CSF ferritin in the clinicopathological progression of Alzheimer's disease and associations with APOE and inflammation biomarkers.

BACKGROUND: A putative role for iron in driving Alzheimer's disease (AD) progression is complicated by previously reported associations with neuroinflammation, apolipoprotein E and AD proteinopathy. To establish how iron interacts with clinicopathological features of AD and at what disease stage iron influences cognitive outcomes, we investigated the association of cerebrospinal fluid (CSF) biomarkers of iron (ferritin), inflammation (acute phase response proteins) and apolipoproteins with pathological biomarkers (CSF Aß42/t-tau, p-tau181), clinical staging and longitudinal cognitive deterioration in subjects from the BioFINDER cohort, with replication of key results in the Alzheimer's Disease Neuroimaging Initiative (ADNI) cohort. METHODS: Ferritin, acute phase response proteins (n=9) and apolipoproteins (n=6) were measured in CSF samples from BioFINDER (n=1239; 4 years cognitive follow-up) participants stratified by cognitive status (cognitively unimpaired, mild cognitive impairment, AD) and for the presence of amyloid and tangle pathology using CSF Aß42/t-tau (A+) and p-tau181 (T+). The ferritin and apolipoprotein E associations were replicated in the ADNI (n=264) cohort. RESULTS: In both cohorts, ferritin and apoE were elevated in A-T+ and A+T+ subjects (16%-40%), but not clinical diagnosis. Other apolipoproteins and acute phase response proteins increased with clinical diagnosis, not pathology. CSF ferritin was positively associated with p-tau181, which was mediated by apolipoprotein E. An optimised threshold of ferritin predicted cognitive deterioration in mild cognitive impairment subjects in the BioFINDER cohort, especially those people classified as A-T- and A+T-. CONCLUSIONS: CSF markers of iron and neuroinflammation have distinct associations with disease stages, while iron may be more intimately associated with apolipoprotein E and tau pathology.

Apolipoprotein E potently inhibits ferroptosis by blocking ferritinophagy.

Allelic variation to the APOE gene confers the greatest genetic risk for sporadic Alzheimer's disease (AD). Independent of genotype, low abundance of apolipoprotein E (apoE), is characteristic of AD CSF, and predicts cognitive decline. The mechanisms underlying the genotype and apoE level risks are uncertain. Recent fluid and imaging biomarker studies have revealed an unexpected link between apoE and brain iron, which also forecasts disease progression, possibly through ferroptosis, an iron-dependent regulated cell death pathway. Here, we report that apoE is a potent inhibitor of ferroptosis (EC50 ≈ 10 nM; N27 neurons). We demonstrate that apoE signals to activate the PI3K/AKT pathway that then inhibits the autophagic degradation of ferritin (ferritinophagy), thus averting iron-dependent lipid peroxidation. Using postmortem inferior temporal brain cortex tissue from deceased subjects from the Rush Memory and Aging Project (MAP) (N = 608), we found that the association of iron with pathologically confirmed clinical Alzheimer's disease was stronger among those with the adverse APOE-ε4 allele. While protection against ferroptosis did not differ between apoE isoforms in vitro, other features of ε4 carriers, such as low abundance of apoE protein and higher levels of polyunsaturated fatty acids (which fuel ferroptosis) could mediate the ε4 allele's heighted risk of AD. These data support ferroptosis as a putative pathway to explain the major genetic risk associated with late onset AD.

Ferroptosis as a mechanism of neurodegeneration in Alzheimer's disease.

Alzheimer's disease (AD) is the most prevalent form of dementia, with complex pathophysiology that is not fully understood. While ß-amyloid plaque and neurofibrillary tangles define the pathology of the disease, the mechanism of neurodegeneration is uncertain. Ferroptosis is an iron-mediated programmed cell death mechanism characterised by phospholipid peroxidation that has been observed in clinical AD samples. This review will outline the growing molecular and clinical evidence implicating ferroptosis in the pathogenesis of AD, with implications for disease-modifying therapies.

Pulsed electron paramagnetic resonance spectroscopy of (33)S-labeled molybdenum cofactor in catalytically active bioengineered sulfite oxidase.

Molybdenum enzymes contain at least one pyranopterin dithiolate (molybdopterin, MPT) moiety that coordinates Mo through two dithiolate (dithiolene) sulfur atoms. For sulfite oxidase (SO), hyperfine interactions (hfi) and nuclear quadrupole interactions (nqi) of magnetic nuclei (I ≠ 0) near the Mo(V) (d(1)) center have been measured using high-resolution pulsed electron paramagnetic resonance (EPR) methods and interpreted with the help of density functional theory (DFT) calculations. These have provided important insights about the active site structure and the reaction mechanism of the enzyme. However, it has not been possible to use EPR to probe the dithiolene sulfurs directly since naturally abundant (32)S has no nuclear spin (I = 0). Here we describe direct incorporation of (33)S (I = 3/2), the only stable magnetic sulfur isotope, into MPT using controlled in vitro synthesis with purified proteins. The electron spin echo envelope modulation (ESEEM) spectra from (33)S-labeled MPT in this catalytically active SO variant are dominated by the "interdoublet" transition arising from the strong nuclear quadrupole interaction, as also occurs for the (33)S-labeled exchangeable equatorial sulfite ligand [ Klein, E. L., et al. Inorg. Chem. 2012 , 51 , 1408 - 1418 ]. The estimated experimental hfi and nqi parameters for (33)S (aiso = 3 MHz and e(2)Qq/h = 25 MHz) are in good agreement with those predicted by DFT. In addition, the DFT calculations show that the two (33)S atoms are indistinguishable by EPR and reveal a strong intermixing between their out-of-plane pz orbitals and the dxy orbital of Mo(V).

Molybdenum cofactor deficiency: metabolic link between taurine and S-sulfocysteine.

Molybdenum cofactor deficiency (MoCD) is a rare inherited metabolic disorder characterized by severe and progressive neurologic damage mainly caused by the loss of sulfite oxidase activity. Elevated urinary levels of sulfite, thiosulfate, and S-sulfocysteine (SSC) are hallmarks in the diagnosis of both MoCD and sulfite oxidase deficiency. Sulfite is generated throughout the catabolism of sulfur-containing amino acids cysteine and methionine. Accumulated sulfite reacts with cystine, thus leading to the formation of SSC, a glutamate analogue, which is assumed to cause N-methyl-D-aspartate receptor-mediated neurodegeneration in MoCD patients. Recently, we described a fast and sensitive HPLC method for diagnostic and treatment monitoring of MoCD patients based on SSC quantification. In this study, we extend the HPLC method to the analysis of hypotaurine and taurine in urine samples and no interference with other compounds was found. Besides the known elevation of SSC and taurine, also hypotaurine shows strong accumulation in MoCD patients, for which the molecular basis is not understood. SSC, hypotaurine, and taurine urinary excretion values from control individuals as well as MoCD patients are reported and over 20-fold increase in taurine urinary excretion was determined for MoCD patients demonstrating a direct link between sulfite toxicity and taurine biosynthesis in MoCD.

Selective ferroptosis vulnerability due to familial Alzheimer's disease presenilin mutations.

Mutations in presenilin 1 and 2 (PS1 and PS2) cause autosomal dominant familial Alzheimer's disease (FAD). Ferroptosis has been implicated as a mechanism of neurodegeneration in AD since neocortical iron burden predicts Alzheimer's disease (AD) progression. We found that loss of the presenilins dramatically sensitizes multiple cell types to ferroptosis, but not apoptosis. FAD causal mutations of presenilins similarly sensitizes cells to ferroptosis. The presenilins promote the expression of GPX4, the selenoprotein checkpoint enzyme that blocks ferroptosis by quenching the membrane propagation of lethal hydroperoxyl radicals. Presenilin γ-secretase activity cleaves Notch-1 to signal LRP8 expression, which then controls GPX4 expression by regulating the supply of selenium into the cell since LRP8 is the uptake receptor for selenoprotein P. Selenium uptake is thus disrupted by presenilin FAD mutations, suppressing GPX4 expression. Therefore, presenilin mutations may promote neurodegeneration by derepressing ferroptosis, which has implications for disease-modifying therapeutics.

Irregular RNA splicing curtails postsynaptic gephyrin in the cornu ammonis of patients with epilepsy.

Anomalous hippocampal inhibition is involved in temporal lobe epilepsy, and reduced gephyrin immunoreactivity in the temporal lobe epilepsy hippocampus has been reported recently. However, the mechanisms responsible for curtailing postsynaptic gephyrin scaffolds are poorly understood. Here, we have investigated gephyrin expression in the hippocampus of patients with intractable temporal lobe epilepsy. Immunohistochemical and western blot analyses revealed irregular gephyrin expression in the cornu ammonis of patients with temporal lobe epilepsy and four abnormally spliced gephyrins lacking several exons in their G-domains were isolated. Identified temporal lobe epilepsy gephyrins have oligomerization deficits and they curtail hippocampal postsynaptic gephyrin and GABA(A) receptor α2 while interacting with regularly spliced gephyrins. We found that cellular stress (alkalosis and hyperthermia) induces exon skipping in gephyrin messenger RNA, which is responsible for curtailed postsynaptic gephyrin and GABA(A) receptor α2 scaffolds. Accordingly, we did not obtain evidence for gephyrin gene mutations in patients with temporal lobe epilepsy. Cellular stress such as alkalosis, for example arising from seizure activity, could thus facilitate the development of temporal lobe epilepsy by reducing GABA(A) receptor α2-mediated hippocampal synaptic transmission selectively in the cornu ammonis.

Characterization of Selenium Compounds for Anti-ferroptotic Activity in Neuronal Cells and After Cerebral Ischemia-Reperfusion Injury.

The emergence of ferroptosis as a cell death pathway associated with brain disorders including stroke and neurodegenerative diseases emphasizes the need to develop therapeutics able to target the brain and to protect neurons from ferroptotic death. Selenium plays an essential role in reducing lipid peroxidation generated during ferroptosis through its incorporation into the catalytic site of glutathione peroxidase 4. Here, we compared the anti-ferroptotic activity of several organic and inorganic selenium compounds: methylselenocysteine, selenocystine, selenomethionine, selenocystamine, ebselen, sodium selenite, and sodium selenate. All were effective against erastin- and RSL3-induced ferroptosis in vitro. We characterized the ability of the selenium compounds to release selenium and boost glutathione peroxidase expression and activity. Based on our results, we selected organic selenium compounds of similar characteristics and investigated their effectiveness in protecting against neuronal death in vivo using the cerebral ischemia-reperfusion injury mouse model. We found that pretreatment with methylselenocysteine or selenocystamine provided protection from ischemia-reperfusion neuronal damage in vivo. These data support the use of ferroptosis inhibitors for treatment and select selenium compounds for prevention of neuronal damage in ischemic stroke and other diseases of the brain where ferroptosis is implicated.

Ferroptosis: mechanisms and links with diseases.

Ferroptosis is an iron-dependent cell death, which is different from apoptosis, necrosis, autophagy, and other forms of cell death. The process of ferroptotic cell death is defined by the accumulation of lethal lipid species derived from the peroxidation of lipids, which can be prevented by iron chelators (e.g., deferiprone, deferoxamine) and small lipophilic antioxidants (e.g., ferrostatin, liproxstatin). This review summarizes current knowledge about the regulatory mechanism of ferroptosis and its association with several pathways, including iron, lipid, and cysteine metabolism. We have further discussed the contribution of ferroptosis to the pathogenesis of several diseases such as cancer, ischemia/reperfusion, and various neurodegenerative diseases (e.g., Alzheimer's disease and Parkinson's disease), and evaluated the therapeutic applications of ferroptosis inhibitors in clinics.

Cellular Senescence and Iron Dyshomeostasis in Alzheimer's Disease.

Iron dyshomeostasis is a feature of Alzheimer's disease (AD). The impact of iron on AD is attributed to its interactions with the central proteins of AD pathology (amyloid precursor protein and tau) and/or through the iron-mediated generation of prooxidant molecules (e.g., hydroxyl radicals). However, the source of iron accumulation in pathologically relevant regions of the brain and its contribution to AD remains unclear. One likely contributor to iron accumulation is the age-associated increase in tissue-resident senescent cells that drive inflammation and contribute to various pathologies associated with advanced age. Iron accumulation predisposes ageing tissue to oxidative stress that can lead to cellular dysfunction and to iron-dependent cell death modalities (e.g., ferroptosis). Further, elevated brain iron is associated with the progression of AD and cognitive decline. Elevated brain iron presents a feature of AD that may be modified pharmacologically to mitigate the effects of age/senescence-associated iron dyshomeostasis and improve disease outcome.

S-sulfocysteine/NMDA receptor-dependent signaling underlies neurodegeneration in molybdenum cofactor deficiency.

Molybdenum cofactor deficiency (MoCD) is an autosomal recessive inborn error of metabolism characterized by neurodegeneration and death in early childhood. The rapid and progressive neurodegeneration in MoCD presents a major clinical challenge and may relate to the poor understanding of the molecular mechanisms involved. Recently, we reported that treating patients with cyclic pyranopterin monophosphate (cPMP) is a successful therapy for a subset of infants with MoCD and prevents irreversible brain damage. Here, we studied S-sulfocysteine (SSC), a structural analog of glutamate that accumulates in the plasma and urine of patients with MoCD, and demonstrated that it acts as an N-methyl D-aspartate receptor (NMDA-R) agonist, leading to calcium influx and downstream cell signaling events and neurotoxicity. SSC treatment activated the protease calpain, and calpain-dependent degradation of the inhibitory synaptic protein gephyrin subsequently exacerbated SSC-mediated excitotoxicity and promoted loss of GABAergic synapses. Pharmacological blockade of NMDA-R, calcium influx, or calpain activity abolished SSC and glutamate neurotoxicity in primary murine neurons. Finally, the NMDA-R antagonist memantine was protective against the manifestation of symptoms in a tungstate-induced MoCD mouse model. These findings demonstrate that SSC drives excitotoxic neurodegeneration in MoCD and introduce NMDA-R antagonists as potential therapeutics for this fatal disease.

The Complex Role of Apolipoprotein E in Alzheimer's Disease: an Overview and Update.

Apolipoprotein E (ApoE) plays a crucial role in the homeostatic control of lipids in both the periphery and the central nervous system (CNS). In humans, ApoE exists in three different isoforms: ε2, ε3 and ε4. ApoE ε3 is the most common isoform, while the ε4 isoform confers the greatest genetic risk for Alzheimer's disease (AD). However, the mechanisms underlying how ApoE contributes to the pathogenesis of AD are still debated. ApoE has been shown to impact amyloid ß (Aß) deposition and clearance in the brain. ApoE also has Aß-independent pathways in AD, which has led to the discovery of new roles of ApoE ranging from mitochondria dysfunction to, most recently, iron metabolism. Here, we review the role of ApoE in health and in AD, with the view of identifying therapeutic approaches that could prevent the risk associated with the ε4 isoform.

Low-molecular weight heparin increases circulating sFlt-1 levels and enhances urinary elimination.

RATIONALE: Preeclampsia is a devastating medical complication of pregnancy which leads to maternal and fetal morbidity and mortality. While the etiology of preeclampsia is unclear, human and animal studies suggest that excessive circulating levels of soluble fms-like tyrosine-kinase-1 (sFlt-1), an alternatively spliced variant of VEGF-receptor1, contribute to the signs and symptoms of preeclampsia. Since sFlt-1 binds to heparin and heparan sulfate proteoglycans, we hypothesized that the anticoagulant heparin, which is often used in pregnancy, may interfere with the levels, distribution and elimination of sFlt-1 in vivo. OBJECTIVE: We systematically determined serum and urine levels of angiogenic factors in preeclamptic women before and after administration of low molecular weight heparin and further characterized the interaction with heparin in biochemical studies. METHODS AND RESULTS: Serum and urine samples were used to measure sFlt-1 levels before and after heparin administration. Serum levels of sFlt-1 increased by 25% after heparin administration in pregnant women. The magnitude of the increase in circulating sFlt-1 correlated with initial sFlt-1 serum levels. Urinary sFlt-1 levels were also elevated following heparin administration and levels of elimination were dependent on the underlying integrity of the glomerular filtration barrier. Biochemical binding studies employing cation exchange chromatography revealed that heparin bound sFlt-1 had decreased affinity to negatively charged surfaces when compared to sFlt-1 alone. CONCLUSION: Low molecular weight heparin administration increased circulating sFlt1 levels and enhanced renal elimination. We provide evidence that both effects may be due to heparin binding to sFlt1 and masking the positive charges on sFlt1 protein.

Molybdenum cofactor deficiency: a new HPLC method for fast quantification of s-sulfocysteine in urine and serum.

Molybdenum cofactor deficiency (MoCD) is a rare inherited metabolic disorder characterized by severe and progressive neurological damage mainly caused by the loss of sulfite oxidase activity. Elevated urinary levels of sulfite, thiosulfate, and S-sulfocysteine (SSC) are hallmarks in the diagnosis of MoCD and sulfite oxidase deficiency (SOD). Recently, a first successful treatment of a human MoCD type A patient based on a substitution therapy with the molybdenum cofactor precursor cPMP has been reported, resulting in nearly complete normalization of MoCD biomarkers. Knowing the rapid progression of the disease symptoms in nontreated patients, an early diagnosis of MoCD as well as a sensitive method to monitor daily changes in SSC levels, a key marker of sulfite toxicity, is crucial for treatment outcome. Here, we describe a fast and sensitive method for the analysis of SSC in human urine samples using high performance liquid chromatography (HPLC). The analysis is based on precolumn derivatization with O-phthaldialdehyde (OPA) and separation on a C18 reverse phase column coupled to UV detection. The method was extended to human serum analysis and no interference with endogenous amino acids was found. Finally, SSC values from 45 pediatric urine, 75 adult urine, and 24 serum samples from control individuals as well as MoCD patients are reported. Our method represents a cost-effective technique for routine diagnosis of MoCD and SOD, and can be used also to monitor treatment efficiency in those sulfite toxicity disorders on a daily basis.