Metal dyshomeostasis in the substantia nigra of patients with Parkinson's disease or multiple sclerosis.

Abnormal metal distribution in vulnerable brain regions is involved in the pathogenesis of most neurodegenerative diseases, suggesting common molecular mechanisms of metal dyshomeostasis. This study aimed to compare the intra- and extra-neuronal metal content and the expression of proteins related to metal homeostasis in the substantia nigra (SN) from patients with Parkinson's disease (PD), multiple sclerosis (MS), and control subjects. Metal quantification was performed via ion-beam micro-analysis in neuromelanin-positive neurons and the surrounding tissue. For proteomic analysis, SN tissue lysates were analyzed on a nanoflow chromatography system hyphenated to a hybrid triple-quadrupole time-of-flight mass spectrometer. We found increased amounts of iron in neuromelanin-positive neurons and surrounding tissue in patients with PD and MS compared to controls (4- to 5-fold higher) that, however, also showed large inter-individual variations. Copper content was systematically lower (-2.4-fold) in neuromelanin-positive neurons of PD patients compared with controls, whereas it remained unchanged in MS. Protein-protein interaction (PPI) network analyses revealed clusters related to Fe and Cu homeostasis among PD-deregulated proteins. An enrichment for the term "metal homeostasis" was observed for MS-deregulated proteins. Important deregulated hub proteins included hemopexin and transferrin in PD, and calreticulin and ferredoxin reductase in MS. Our findings show that PD and MS share commonalities in terms of iron accumulation in the SN. Concomitant proteomics experiments revealed PPI networks related to metal homeostasis, substantiating the results of metal quantification.

Multi-omic landscaping of human midbrains identifies disease-relevant molecular targets and pathways in advanced-stage Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is the second most common neurodegenerative disorder whose prevalence is rapidly increasing worldwide. The molecular mechanisms underpinning the pathophysiology of sporadic PD remain incompletely understood. Therefore, causative therapies are still elusive. To obtain a more integrative view of disease-mediated alterations, we investigated the molecular landscape of PD in human post-mortem midbrains, a region that is highly affected during the disease process. METHODS: Tissue from 19 PD patients and 12 controls were obtained from the Parkinson's UK Brain Bank and subjected to multi-omic analyses: small and total RNA sequencing was performed on an Illumina's HiSeq4000, while proteomics experiments were performed in a hybrid triple quadrupole-time of flight mass spectrometer (TripleTOF5600+) following quantitative sequential window acquisition of all theoretical mass spectra. Differential expression analyses were performed with customized frameworks based on DESeq2 (for RNA sequencing) and with Perseus v.1.5.6.0 (for proteomics). Custom pipelines in R were used for integrative studies. RESULTS: Our analyses revealed multiple deregulated molecular targets linked to known disease mechanisms in PD as well as to novel processes. We have identified and experimentally validated (quantitative real-time polymerase chain reaction/western blotting) several PD-deregulated molecular candidates, including miR-539-3p, miR-376a-5p, miR-218-5p and miR-369-3p, the valid miRNA-mRNA interacting pairs miR-218-5p/RAB6C and miR-369-3p/GTF2H3, as well as multiple proteins, such as CHI3L1, HSPA1B, FNIP2 and TH. Vertical integration of multi-omic analyses allowed validating disease-mediated alterations across different molecular layers. Next to the identification of individual molecular targets in all explored omics layers, functional annotation of differentially expressed molecules showed an enrichment of pathways related to neuroinflammation, mitochondrial dysfunction and defects in synaptic function. CONCLUSIONS: This comprehensive assessment of PD-affected and control human midbrains revealed multiple molecular targets and networks that are relevant to the disease mechanism of advanced PD. The integrative analyses of multiple omics layers underscore the importance of neuroinflammation, immune response activation, mitochondrial and synaptic dysfunction as putative therapeutic targets for advanced PD.

Brain iron enrichment attenuates α-synuclein spreading after injection of preformed fibrils.

Regional iron accumulation and α-synuclein (α-syn) spreading pathology within the central nervous system are common pathological findings in Parkinson's disease (PD). Whereas iron is known to bind to α-syn, facilitating its aggregation and regulating α-syn expression, it remains unclear if and how iron also modulates α-syn spreading. To elucidate the influence of iron on the propagation of α-syn pathology, we investigated α-syn spreading after stereotactic injection of α-syn preformed fibrils (PFFs) into the striatum of mouse brains after neonatal brain iron enrichment. C57Bl/6J mouse pups received oral gavage with 60, 120, or 240 mg/kg carbonyl iron or vehicle between postnatal days 10 and 17. At 12 weeks of age, intrastriatal injections of 5-µg PFFs were performed to induce seeding of α-syn aggregates. At 90 days post-injection, PFFs-injected mice displayed long-term memory deficits, without affection of motor behavior. Interestingly, quantification of α-syn phosphorylated at S129 showed reduced α-syn pathology and attenuated spreading to connectome-specific brain regions after brain iron enrichment. Furthermore, PFFs injection caused intrastriatal microglia accumulation, which was alleviated by iron in a dose-dependent way. In primary cortical neurons in a microfluidic chamber model in vitro, iron application did not alter trans-synaptic α-syn propagation, possibly indicating an involvement of non-neuronal cells in this process. Our study suggests that α-syn PFFs may induce cognitive deficits in mice independent of iron. However, a redistribution of α-syn aggregate pathology and reduction of striatal microglia accumulation in the mouse brain may be mediated via iron-induced alterations of the brain connectome.

A Workflow for the Performance of the Differential Ovarian Follicle Count Using Deep Neuronal Networks.

In order to automate the counting of ovarian follicles required in multigeneration reproductive studies performed in the rat according to Organization for Economic Co-operation and Development guidelines 443 and 416, the application of deep neural networks was tested. The manual evaluation of the differential ovarian follicle count is a tedious and time-consuming task that requires highly trained personnel. In this regard, deep learning outputs provide overlay pictures for a more detailed documentation, together with an increased reproducibility of the counts. To facilitate the planned good laboratory practice (GLP) validation a workflow was set up using MLFlow to make all steps from generating of scans, training of the neural network, uploading of study images to the neural network, generation and storage of the results in a compliant manner controllable and reproducible. PyTorch was used as main framework to build the Faster region-based convolutional neural network for the training. We compared the performances of different depths of ResNet models with specific regard to the sensitivity, specificity, accuracy of the models. In this paper, we describe all steps from data labeling, training of networks, and the performance metrics chosen to evaluate different network architectures. We also make recommendation on steps, which should be taken into consideration when GLP validation is aimed for.

Imaging of neuronal tissues by x-ray diffraction and x-ray fluorescence microscopy: evaluation of contrast and biomarkers for neurodegenerative diseases.

We have used scanning X-ray diffraction (XRD) and X-ray fluorescence (XRF) with micro-focused synchrotron radiation to study histological sections from human substantia nigra (SN). Both XRF and XRD mappings visualize tissue properties, which are inaccessible by conventional microscopy and histology. We propose to use these advanced tools to characterize neuronal tissue in neurodegeneration, in particular in Parkinson's disease (PD). To this end, we take advantage of the recent experimental progress in x-ray focusing, detection, and use automated data analysis scripts to enable quantitative analysis of large field of views. XRD signals are recorded and analyzed both in the regime of small-angle (SAXS) and wide-angle x-ray scattering (WAXS). The SAXS signal was analyzed in view of the local myelin structure, while WAXS was used to identify crystalline deposits. PD tissue scans exhibited increased amounts of crystallized cholesterol. The XRF analysis showed increased amounts of iron and decreased amounts of copper in the PD tissue compared to the control.

Deferiprone Rescues Behavioral Deficits Induced by Mild Iron Exposure in a Mouse Model of Alpha-Synuclein Aggregation.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder, and its causes remain unknown. A major hallmark of the disease is the increasing presence of aggregated alpha-synuclein (aSyn). Furthermore, there is a solid consensus on iron (Fe) accumulation in several regions of PD brains during disease progression. In our study, we focused on the interaction of Fe and aggregating aSyn in vivo in a transgenic mouse model overexpressing human aSyn bearing the A53T mutation (prnp.aSyn.A53T). We utilized a neonatal iron-feeding model to exacerbate the motor phenotype of the transgenic mouse model. Beginning from day 100, mice were treated with deferiprone (DFP), a ferric chelator that is able to cross the blood-brain barrier and is currently used in clinics as treatment for hemosiderosis. Our paradigm resulted in an impairment of the learning abilities in the rotarod task and the novel object recognition test. DFP treatment significantly improved the performance in both tasks. Although this was not accompanied by alterations in aSyn aggregation, our results support DFP as possible therapeutic option in PD.

Alpha-synuclein and iron: two keys unlocking Parkinson's disease.

Current therapies for Parkinson's disease (PD) confer symptomatic relief and are particularly efficient in the treatment of motor symptoms in earlier disease stages. However, we are still unable to treat the causes of neurodegeneration by modification of the underlying mechanisms, which is partially due to their insufficient understanding. In this short review, we focus on two pivotal disease mechanisms: alpha-synuclein pathology and dysfunction of iron homeostasis as well as their intricate interaction. Both pathomechanisms have been extensively studied in the past and represent valid targets for disease-modifying pharmacological treatment approaches for PD. We summarize the current attempts to exploit iron chelation and modification of alpha-synuclein pathology as translational therapies in PD and discuss the chances and challenges of prospective disease-modifying approaches.

α-Synuclein Over-Expression Induces Increased Iron Accumulation and Redistribution in Iron-Exposed Neurons.

Parkinson's disease is the most common α-synucleinopathy, and increased levels of iron are found in the substantia nigra of Parkinson's disease patients, but the potential interlink between both molecular changes has not been fully understood. Metal to protein binding assays have shown that α-synuclein can bind iron in vitro; therefore, we hypothesized that iron content and iron distribution could be modified in cellulo, in cells over-expressing α-synuclein. Owing to particle-induced X-ray emission and synchrotron X-ray fluorescence chemical nano-imaging, we were able to quantify and describe the iron distribution at the subcellular level. We show that, in neurons exposed to excess iron, the mere over-expression of human α-synuclein results in increased levels of intracellular iron and in iron redistribution from the cytoplasm to the perinuclear region within α-synuclein-rich inclusions. Reproducible results were obtained in two distinct recombinant expression systems, in primary rat midbrain neurons and in a rat neuroblastic cell line (PC12), both infected with viral vectors expressing human α-synuclein. Our results link two characteristic molecular features found in Parkinson's disease, the accumulation of α-synuclein and the increased levels of iron in the substantia nigra.

Alpha-Synuclein Regulates Neuronal Levels of Manganese and Calcium.

Manganese (Mn) may foster aggregation of alpha-synuclein (αSyn) contributing to the pathogenesis of PD. Here, we examined the influence of αSyn overexpression on distribution and oxidation states of Mn in frozen-hydrated primary midbrain neurons (PMNs) by synchrotron-based X-ray fluorescence (XRF) and X-ray absorption near edge structure spectroscopy (XANES). Overexpression of αSyn increased intracellular Mn levels, whereas levels of Ca, Zn, K, P, and S were significantly decreased. Mn oxidation states were not altered. A strong correlation between Cu-/Mn-levels as well as Fe-/Mn-levels was observed in αSyn-overexpressing cells. Subcellular resolution revealed a punctate or filament-like perinuclear and neuritic distribution of Mn, which resembled the expression of DMT1 and MnSOD. While overexpression of αSyn did not significantly alter the expression patterns of the most-expressed Mn transport proteins (DMT1, VGCC, Fpn1), it attenuated the Mn release from Mn-treated neurons. Thus, these data suggest that αSyn may act as an intracellular Mn store. In total, neurotoxicity in PD could be mediated via regulation of transition metal levels and the metal-binding capacity of αSyn, which could represent a promising therapeutic target for this neurodegenerative disorder.

Insights on the interaction of alpha-synuclein and metals in the pathophysiology of Parkinson's disease.

Parkinson's disease (PD) is the most frequent neurodegenerative movement disorder with severe consequences for patients and caregivers. In the last twenty years of research, alpha-synuclein (αSyn) emerged as a main regulator of PD pathology, both in genetic and sporadic cases. Most importantly, oligomeric and aggregated species of αSyn appear to be pathogenic. In addition, transition metals have been implicated in the disease pathogenesis of PD already for decades. The interaction of metals with αSyn has been shown to trigger the aggregation of this protein. Furthermore, metals can exert cellular toxicity due to their red-ox potential, which leads to the formation of reactive oxygen species, exacerbating the noxious effects of αSyn. Here we give a brief overview on αSyn pathology and the role of metals in the brain and then address in more detail the interaction of αSyn with three disease-relevant transition metals, iron (Fe), copper (Cu) and manganese (Mn). We also discuss possible therapeutic approaches for PD, which are based on these interactions, e.g. chelation therapy and anti-oxidative treatments. Not all mechanisms of alpha-synuclein-mediated toxicity and roles of metals are sufficiently understood. We discuss several aspects, which deserve further investigation in order to shed light on the etiopathology of the disease and enable the development of more specific, innovative drugs for the treatment of PD and other synucleinopathies.