Iron, Neuroinflammation and Neurodegeneration.

Disturbance of the brain homeostasis, either directly via the formation of abnormal proteins or cerebral hypo-perfusion, or indirectly via peripheral inflammation, will activate microglia to synthesise a variety of pro-inflammatory agents which may lead to inflammation and cell death. The pro-inflammatory cytokines will induce changes in the iron proteins responsible for maintaining iron homeostasis, such that increased amounts of iron will be deposited in cells in the brain. The generation of reactive oxygen and nitrogen species, which is directly involved in the inflammatory process, can significantly affect iron metabolism via their interaction with iron-regulatory proteins (IRPs). This underlies the importance of ensuring that iron is maintained in a form that can be kept under control; hence, the elegant mechanisms which have become increasingly well understood for regulating iron homeostasis. Therapeutic approaches to minimise the toxicity of iron include N-acetyl cysteine, non-steroidal anti-inflammatory compounds and iron chelation.

Iron and inflammation: in vivo and post-mortem studies in Parkinson's disease.

In these present studies, in vivo and and post-mortem studies have investigated the association between iron and inflammation. Early-stage Parkinson's disease (PD) patients, of less than 5 years disease duration, showed associations of plasmatic ferritin concentrations with both proinflammatory cytokine interleukin-6 and hepcidin, a regulator of iron metabolism as well as clinical measures. In addition ratios of plasmatic ferritin and iron accumulation in deep grey matter nuclei assessed with relaxometry T2* inversely correlated with disease severity and duration of PD. On the hand, post-mortem material of the substantia nigra compacta (SNc) divided according to Braak and Braak scores, III-IV and V-VI staging, exhibited comparable microgliosis, with a variety of phenotypes present. There was an association between the intensity of microgliosis and iron accumulation as assayed by Perl's staining in the SNc sections. In conclusion, markers of inflammation and iron metabolism in both systemic and brain systems are closely linked in PD, thus offering a potential biomarker for progression of the disease.

Is Chelation Therapy a Potential Treatment for Parkinson's Disease?

Iron loading in some brain regions occurs in Parkinson's Disease (PD), and it has been considered that its removal by iron chelators could be an appropriate therapeutic approach. Since neuroinflammation with microgliosis is also a common feature of PD, it is possible that iron is sequestered within cells as a result of the "anaemia of chronic disease" and remains unavailable to the chelator. In this review, the extent of neuroinflammation in PD is discussed together with the role played by glia cells, specifically microglia and astrocytes, in controlling iron metabolism during inflammation, together with the results of MRI studies. The current use of chelators in clinical medicine is presented together with a discussion of two clinical trials of PD patients where an iron chelator was administered and showed encouraging results. It is proposed that the use of anti-inflammatory drugs combined with an iron chelator might be a better approach to increase chelator efficacy.

Novel 1-hydroxypyridin-2-one metal chelators prevent and rescue ubiquitin proteasomal-related neuronal injury in an in vitro model of Parkinson's disease.

Ubiquitin proteasome system (UPS) impairment, excessive cellular oxidative stress, and iron dyshomeostasis are key to substantia nigra dopaminergic neuronal degeneration in Parkinson's disease (PD); however, a link between these features remains unconfirmed. Using the proteasome inhibitor lactacystin we confirm that nigral injury via UPS impairment disrupts iron homeostasis, in turn increasing oxidative stress and promoting protein aggregation. We demonstrate the neuroprotective potential of two novel 1-hydroxy-2(1H)-pyridinone (1,2-HOPO) iron chelators, compounds C6 and C9, against lactacystin-induced cell death. We demonstrate that this cellular preservation relates to the compounds' iron chelating capabilities and subsequent reduced capacity of iron to form reactive oxygen species (ROS), where we also show that the ligands act as antioxidant agents. Our results also demonstrate the ability of C6 and C9 to reduce intracellular lactacystin-induced α-synuclein burden. Stability constant measurements confirmed a high affinity of C6 and C9 for Fe3+ and display a 3:1 HOPO:Fe3+ complex formation at physiological pH. Reducing iron reactivity could prevent the demise of nigral dopaminergic neurons. We provide evidence that the lactacystin model presents with several neuropathological hallmarks of PD related to iron dyshomeostasis and that the novel chelating compounds C6 and C9 can protect against lactacystin-related neurotoxicity.

The histone deacetylase inhibitor nicotinamide exacerbates neurodegeneration in the lactacystin rat model of Parkinson's disease.

Histone hypoacetylation is associated with dopaminergic neurodegeneration in Parkinson's disease (PD), because of an imbalance in the activities of the enzymes responsible for histone (de)acetylation. Correction of this imbalance, with histone deacetylase (HDAC) inhibiting agents, could be neuroprotective. We therefore hypothesize that nicotinamide, being a selective inhibitor of HDAC class III as well as having modulatory effects on mitochondrial energy metabolism, would be neuroprotective in the lactacystin rat model of PD, which recapitulates the formation of neurotoxic accumulation of altered proteins within the substantia nigra to cause progressive dopaminergic cell death. Rats received nicotinamide for 28 days, starting 7 days after unilateral injection of the irreversible proteasome inhibitor, lactacystin, into the substantia nigra. Longitudinal motor behavioural testing and structural magnetic resonance imaging were used to track changes in this model of PD, and assessment of nigrostriatal integrity, histone acetylation and brain gene expression changes post-mortem used to quantify nicotinamide-induced neuroprotection. Counterintuitively, nicotinamide dose-dependently exacerbated neurodegeneration of dopaminergic neurons, behavioural deficits and structural brain changes in the lactacystin-lesioned rat. Nicotinamide treatment induced histone hyperacetylation and over-expression of numerous neurotrophic and anti-apoptotic factors in the brain, yet failed to result in neuroprotection, rather exacerbated dopaminergic pathology. These findings highlight the importance of inhibitor specificity within HDAC isoforms for therapeutic efficacy in PD, demonstrating the contrasting effects of HDAC class III inhibition upon cell survival in this animal model of the disease. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Label-Free Time-of-Flight Secondary Ion Mass Spectrometry Imaging of Sulfur-Producing Enzymes inside Microglia Cells following Exposure to Silver Nanowires.

There are no methods sensitive enough to detect enzymes within cells, without the use of analyte labeling. Here we show that it is possible to detect protein ion signals of three different H2S-synthesizing enzymes inside microglia after pretreatment with silver nanowires (AgNW) using time-of-flight secondary ion mass spectrometry (TOF-SIMS). Protein fragment ions, including the fragment of amino acid (C4H8N+ = 70 amu), fragments of the sulfur-producing cystathionine-containing enzymes, and the Ag+ ion signal could be detected without the use of any labels; the cells were mapped using the C4H8N+ amino acid fragment. Scanning electron microscopy imaging and energy-dispersive X-ray chemical analysis showed that the AgNWs were inside the same cells imaged by TOF-SIMS and transformed chemically into crystalline Ag2S within cells in which the sulfur-producing proteins were detected. The presence of these sulfur-producing cystathionine-containing enzymes within the cells was confirmed by Western blots and confocal microscopy images of fluorescently labeled antibodies against the sulfur-producing enzymes. Label-free TOF-SIMS is very promising for the label-free identification of H2S-contributing enzymes and their cellular localization in biological systems. The technique could in the future be used to identify which of these enzymes are most contributory.

L-DOPA functionalized, multi-branched gold nanoparticles as brain-targeted nano-vehicles.

The blood-brain barrier (BBB) is a protective endothelial barrier lining the brain microvasculature which prevents brain delivery of therapies against brain diseases. Hence, there is an urgent need to develop vehicles which efficiently penetrate the BBB to deliver therapies into the brain. The drug L-DOPA efficiently and specifically crosses the BBB via the large neutral amino acid transporter (LAT)-1 protein to enter the brain. Thus, we synthesized L-DOPA-functionalized multi-branched nanoflower-like gold nanoparticles (L-DOPA-AuNFs) using a seed-mediated method involving catechols as a direct reducing-cum-capping agent, and examined their ability to cross the BBB to act as brain-penetrating nanovehicles. We show that L-DOPA-AuNFs efficiently penetrate the BBB compared to similarly sized and shaped AuNFs functionalized with a non-targeting ligand. Furthermore, we show that L-DOPA-AuNFs are efficiently internalized by brain macrophages without inducing inflammation. These results demonstrate the application of L-DOPA-AuNFs as a non-inflammatory BBB-penetrating nanovehicle to efficiently deliver therapies into the brain.

Changes in Pro-Inflammatory Markers in Detoxifying Chronic Alcohol Abusers, Divided by Lesch Typology, Reflect Cognitive Dysfunction.

AIM: To investigate pro-inflammatory markers in the blood and associate with cognitive impairment. METHODS: Il-6 and ferritin were assayed in the blood of 27 patients, divided according to Lesch typology, at the commencement and after 21 days of detoxification, together with a battery of cognitive tests. RESULTS: A significantly higher mean level of IL-6 was present in the blood of patients with Lesch typology 1 compared to the other typologies 2 and 3 on admission to the Detoxification Ward which did not alter significantly after detoxification. The mean level of IL-6 was initially elevated in Lesch typology 2 alcohol abusers and declined to the reference range after detoxification. Lesch typology 3 alcohol abusers showed normal levels of IL-6 at both time points. Only in Lesch typology 1 were the levels of ferritin and IL-10 significantly elevated at the start of the detoxification process. Cognitive impairment, as ascertained by Stroop test and Brown-Peterson procedure was greater in Lesch typology 1 than the other 2 patient groups. CONCLUSION: Such data might indicate a greater degree of neuroinflammation in Lesch typology 1 alcoholic patients. SHORT SUMMARY: Dividing a heterogeneous group of alcoholic subjects into homogenous groups according to Lesch typology, identifies a greater pro-inflammatory profile in Lesch typology 1 patients who also showed greater cognitive impairment.

Transmission of multiple system atrophy prions to transgenic mice.

Prions are proteins that adopt alternative conformations, which become self-propagating. Increasing evidence argues that prions feature in the synucleinopathies that include Parkinson's disease, Lewy body dementia, and multiple system atrophy (MSA). Although TgM83(+/+) mice homozygous for a mutant A53T α-synuclein transgene begin developing CNS dysfunction spontaneously at ∼10 mo of age, uninoculated TgM83(+/-) mice (hemizygous for the transgene) remain healthy. To determine whether MSA brains contain α-synuclein prions, we inoculated the TgM83(+/-) mice with brain homogenates from two pathologically confirmed MSA cases. Inoculated TgM83(+/-) mice developed progressive signs of neurologic disease with an incubation period of ∼100 d, whereas the same mice inoculated with brain homogenates from spontaneously ill TgM83(+/+) mice developed neurologic dysfunction in ∼210 d. Brains of MSA-inoculated mice exhibited prominent astrocytic gliosis and microglial activation as well as widespread deposits of phosphorylated α-synuclein that were proteinase K sensitive, detergent insoluble, and formic acid extractable. Our results provide compelling evidence that α-synuclein aggregates formed in the brains of MSA patients are transmissible and, as such, are prions. The MSA prion represents a unique human pathogen that is lethal upon transmission to Tg mice and as such, is reminiscent of the prion causing kuru, which was transmitted to chimpanzees nearly 5 decades ago.

Deep-brain stimulation associates with improved microvascular integrity in the subthalamic nucleus in Parkinson's disease.

Deep brain stimulation (DBS) of the subthalamic nucleus (STN) has become an accepted treatment for motor symptoms in a subset of Parkinson's disease (PD) patients. The mechanisms why DBS is effective are incompletely understood, but previous studies show that DBS targeted in brain structures other than the STN may modify the microvasculature. However, this has not been studied in PD subjects who have received STN-DBS. Here we investigated the extent and nature of microvascular changes in post-mortem STN samples from STN-DBS PD patients, compared to aged controls and PD patients who had not been treated with STN-DBS. We used immunohistochemical and immunofluorescent methods to assess serial STN-containing brain sections from PD and STN-DBS PD cases, compared to similar age controls using specific antibodies to detect capillaries, an adherens junction and tight junction-associated proteins as well as activated microglia. Cellular features in stained sections were quantified by confocal fluorescence microscopy and stereological methods in conjunction with in vitro imaging tools. We found significant upregulation of microvessel endothelial cell thickness, length and density but lowered activated microglia density and striking upregulation of all analysed adherens junction and tight junction-associated proteins in STN-DBS PD patients compared to non-DBS PD patients and controls. Moreover, in STN-DBS PD samples, expression of an angiogenic factor, vascular endothelial growth factor (VEGF), was significantly upregulated compared to the other groups. Our findings suggest that overexpressed VEGF and downregulation of inflammatory processes may be critical mechanisms underlying the DBS-induced microvascular changes.

Glitazone Treatment and Incidence of Parkinson's Disease among People with Diabetes: A Retrospective Cohort Study.

BACKGROUND: Recent in vitro and animal experiments suggest that peroxisome proliferation-activated receptor gamma (PPARÉ£) agonist medications, such as antidiabetic glitazone (GTZ) drugs, are neuroprotective in models of Parkinson's disease (PD). These findings have not been tested in humans. We hypothesized that individuals prescribed GTZ drugs would have a lower incidence of PD compared to individuals prescribed other treatments for diabetes. METHODS AND FINDINGS: Using primary care data from the United Kingdom Clinical Practice Research Datalink (CPRD), we conducted a retrospective cohort study in which individuals with diabetes who were newly prescribed GTZ (GTZ-exposed group) were matched by age, sex, practice, and diabetes treatment stage with up to five individuals prescribed other diabetes treatments (other antidiabetic drug-exposed group). Patients were followed up from 1999 until the first recording of a PD diagnosis, end of observation in the database, or end of the study (1 August 2013). An incidence rate ratio (IRR) was calculated using conditional Poisson regression, adjusted for possible confounders. 44,597 GTZ exposed individuals were matched to 120,373 other antidiabetic users. 175 GTZ-exposed individuals were diagnosed with PD compared to 517 individuals in the other antidiabetic drug-exposed group. The incidence rate (IR) of PD in the GTZ-exposed group was 6.4 per 10,000 patient years compared with 8.8 per 10,000 patient years in those prescribed other antidiabetic treatments (IRR 0.72, 95% confidence interval [CI] 0.60-0.87). Adjustments for potential confounding variables, including smoking, other medications, head injury, and disease severity, had no material impact (fully adjusted IRR 0.75, 0.59-0.94). The risk was reduced in those with current GTZ prescriptions (current GTZ-exposed IRR 0.59, 0.46-0.77) but not reduced among those with past prescriptions (past GTZ-exposed IRR 0.85, 0.65-1.10). Our study only included patients with diabetes who did not have a PD diagnosis when they were first prescribed GTZ, and thus, it cannot establish whether GTZ use prevents or slows the progression of PD. CONCLUSIONS: In patients with diabetes, a current prescription for GTZ is associated with a reduction in incidence of PD. This suggests PPAR gamma pathways may be a fruitful drug target in PD.

Common mechanisms in neurodegeneration and neuroinflammation: a BrainNet Europe gene expression microarray study.

Neurodegenerative diseases of the central nervous system are characterized by pathogenetic cellular and molecular changes in specific areas of the brain that lead to the dysfunction and/or loss of explicit neuronal populations. Despite exhibiting different clinical profiles and selective neuronal loss, common features such as abnormal protein deposition, dysfunctional cellular transport, mitochondrial deficits, glutamate excitotoxicity, iron accumulation and inflammation are observed in many neurodegenerative disorders, suggesting converging pathways of neurodegeneration. We have generated comparative genome-wide gene expression data, using the Illumina HumanRef 8 Beadchip, for Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, multiple sclerosis, Parkinson's disease, and schizophrenia using an extensive cohort (n = 113) of well-characterized post-mortem brain tissues. The analysis of whole-genome expression patterns across these major disorders offers an outstanding opportunity not only to look into exclusive disease-specific changes, but more importantly to look for potential common molecular pathogenic mechanisms. Surprisingly, no dysregulated gene that passed our selection criteria was found in common across all six diseases. However, 61 dysregulated genes were shared when comparing five and four diseases. The few genes highlighted by our direct gene comparison analysis hint toward common neuronal homeostatic, survival and synaptic plasticity pathways. In addition, we report changes to several inflammation-related genes in all diseases. This work is supportive of a general role of the innate immune system in the pathogenesis and/or response to neurodegeneration.

BrainNet Europe's Code of Conduct for brain banking.

Research utilizing human tissue and its removal at post-mortem has given rise to many controversies in the media and posed many dilemmas in the fields of law and ethics. The law often lacks clear instructions and unambiguous guidelines. The absence of a harmonized international legislation with regard to post-mortem medical procedures and donation of tissue and organs contributes to the complexity of the issue. Therefore, within the BrainNet Europe (BNE) consortium, a consortium of 19 European brain banks, we drafted an ethical Code of Conduct for brain banking that covers basic legal rules and bioethical principles involved in brain banking. Sources include laws, regulations and guidelines (Declarations, Conventions, Recommendations, Guidelines and Directives) issued by international key organizations, such as the Council of Europe, European Commission, World Medical Association and World Health Organization. The Code of Conduct addresses fundamental topics as the rights of the persons donating their tissue, the obligations of the brain bank with regard to respect and observance of such rights, informed consent, confidentiality, protection of personal data, collections of human biological material and their management, and transparency and accountability within the organization of a brain bank. The Code of Conduct for brain banking is being adopted by the BNE network prior to being enshrined in official legislation for brain banking in Europe and beyond.

Neuroprotective and symptomatic effects of targeting group III mGlu receptors in neurodegenerative disease.

Neurodegenerative disorders possess common pathological mechanisms, such as protein aggregation, inflammation, oxidative stress (OS) and excitotoxicity, raising the possibility of shared therapeutic targets. As a result of the selective cellular and regional expression of group III metabotropic glutamate (mGlu) receptors, drugs targeting such receptors have demonstrated both neuroprotective properties and symptomatic improvements in several models of neurodegeneration. In recent years, the discovery and development of subtype-selective ligands for the group III mGlu receptors has gained pace, allowing further research into the functions of these receptors and revealing their roles in health and disease. Activation of this class of receptors results in neuroprotection, with a variety of underlying mechanisms implicated. Group III mGlu receptor stimulation prevents excitotoxicity by inhibiting glutamate release from neurons and microglia and increasing glutamate uptake by astrocytes. It also attenuates the neuroinflammatory response by reducing glial reactivity and encourages neurotrophic phenotypes. This article will review the current literature with regard to the neuroprotective and symptomatic effects of group III mGlu receptor activation and discuss their promise as therapeutic targets in neurodegenerative disease. We review the neuroprotective and symptomatic effects of targeting group III mGlu receptors in neurodegenerative disease: Excess extracellular glutamate causes overactivation of NMDA receptors resulting in excitotoxicity. Externalization of phosphatidylserine stimulates phagocytosis of neurons by activated microglia, which contribute to damage through glutamate and pro-inflammatory factor release. Reactive astrocytes produce cytotoxic factors enhancing neuronal cell death. Activation of group III mGlu receptors by glutamate and/or mGlu receptor ligands results in inhibition of glutamate release from presynaptic terminals and microglia, reducing excitotoxicity. Astrocytic glutamate uptake is increased and microglia produce neurotrophic factors.

Parkinson's disease is associated with altered expression of CaV1 channels and calcium-binding proteins.

In Parkinson's disease oxidative stress and calcium-induced excitotoxicity have been considered important mechanisms leading to cell death for decades, but the factors that make some neurons vulnerable to neurodegeneration while others remain resistant are not fully understood. Studies of the disorder in animal models suggest that the voltage-gated calcium channel subtype Ca(V)1.3 has a role in making neurons susceptible to neurodegeneration and support earlier work in post-mortem human brain that suggested loss of calcium buffering capacity in neurons correlated with areas of neuronal loss in the substantia nigra of parkinsonian brain. This study examined expression of Ca(V)1 subtypes and the calcium-binding proteins calbindin, calmodulin and calreticulin in areas vulnerable and resistant to neurodegeneration in Parkinson's disease, in brain from neurologically normal individuals and patients with Parkinson's disease. In control brain the expression of a specific Ca(V)1 subtype or distribution of each calcium-binding protein did not associate with those regions prone to neurodegeneration in Parkinson's disease. Whereas, alterations in the amount of both Ca(V)1 subtypes and the calcium-binding proteins were found throughout the brain in Parkinson's disease. Some changes reflected the cell loss seen in Parkinson's disease, whereas others represented altered levels of cellular expression, which as they occurred in the absence of cell loss could not be explained as solely compensatory to the neurodegeneration. The finding of increased Ca(V)1.3 subtype expression in the cerebral cortex of early stage Parkinson's disease, before the appearance of pathological changes, supports the view that disturbed calcium homeostasis is an early feature of Parkinson's disease and not just a compensatory consequence to the neurodegenerative process. This interpretation is supported further by the finding that the ratio of Ca(V)1 subtypes differed throughout the brain in patients with Parkinson's disease compared with control subjects, in favour of an increased use of Ca(V)1.3, which would add to the metabolic burden for cells that rely on this Ca(V)1 subtype for electrical activity and could therefore render specific neuronal populations more vulnerable to neurodegeneration.

Twelve Years of Drug Prioritization to Help Accelerate Disease Modification Trials in Parkinson's Disease: The International Linked Clinical Trials Initiative.

In 2011, the UK medical research charity Cure Parkinson's set up the international Linked Clinical Trials (iLCT) committee to help expedite the clinical testing of potentially disease modifying therapies for Parkinson's disease (PD). The first committee meeting was held at the Van Andel Institute in Grand Rapids, Michigan in 2012. This group of PD experts has subsequently met annually to assess and prioritize agents that may slow the progression of this neurodegenerative condition, using a systematic approach based on preclinical, epidemiological and, where possible, clinical data. Over the last 12 years, 171 unique agents have been evaluated by the iLCT committee, and there have been 21 completed clinical studies and 20 ongoing trials associated with the initiative. In this review, we briefly outline the iLCT process as well as the clinical development and outcomes of some of the top prioritized agents. We also discuss a few of the lessons that have been learnt, and we conclude with a perspective on what the next decade may bring, including the introduction of multi-arm, multi-stage clinical trial platforms and the possibility of combination therapies for PD.

Neuroprotective role for RORA in Parkinson's disease revealed by analysis of post-mortem brain and a dopaminergic cell line.

Parkinson's disease (PD) is almost twice as prevalent in men, which has largely been attributed to neuroprotective effect of oestradiol in women. RORA (retinoic acid receptor-related orphan receptor alpha) regulates the transcription of central aromatase, the enzyme responsible for local oestradiol synthesis, simultaneously, RORA expression is regulated by sex hormones. Moreover, RORA protects neurones against oxidative stress, a key mechanism contributing to the loss of dopaminergic neurones in PD. Therefore, we hypothesized that there would be sex differences in RORA expression in the substantia nigra pars compacta (SNpc), which could contribute to sex differences observed in PD prevalence and pathogenesis. In a case control study, qPCR and western blot analyses were used to quantify gene and protein expression in the SNpc of post-mortem brains (n = 14 late-stage PD and 11 age and sex matched controls). The neuroprotective properties of a RORA agonist were then investigated directly using a cell culture toxin-based model of PD coupled with measures of viability, mitochondrial function and apoptosis. RORA was expressed at significantly higher levels in the SNpc from control females' brains compared to males. In PD, we found a significant increase in SNpc RORA expression in male PD compared to female PD. Treatment with a RORA agonist showed a significant neuroprotection in our cell culture model of PD and revealed significant effects on intracellular factors involved in neuronal survival and demise. This study is the first to demonstrate a sex specific pattern of RORA protein and gene expression in the SNpc of controls post-mortem human brains, and to show that this is differentially altered in male and female PD subjects, thus supporting a role for RORA in sex-specific aspects of PD. Furthermore, our in vitro PD model indicates mechanisms whereby a RORA agonist exerts its neuroprotective effect, thereby highlighting the translational potential for RORA ligands in PD.

Selection of novel reference genes for use in the human central nervous system: a BrainNet Europe Study.

The use of an appropriate reference gene to ensure accurate normalisation is crucial for the correct quantification of gene expression using qPCR assays and RNA arrays. The main criterion for a gene to qualify as a reference gene is a stable expression across various cell types and experimental settings. Several reference genes are commonly in use but more and more evidence reveals variations in their expression due to the presence of on-going neuropathological disease processes, raising doubts concerning their use. We conducted an analysis of genome-wide changes of gene expression in the human central nervous system (CNS) covering several neurological disorders and regions, including the spinal cord, and were able to identify a number of novel stable reference genes. We tested the stability of expression of eight novel (ATP5E, AARS, GAPVD1, CSNK2B, XPNPEP1, OSBP, NAT5 and DCTN2) and four more commonly used (BECN1, GAPDH, QARS and TUBB) reference genes in a smaller cohort using RT-qPCR. The most stable genes out of the 12 reference genes were tested as normaliser to validate increased levels of a target gene in CNS disease. We found that in human post-mortem tissue the novel reference genes, XPNPEP1 and AARS, were efficient in replicating microarray target gene expression levels and that XPNPEP1 was more efficient as a normaliser than BECN1, which has been shown to change in expression as a consequence of neuronal cell loss. We provide herein one more suitable novel reference gene, XPNPEP1, with no current neuroinflammatory or neurodegenerative associations that can be used for gene quantitative gene expression studies with human CNS post-mortem tissue and also suggest a list of potential other candidates. These data also emphasise the importance of organ/tissue-specific stably expressed genes as reference genes for RNA studies.

Brain iron metabolism and its perturbation in neurological diseases.

Metal ions are of particular importance in brain function, notably iron. A broad overview of iron metabolism and its homeostasis both at the cellular level (involving regulation at the level of mRNA translation) and the systemic level (involving the peptide 'hormone' hepcidin) is presented. The mechanisms of iron transport both across the blood-brain barrier and within the brain are then examined. The importance of iron in the developing foetus and in early life is underlined. We then review the growing corpus of evidence that many neurodegenerative diseases (NDs) are the consequence of dysregulation of brain iron homeostasis. This results in the production of reactive oxygen species, generating reactive aldehydes, which, together with further oxidative insults, causes oxidative modification of proteins, manifested by carbonyl formation. These misfolded and damaged proteins overwhelm the ubiquitin/proteasome system, accumulating the characteristic inclusion bodies found in many NDs. The involvement of iron in Alzheimer's disease and Parkinson's disease is then examined, with emphasis on recent data linking in particular interactions between iron homeostasis and key disease proteins. We conclude that there is overwhelming evidence for a direct involvement of iron in NDs.

Iron and the immune system.

Iron and immunity are closely linked: firstly by the fact that many of the genes/proteins involved in iron homoeostasis play a vital role in controlling iron fluxes such that bacteria are prevented from utilising iron for growth; secondly, cells of the innate immune system, monocytes, macrophages, microglia and lymphocytes, are able to combat bacterial insults by carefully controlling their iron fluxes, which are mediated by hepcidin and ferroportin. In addition, lymphocytes play an important role in adaptive immunity. Thirdly, a variety of effector molecules, e.g. toll-like receptors, NF-κB, hypoxia factor-1, haem oxygenase, will orchestrate the inflammatory response by mobilising a variety of cytokines, neurotrophic factors, chemokines, and reactive oxygen and nitrogen species. Pathologies, where iron loading and depletion occur, may adversely affect the ability of the cell to respond to the bacterial insult.