the combined presence of an Fgf23-stabilizing mutation and iron deficiency can lead to ADHR.

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.

Memory deficits due to familial British dementia BRI2 mutation are caused by loss of BRI2 function rather than amyloidosis.

Familial dementias, which include Alzheimer disease (AD), familial British dementia (FBD), and familial Danish dementia (FDD), are caused by dominantly inherited autosomal mutations and are characterized by the production of amyloidogenic peptides, neurofibrillary tangles (NFTs) and neurodegeneration (St George-Hyslop and Petit, 2005; Garringer et al., 2009). The prevailing pathogenic theory, the "amyloid cascade hypothesis" (Hardy and Selkoe, 2002), posits that the accumulation of amyloidogenic peptides triggers tauopathy, neurodegeneration, and cognitive and behavioral changes. However, this hypothesis is yet to be validated, and causes of dementia may be multifaceted and involve other mechanisms, such as loss of function due to pathogenic mutations. Mouse models of human dementia invariably use transgenic expression systems (LaFerla and Oddo, 2005; McGowan et al., 2006; Vidal et al., 2009; Coomaraswamy et al., 2010) that do not reflect the genotypes of human disease and cannot replicate loss of function. Therefore, we generated a knock-in (KI) mouse model of FBD (FBD(KI)) genetically congruous with the human disease. FBD is caused by a missense mutation at the stop codon of the BRI2 gene (Vidal et al., 1999) and, like FBD patients, FBD(KI) mice carry this mutation in one of the two murine Bri2 alleles. We report that the British mutation drastically reduces expression of mature BRI2 in both KI mice and human FBD brains. This deficit is associated with severe hippocampal memory deficits in FBD(KI) mice. Remarkably, these animals showed no cerebral amyloidosis and tauopathy. Bri2(+/-) mice present memory deficits similar to those in FBD(KI) animals. Collectively, these results indicate that the British BRI2 mutation underlies abnormal memory due to loss of BRI2 function and independently of histopathological alterations typically evident in advanced neurodegenerative disease.