Holly J. Garringer

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 176RXXR179/S180 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.

Cryo-EM structures of tau filaments from Alzheimer's disease.

Alzheimer’s disease is the most common neurodegenerative disease, and there are no mechanism-based therapies. The disease is defined by the presence of abundant neurofibrillary lesions and neuritic plaques in the cerebral cortex. Neurofibrillary lesions comprise paired helical and straight tau filaments, whereas tau filaments with different morphologies characterize other neurodegenerative diseases. No high-resolution structures of tau filaments are available. Here we present cryo-electron microscopy (cryo-EM) maps at 3.4–3.5 Å resolution and corresponding atomic models of paired helical and straight filaments from the brain of an individual with Alzheimer’s disease. Filament cores are made of two identical protofilaments comprising residues 306–378 of tau protein, which adopt a combined cross-β/β-helix structure and define the seed for tau aggregation. Paired helical and straight filaments differ in their inter-protofilament packing, showing that they are ultrastructural polymorphs. These findings demonstrate that cryo-EM allows atomic characterization of amyloid filaments from patient-derived material, and pave the way for investigation of a range of neurodegenerative diseases.

Abnormal iron metabolism and oxidative stress in mice expressing a mutant form of the ferritin light polypeptide gene

Insertional mutations in exon 4 of the ferritin light chain (FTL) gene are associated with hereditary ferritinopathy (HF) or neuroferritinopathy, an autosomal dominant neurodegenerative disease characterized by progressive impairment of motor and cognitive functions. To determine the pathogenic mechanisms by which mutations in FTL lead to neurodegeneration, we investigated iron metabolism and markers of oxidative stress in the brain of transgenic (Tg) mice that express the mutant human FTL498‐499InsTC cDNA. Compared with wild‐type mice, brain extracts from Tg (FTL‐Tg) mice showed an increase in the cytoplasmic levels of both FTL and ferritin heavy chain polypeptides, a decrease in the protein and mRNA levels of transferrin receptor‐1, and a significant increase in iron levels. Transgenic mice also showed the presence of markers for lipid peroxidation, protein carbonyls, and nitrone–protein adducts in the brain. However, gene expression analysis of iron management proteins in the liver of Tg mice indicates that the FTL‐Tg mouse liver is iron deficient. Our data suggest that disruption of iron metabolism in the brain has a primary role in the process of neurodegeneration in HF and that the pathogenesis of HF is likely to result from a combination of reduction in iron storage function and enhanced toxicity associated with iron‐induced ferritin aggregates in the brain.

Phenotypic spectrum of mosaic trisomy 18: Two new patients, a literature review, and counseling issues†

Mosaic trisomy 18 occurs when two different cell lines exist in the same individual; one cell line has two copies of chromosome 18, while the other has three copies. Here we present two new patients with mosaic trisomy 18, summarize 31 reported cases from the literature, and discuss management and counseling themes. Our first patient is an 8½‐year‐old female with normal intelligence and no significant dysmorphic features other than short stature and cubitus valgus. The second patient is a 21‐month‐old male with developmental delay, several dysmorphic features, including a patent ductus arteriosus, and normal growth. In general, the phenotype of individuals with mosaic trisomy 18 varies greatly. Some individuals have the complete trisomy 18, Edwards syndrome phenotype with early death while others are phenotypically completely normal. The latter group is exemplified by four normal appearing adults with mosaic trisomy 18 who were identified only after giving birth to children with complete trisomy 18. Further, a wide range of anomalies have been reported, most at low frequencies, including microcephaly, delayed bone age, brachydactyly, congenital heart defects, developmental delay, short stature, and premature ovarian failure. Intellectual capabilities range from profound mental retardation to above average intelligence. There appears to be no correlation with the percentage of trisomic cells in either fibroblasts or leukocytes and the individuals phenotype or intellectual function. We also discuss a variety of counseling issues including long‐term survival, reproductive capacity of individuals with mosaic trisomy 18, and recurrence risks.

Two novel GALNT3 mutations in familial tumoral calcinosis.

Familial tumoral calcinosis (TC) is characterized by elevated serum phosphate concentrations, normal or elevated 1,25(OH)2 vitamin D, as well as periarticular and vascular calcifications. Recessive mutations in the mucin‐like glycosyltransferase GalNAc transferase‐3 (GALNT3) and the phosphaturic hormone fibroblast growth factor‐23 (FGF23) have been shown to result in TC. In the present study, mutational analyses were performed on two patients with TC to determine the molecular basis of their diseases. Analysis of the first patient revealed a novel, homozygous base insertion (1102_1103insT) in GALNT3 exon 5 that results in a frameshift and premature stop codon (E375X). The second patient had a novel homozygous transition (1460 g>a) in GALNT3 exon 7, which caused a nonsense mutation (W487X). Both mutations are predicted to markedly truncate the mature GALNT3 protein product. Although the patients carry GALNT3 mutations, these individuals presented with low‐normal serum concentrations of intact biologically active FGF23 and high levels of C‐terminal FGF23. In order to discern a possible relationship between GALNT3 and FGF23 in TC, a comprehensive assessment of the reported TC mutations was also performed. In summary, we have detected novel GALNT3 mutations that result in familial TC, and show that disturbed serum FGF23 concentrations are present in our TC cases as well as in previously reported cases. These studies expand our current genetic understanding of familial TC, and support a pathophysiological association between GALNT3 and FGF23.

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 Å 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.

Modeling familial British and Danish dementia

Familial British dementia (FBD) and familial Danish dementia (FDD) are two autosomal dominant neurodegenerative diseases caused by mutations in the BRI2 gene. FBD and FDD are characterized by widespread cerebral amyloid angiopathy (CAA), parenchymal amyloid deposition, and neurofibrillary tangles. Transgenic mice expressing wild-type and mutant forms of the BRI2 protein, Bri2 knock-in mutant mice, and Bri2 gene knock-out mice have been developed. Transgenic mice expressing a human FDD-mutated form of the BRI2 gene have partially reproduced the neuropathological lesions observed in FDD. These mice develop extensive CAA, parenchymal amyloid deposition, and neuroinflammation in the central nervous system. These animal models allow the study of the molecular mechanism(s) underlying the neuronal dysfunction in these diseases and allow the development of potential therapeutic approaches for these and related neurodegenerative conditions. In this review, a comprehensive account of the advances in the development of animal models for FBD and FDD and of their relevance to the study of Alzheimer disease is presented.

Increased Tau Phosphorylation and Tau Truncation, and Decreased Synaptophysin Levels in Mutant BRI2/Tau Transgenic Mice

Familial Danish dementia (FDD) is an autosomal dominant neurodegenerative disease caused by a 10-nucleotide duplication-insertion in the BRI2 gene. FDD is clinically characterized by loss of vision, hearing impairment, cerebellar ataxia and dementia. The main neuropathologic findings in FDD are the deposition of Danish amyloid (ADan) and the presence of neurofibrillary tangles (NFTs). Here we investigated tau accumulation and truncation in double transgenic (Tg-FDD-Tau) mice generated by crossing transgenic mice expressing human Danish mutant BRI2 (Tg-FDD) with mice expressing human 4-repeat mutant Tau-P301S (Tg-Tau). Compared to Tg-Tau mice, we observed a significant enhancement of tau deposition in Tg-FDD-Tau mice. In addition, a significant increase in tau cleaved at aspartic acid (Asp) 421 was observed in Tg-FDD-Tau mice. Tg-FDD-Tau mice also showed a significant decrease in synaptophysin levels, occurring before widespread deposition of fibrillar ADan and tau can be observed. Thus, the presence of soluble ADan/mutant BRI2 can lead to significant changes in tau metabolism and synaptic dysfunction. Our data provide new in vivo insights into the pathogenesis of FDD and the pathogenic pathway(s) by which amyloidogenic peptides, regardless of their primary amino acid sequence, can cause neurodegeneration.

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.

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 β‐amyloidosis, one of the hallmarks of Alzheimer disease (AD) pathology, based on the overexpression of a mutated cDNA of the amyloid‐β precursor protein (AβPP) or by knock‐in of the murine Aβpp 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 AβPP and the L166P presenilin 1 mutation. At ∼6 mo of age, double‐mutant mice develop amyloid pathology, with signs of neuritic dystrophy, intracellular Aβ accumulation, and glial inflammation, an increase in AβPP C‐terminal fragments, and an 8 times increase in Aβ42 levels with a 40% decrease in Aβ40 levels, leading to a significant increase (14 times) of Aβ42/Aβ40 ratios, with minimal effects on presenilin or the Notch1 pathway in the brain. We conclude that in mice, neither mutations in AβPP nor overexpression of an AβPP isoform are a prerequisite for Aβ 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 AβPP isoform using exogenous promoters.—Vidal, R., Sammeta, N., Garringer, H. J., Sambamurti, K., Miravalle, L., Lamb B. T., Ghetti, B. The Psen1‐L166P‐knock‐in mutation leads to amyloid deposition in human wild‐type amyloid precursor protein YAC transgenic mice. FASEB J. 26, 2899–2910 (2012). www.fasebj.org