Microglial ferroptotic stress causes non-cell autonomous neuronal death.

BACKGROUND: Ferroptosis is a form of regulated cell death characterised by lipid peroxidation as the terminal endpoint and a requirement for iron. Although it protects against cancer and infection, ferroptosis is also implicated in causing neuronal death in degenerative diseases of the central nervous system (CNS). The precise role for ferroptosis in causing neuronal death is yet to be fully resolved. METHODS: To elucidate the role of ferroptosis in neuronal death we utilised co-culture and conditioned medium transfer experiments involving microglia, astrocytes and neurones. We ratified clinical significance of our cell culture findings via assessment of human CNS tissue from cases of the fatal, paralysing neurodegenerative condition of amyotrophic lateral sclerosis (ALS). We utilised the SOD1G37R mouse model of ALS and a CNS-permeant ferroptosis inhibitor to verify pharmacological significance in vivo. RESULTS: We found that sublethal ferroptotic stress selectively affecting microglia triggers an inflammatory cascade that results in non-cell autonomous neuronal death. Central to this cascade is the conversion of astrocytes to a neurotoxic state. We show that spinal cord tissue from human cases of ALS exhibits a signature of ferroptosis that encompasses atomic, molecular and biochemical features. Further, we show the molecular correlation between ferroptosis and neurotoxic astrocytes evident in human ALS-affected spinal cord is recapitulated in the SOD1G37R mouse model where treatment with a CNS-permeant ferroptosis inhibitor, CuII(atsm), ameliorated these markers and was neuroprotective. CONCLUSIONS: By showing that microglia responding to sublethal ferroptotic stress culminates in non-cell autonomous neuronal death, our results implicate microglial ferroptotic stress as a rectifiable cause of neuronal death in neurodegenerative disease. As ferroptosis is currently primarily regarded as an intrinsic cell death phenomenon, these results introduce an entirely new pathophysiological role for ferroptosis in disease.

Fundamental Neurochemistry Review: Copper availability as a potential therapeutic target in progressive supranuclear palsy: Insight from other neurodegenerative diseases.

Since the first description of Parkinson's disease (PD) over two centuries ago, the recognition of rare types of atypical parkinsonism has introduced a spectrum of related PD-like diseases. Among these is progressive supranuclear palsy (PSP), a neurodegenerative condition that clinically differentiates through the presence of additional symptoms uncommon in PD. As with PD, the initial symptoms of PSP generally present in the sixth decade of life when the underpinning neurodegeneration is already significantly advanced. The causal trigger of neuronal cell loss in PSP is unknown and treatment options are consequently limited. However, converging lines of evidence from the distinct neurodegenerative conditions of PD and amyotrophic lateral sclerosis (ALS) are beginning to provide insights into potential commonalities in PSP pathology and opportunity for novel therapeutic intervention. These include accumulation of the high abundance cuproenzyme superoxide dismutase 1 (SOD1) in an aberrant copper-deficient state, associated evidence for altered availability of the essential micronutrient copper, and evidence for neuroprotection using compounds that can deliver available copper to the central nervous system. Herein, we discuss the existing evidence for SOD1 pathology and copper imbalance in PSP and speculate that treatments able to provide neuroprotection through manipulation of copper availability could be applicable to the treatment of PSP.

The Compound ATH434 Prevents Alpha-Synuclein Toxicity in a Murine Model of Multiple System Atrophy.

BACKGROUND: An elevation in iron levels, together with an accumulation of α-synuclein within the oligodendrocytes, are features of the rare atypical parkinsonian disorder, Multiple System Atrophy (MSA). We have previously tested the novel compound ATH434 (formally called PBT434) in preclinical models of Parkinson's disease and shown that it is brain-penetrant, reduces iron accumulation and iron-mediated redox activity, provides neuroprotection, inhibits alpha synuclein aggregation and lowers the tissue levels of alpha synuclein. The compound was also well-tolerated in a first-in-human oral dosing study in healthy and older volunteers with a favorable, dose-dependent pharmacokinetic profile. OBJECTIVE: To evaluate the efficacy of ATH434 in a mouse MSA model. METHODS: The PLP-α-syn transgenic mouse overexpresses α-synuclein, demonstrates oligodendroglial pathology, and manifests motor and non-motor aspects of MSA. Animals were provided ATH434 (3, 10, or 30 mg/kg/day spiked into their food) or control food for 4 months starting at 12 months of age and were culled at 16 months. Western blot was used to assess oligomeric and urea soluble α-synuclein levels in brain homogenates, whilst stereology was used to quantitate the number of nigral neurons and glial cell inclusions (GCIs) present in the substantia nigra pars compacta. RESULTS: ATH434 reduced oligomeric and urea soluble α-synuclein aggregation, reduced the number of GCIs, and preserved SNpc neurons. In vitro experiments suggest that ATH434 prevents the formation of toxic oligomeric "species of synuclein". CONCLUSION: ATH434 is a promising small molecule drug candidate that has potential to move forward to trial for treating MSA.

l-3,4-dihydroxyphenylalanine (l-DOPA) modulates brain iron, dopaminergic neurodegeneration and motor dysfunction in iron overload and mutant alpha-synuclein mouse models of Parkinson's disease.

Treatment with the dopamine (DA) precursor l-3,4-dihydroxyphenylalanine (l-DOPA) provides symptomatic relief arising from DA denervation in Parkinson's disease. Mounting evidence that DA autooxidation to neurotoxic quinones is involved in Parkinson's disease pathogenesis has raised concern about potentiation of oxidative stress by l-DOPA. The rate of DA quinone formation increases in the presence of excess redox-active iron (Fe), which is a pathological hallmark of Parkinson's disease. Conversely, l-DOPA has pH-dependent Fe-chelating properties, and may act to 'redox silence' Fe and partially allay DA autoxidation. We examined the effects of l-DOPA in three murine models of parkinsonian neurodegeneration: early-life Fe overexposure in wild-type mice, transgenic human (h)A53T mutant α-synuclein (α-syn) over-expression, and a combined 'multi-hit' model of Fe-overload in hA53T mice. We found that l-DOPA was neuroprotective and prevented age-related Fe accumulation in the substantia nigra pars compacta (SNc), similar to the mild-affinity Fe chelator clioquinol. Chronic l-DOPA treatment showed no evidence of increased oxidative stress in wild-type midbrain and normalized motor performance, when excess Fe was present. Similarly, l-DOPA also did not exacerbate protein oxidation levels in hA53T mice, with or without excess nigral Fe, and showed evidence of neuroprotection. The effects of l-DOPA in Fe-fed hA53T mice were somewhat muted, suggesting that Fe-chelation alone is insufficient to attenuate neuron loss in an animal model also recapitulating altered DA metabolism. In summary, we found no evidence in any of our model systems that l-DOPA treatment accentuated neurodegeneration, suggesting DA replacement therapy does not contribute to oxidative stress in the Parkinson's disease brain.

The novel compound PBT434 prevents iron mediated neurodegeneration and alpha-synuclein toxicity in multiple models of Parkinson's disease.

Elevated iron in the SNpc may play a key role in Parkinson's disease (PD) neurodegeneration since drug candidates with high iron affinity rescue PD animal models, and one candidate, deferirpone, has shown efficacy recently in a phase two clinical trial. However, strong iron chelators may perturb essential iron metabolism, and it is not yet known whether the damage associated with iron is mediated by a tightly bound (eg ferritin) or lower-affinity, labile, iron pool. Here we report the preclinical characterization of PBT434, a novel quinazolinone compound bearing a moderate affinity metal-binding motif, which is in development for Parkinsonian conditions. In vitro, PBT434 was far less potent than deferiprone or deferoxamine at lowering cellular iron levels, yet was found to inhibit iron-mediated redox activity and iron-mediated aggregation of α-synuclein, a protein that aggregates in the neuropathology. In vivo, PBT434 did not deplete tissue iron stores in normal rodents, yet prevented loss of substantia nigra pars compacta neurons (SNpc), lowered nigral α-synuclein accumulation, and rescued motor performance in mice exposed to the Parkinsonian toxins 6-OHDA and MPTP, and in a transgenic animal model (hA53T α-synuclein) of PD. These improvements were associated with reduced markers of oxidative damage, and increased levels of ferroportin (an iron exporter) and DJ-1. We conclude that compounds designed to target a pool of pathological iron that is not held in high-affinity complexes in the tissue can maintain the survival of SNpc neurons and could be disease-modifying in PD.

Clioquinol Improves Cognitive, Motor Function, and Microanatomy of the Alpha-Synuclein hA53T Transgenic Mice.

The abnormal accumulation of alpha-synuclein (α-syn) has been linked to a number of neurodegenerative disorders, the most noteworthy of which is Parkinson's disease. Alpha-synuclein itself is not toxic and fulfills various physiological roles in the central nervous system. However, specific types of aggregates have been shown to be toxic, and metals have been linked to the assembly of these toxic aggregates. In this paper, we have characterized a transgenic mouse that overexpresses the A53T mutation of human α-syn, specifically assessing cognition, motor performance, and subtle anatomical markers that have all been observed in synucleinopathies in humans. We hypothesized that treatment with the moderate-affinity metal chelator, clioquinol (CQ), would reduce the interaction between metals and α-syn to subsequently improve the phenotype of the A53T animal model. We showed that CQ prevents an iron-synuclein interaction, the formation of urea-soluble α-syn aggregates, α-syn-related substantia nigra pars compacta cell loss, reduction in dendritic spine density of hippocampal and caudate putamen medium spiny neurons, and the decline in motor and cognitive function. In conclusion, our data suggests that CQ is capable of mitigating the pathological metal/α-syn interactions, suggesting that the modulation of metal ions warrants further study as a therapeutic approach for the synucleinopathies.

Effects of Neonatal Iron Feeding and Chronic Clioquinol Administration on the Parkinsonian Human A53T Transgenic Mouse.

Increased nigral iron (Fe) is a cardinal feature of Parkinson's disease, as is the accumulation of aggregates comprising α-synuclein. We used wild-type mice and transgenic mice overexpressing the human A53T mutation to α-synuclein to examine the influence of increased Fe (days 10-17 postpartum) on the parkinsonian development phenotype of these animals (including abnormal nigral Fe levels and deficits in both cell numbers and locomotor activity), and to explore the impact of the Fe chelator clioquinol in the model. Both untreated and Fe-loaded A53T mice showed similar levels of nigral cell loss, though 5 months of clioquinol treatment was only able to prevent the loss in the non-Fe-loaded A53T group. Iron levels in the Fe-loaded A53T mice returned to normal at 8 months, though effects of dopamine denervation remained, demonstrated by limited locomotor activity and sustained neuron loss. These data suggest that Fe exposure during a critical developmental window, combined with the overexpression mutant α-synuclein, presents a disease phenotype resistant to intervention using clioquinol later in life.

Correction: Visualising mouse neuroanatomy and function by metal distribution using laser ablation-inductively coupled plasma-mass spectrometry imaging.

[This corrects the article DOI: 10.1039/C5SC02231B.].

Comparative Study of Metal Quantification in Neurological Tissue Using Laser Ablation-Inductively Coupled Plasma-Mass Spectrometry Imaging and X-ray Fluorescence Microscopy.

Redox-active metals in the brain mediate numerous biochemical processes and are also implicated in a number of neurodegenerative diseases. A number of different approaches are available for quantitatively measuring the spatial distribution of biometals at an image resolution approaching the subcellular level. Measured biometal levels obtained using laser ablation-inductively coupled plasma mass spectrometry (LA-ICPMS; spatial resolution 15 µm × 15 µm) were within the range of those obtained using X-ray fluorescence microscopy (XFM; spatial resolution 2 µm × 7 µm) and regional changes in metal concentration across discrete brain regions were replicated to the same degree. Both techniques are well suited to profiling changes in regional biometal distribution between healthy and diseased brain tissues, but absolute quantitation of metal levels varied significantly between methods, depending on the metal of interest. Where all possible variables affect metal levels, independent of a treatment/phenotype are controlled, either method is suitable for examining differences between experimental groups, though, as with any method for imaging post mortem brain tissue, care should be taken when interpreting the total metal levels with regard to physiological concentrations.

Visualising mouse neuroanatomy and function by metal distribution using laser ablation-inductively coupled plasma-mass spectrometry imaging.

Metals have a number of important roles within the brain. We used laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) to map the three-dimensional concentrations and distributions of transition metals, in particular iron (Fe), copper (Cu) and zinc (Zn) within the murine brain. LA-ICP-MS is one of the leading analytical tools for measuring metals in tissue samples. Here, we present a complete data reduction protocol for measuring metals in biological samples, including the application of a pyramidal voxel registration technique to reproducibly align tissue sections. We used gold (Au) nanoparticle and ytterbium (Yb)-tagged tyrosine hydroxylase antibodies to assess the co-localisation of Fe and dopamine throughout the entire mouse brain. We also examined the natural clustering of metal concentrations within the murine brain to elucidate areas of similar composition. This clustering technique uses a mathematical approach to identify multiple 'elemental clusters', avoiding user bias and showing that metal composition follows a hierarchical organisation of neuroanatomical structures. This work provides new insight into the distinct compartmentalisation of metals in the brain, and presents new avenues of exploration with regard to region-specific, metal-associated neurodegeneration observed in several chronic neurodegenerative diseases.

Correction: Visualising mouse neuroanatomy and function by metal distribution using laser ablation-inductively coupled plasma-mass spectrometry imaging.

[This corrects the article DOI: 10.1039/C5SC02231B.].