Metal Ion Regulation in the Central Nervous System and in Glutamatergic Synapses: Role of the Cellular Prion Protein

Despite many efforts, the molecular mechanisms underlying the pathophysiology of neurodegenerative disorders have not been fully understood. Results published in literature highlight that different neurodegenerative diseases share common features: protein aggregation in neuronal tissue; oxidation of neuronal tissue mediated by redox-active metal ions interaction with a target protein; and functional demise. So, unveiling the physiological function of protein that aggregate in neurodegenerating tissues, as well as their interplay with metal ions, becomes a prominent issue, in order to understand the etiological trigger and to define a possible therapeutic strategy. Metal ions are essential elements for cellular processes, but at the same they are potentially dangerous since they can give rise to Fenton reaction and oxidative/nitrosative stress. So, their homeostasis is strictly regulated in each district of the organism, but in particular in the brain. The brain, having the highest metabolic rate and depending predominantly on oxidative metabolism for its energy, has developed fine mechanisms to compartmentalize, distribute, uptake and excrete the different ionic species. Alterations in one of these mechanisms can lead to great neuronal damages, and maybe neurodegenerative disorders. This work has been focused on the cellular prion protein (PrPC), whose conformational isoform, the scrapie prion protein (PrPSc) is the causative agent of prion disordes and whose function has not been clearly defined, yet. Metal ions are a common denominator to all the cellular pathways in which PrPC seems to be actively involved. In particular, metal ions homeostasis maintainance, neuroprotection in excitotoxic condition and ionotropic receptor modulation have been studied. In the first part of the project, PrPC role in metal ion homeostais maintainance has been investigated. To this aim, copper, manganese, zinc and iron content, as well as metal binding proteins expression have been measured in a PrP knockout murine model, compared to wild-type. The results describe the global rearrangement occurring in the expression of metal binding proteins to maintain trace metals homeostasis, trying to compensate PrPC absence. At the same time, a pronounced decrease in Ceruloplasmin ferroxidase activity has been detected in PrP null mouse serum, pointing out a global impairment in copper metabolism in PrPC absence. In the second part of the project, the importance of the interaction between PrPC and copper ions in excitotoxic conditions and in synapses functionality has been studied. It has been published that PrP null mice show higher levels of neuronal cell death in stressful conditions and when subjected to toxic treatment with glutamate receptor agonists. Moreover, these mice show altered kinetics of N-methyl-D-aspartate (NMDA) receptor current. These alterations appears to be due to an inhibitory regulation that PrPC exerts on NMDA receptors via copper ions, lacking in PrP null hippocampi. First, the enhanced suceptibility to excitotoxicity of PrP knockout mice has been verified and characterised in organotypic hippocampal cultures upon treatment with NMDA. Higher neuronal cell death levels have been detected in all the investigated hippocampal regions. To identify which cellular regulatory mechanism is alterd in PrPC absence, the expression of the proteins mainly involved in excitotoxicity has been compared between PrP knockout and wild-type hippocampi. Among other minor differences, a different modulation of calcium transporters expression has been identified in PrP knockout hippocampi and brains. This global alteration appears to be necessary to maintain calcium homeostasis, since calcium content measurements did not reveal any strong difference between PrP null and wild-type samples. NMDA receptors can be S-nitrosylated on extracellular cysteines and this reaction is always inhibitory. S-nytrosilation requires an electron acceptor to occur, for this reason copper ions are often involved in these kind of reactions. Moreover, copper ions are known to modulate NMDA receptor activity, but the precise mechanism has not been described, yet. Since PrPC is known to support the S-nitrosylation of other membrane proteins, the S-nitrosylation levels of NMDA receptor subunits GluN1 and GluN2A have been measured in PrP knockout hippocampi from adult mice and compared to wild-type ones. Results show that the S-nitrosylated fractions of both GluN1 and GluN2A are reduced in PrP absence. So, this reveals that PrPC modulates NMDA receptor activity providing the copper ions necessary to support their inhibitory S-nitrosylation reaction. Through this mechanism, PrPC contributes to inhibit NMDA receptor currents, as well as to protect neurons in excitotoxic conditions

Prion protein and aging

The cellular prion protein (PrP(C)) has been widely investigated ever since its conformational isoform, the prion (or PrP(Sc)), was identified as the etiological agent of prion disorders. The high homology shared by the PrP(C)-encoding gene among mammals, its high turnover rate and expression in every tissue strongly suggest that PrP(C) may possess key physiological functions. Therefore, defining PrP(C) roles, properties and fate in the physiology of mammalian cells would be fundamental to understand its pathological involvement in prion diseases. Since the incidence of these neurodegenerative disorders is enhanced in aging, understanding PrP(C) functions in this life phase may be of crucial importance. Indeed, a large body of evidence suggests that PrP(C) plays a neuroprotective and antioxidant role. Moreover, it has been suggested that PrP(C) is involved in Alzheimer disease, another neurodegenerative pathology that develops predominantly in the aging population. In prion diseases, PrP(C) function is likely lost upon protein aggregation occurring in the course of the disease. Additionally, the aging process may alter PrP(C) biochemical properties, thus influencing its propensity to convert into PrP(Sc). Both phenomena may contribute to the disease development and progression. In Alzheimer disease, PrP(C) has a controversial role because its presence seems to mediate β-amyloid toxicity, while its down-regulation correlates with neuronal death. The role of PrP(C) in aging has been investigated from different perspectives, often leading to contrasting results. The putative protein functions in aging have been studied in relation to memory, behavior and myelin maintenance. In aging mice, PrP(C) changes in subcellular localization and post-translational modifications have been explored in an attempt to relate them to different protein roles and propensity to convert into PrP(Sc). Here we provide an overview of the most relevant studies attempting to delineate PrP(C) functions and fate in aging

Prion protein and copper cooperatively protect neurons by modulating NMDA receptor through S-nitrosylation

AIMS: Several neurodegenerative disorders show alterations in glutamatergic synapses and increased susceptibility to excitotoxicity. Mounting evidence suggests a central role for the cellular prion protein (PrP(C)) in neuroprotection. Therefore, the loss of PrP(C) function occurring in prion disorders may contribute to the disease progression and neurodegeneration. Indeed, PrP(C) modulates N-methyl-d-aspartate receptors (NMDAR), thus preventing cell death. In this study, we show that PrP(C) and copper cooperatively inhibit NMDAR through S-nitrosylation, a post-translational modification resulting from the chemical reaction of nitric oxide (NO) with cysteines. RESULTS: Comparing wild-type Prnp (Prnp(+/+)) and PrP(C) knockout (Prnp(0/0)) mouse hippocampi, we found that GluN1 and GluN2A S-nitrosylation decrease in Prnp(0/0). Using organotypic hippocampal cultures, we found that copper chelation decreases NMDAR S-nitrosylation in Prnp(+/+) but not in Prnp(0/0). This suggests that PrP(C) requires copper to support the chemical reaction between NO and thiols. We explored PrP(C)-Cu neuroprotective role by evaluating neuron susceptibility to excitotoxicity in Prnp(+/+) and Prnp(0/0) cultures. We found that (i) PrP(C)-Cu modulates GluN2A-containing NMDAR, those inhibited by S-nitrosylation; (ii) PrP(C) and copper are interdependent to protect neurons from insults; (iii) neuronal NO synthase inhibition affects susceptibility in wild-type but not in Prnp(0/0), while (iv) the addition of a NO donor enhances Prnp(0/0) neurons survival. INNOVATION AND CONCLUSIONS: Our results show that PrP(C) and copper support NMDAR S-nitrosylation and cooperatively exert neuroprotection. In addition to NMDAR, PrP(C) may also favor the S-nitrosylation of other proteins. Therefore, this mechanism may be investigated in the context of the different cellular processes in which PrP(C) is involved

TRPM8 and Nav1.8 sodium channels are required for transthyretin-induced calcium influx in growth cones of small-diameter TrkA-positive sensory neurons

<p>Abstract</p> <p>Background</p> <p>Familial amyloidotic polyneuropathy (FAP) is a peripheral neuropathy caused by the extracellular accumulation and deposition of insoluble transthyretin (TTR) aggregates. However the molecular mechanism that underlies TTR toxicity in peripheral nerves is unclear. Previous studies have suggested that amyloidogenic proteins can aggregate into oligomers which disrupt intracellular calcium homeostasis by increasing the permeability of the plasma membrane to extracellular calcium. The aim of the present study was to examine the effect of TTR on calcium influx in dorsal root ganglion neurons.</p> <p>Results</p> <p>Levels of intracellular cytosolic calcium were monitored in dorsal root ganglion (DRG) neurons isolated from embryonic rats using the calcium-sensitive fluorescent indicator Fluo4. An amyloidogenic mutant form of TTR, L55P, induced calcium influx into the growth cones of DRG neurons, whereas wild-type TTR had no significant effect. Atomic force microscopy and dynamic light scattering studies confirmed that the L55P TTR contained oligomeric species of TTR. The effect of L55P TTR was decreased by blockers of voltage-gated calcium channels (VGCC), as well as by blockers of Na_v1.8 voltage-gated sodium channels and transient receptor potential M8 (TRPM8) channels. siRNA knockdown of TRPM8 channels using three different TRPM8 siRNAs strongly inhibited calcium influx in DRG growth cones.</p> <p>Conclusions</p> <p>These data suggest that activation of TRPM8 channels triggers the activation of Na_v1.8 channels which leads to calcium influx through VGCC. We suggest that TTR-induced calcium influx into DRG neurons may contribute to the pathophysiology of FAP. Furthermore, we speculate that similar mechanisms may mediate the toxic effects of other amyloidogenic proteins such as the β-amyloid protein of Alzheimer's disease.</p

A new method for targeted and sustained induction of type 2 diabetes in rodents

Type 2 diabetes is a chronic metabolic disorder that is becoming a leading cause of morbidity and mortality. The prolonged time-course of human type 2 diabetes makes modelling of the disease difficult and additional animal models and methodologies are needed. The goal of this study was to develop and characterise a new method that allows controlled, targeted and sustained induction of discrete stages of type 2 diabetes in rodents. Using adult, male rats, we employed a three-week high fat-diet regimen and confirmed development of obesity-associated glucose intolerance, a key feature of human type 2 diabetes. Next, we utilised osmotic mini-pumps to infuse streptozotocin (STZ; doses ranging 80-200&thinsp;mg/kg) over the course of 14-days to decrease insulin-producing capacity thus promoting hyperglycemia. Using this new approach, we demonstrate a dose-dependent effect of STZ on circulating glucose and insulin levels as well as glucose tolerance, while retaining a state of obesity. Importantly, we found that insulin secretion in response to a glucose load was present, but reduced in a dose-dependent manner by increasing STZ. In conclusion, we demonstrate a novel method that enables induction of discrete stages of type 2 diabetes in rodents that closely mirrors the different stages of type 2 diabetes in humans

HuD is a neural translation enhancer acting on mTORC1-responsive genes and counteracted by the Y3 small non-coding RNA

The RNA-binding protein HuD promotes neurogenesis and favors recovery from peripheral axon injury. HuD interacts with many mRNAs, altering both stability and translation efficiency. We generated a nucleotide resolution map of the HuD RNA interactome in motor neuron-like cells, identifying HuD target sites in 1,304 mRNAs, almost exclusively in the 3' UTR. HuD binds many mRNAs encoding mTORC1-responsive ribosomal proteins and translation factors. Altered HuD expression correlates with the translation efficiency of these mRNAs and overall protein synthesis, in a mTORC1-independent fashion. The predominant HuD target is the abundant, small non-coding RNA Y3, amounting to 70% of the HuD interaction signal. Y3 functions as a molecular sponge for HuD, dynamically limiting its recruitment to polysomes and its activity as a translation and neuron differentiation enhancer. These findings uncover an alternative route to the mTORC1 pathway for translational control in motor neurons that is tunable by a small non-coding RNA

In Absence of the Cellular Prion Protein, Alterations in Copper Metabolism and Copper-Dependent Oxidase Activity Affect Iron Distribution

Essential elements as copper and iron modulate a wide range of physiological functions. Their metabolism is strictly regulated by cellular pathways, since dysregulation of metal homeostasis is responsible for many detrimental effects. Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and prion diseases are characterized by alterations of metal ions. These neurodegenerative maladies involve proteins that bind metals and mediate their metabolism through not well-defined mechanisms. Prion protein, for instance, interacts with divalent cations via multiple metal-binding sites and it modulates several metal-dependent physiological functions, such as S-nitrosylation of NMDA receptors. In this work we focused on the effect of prion protein absence on copper and iron metabolism during development and adulthood. In particular, we investigated copper and iron functional values in serum and several organs such as liver, spleen, total brain and isolated hippocampus. Our results show that iron content is diminished in prion protein-null mouse serum, while it accumulates in liver and spleen. Our data suggest that these alterations can be due to impairments in copper-dependent cerulopalsmin activity which is known to affect iron mobilization. In prion protein-null mouse total brain and hippocampus, metal ion content shows a fluctuating trend, suggesting the presence of homeostatic compensatory mechanisms. However, copper and iron functional values are likely altered also in these two organs, as indicated by the modulation of metal-binding protein expression levels. Altogether, these results reveal that the absence of the cellular prion protein impairs copper metabolism and copper-dependent oxidase activity, with ensuing alteration of iron mobilization from cellular storage compartments

Proteomics of rat hypothalamus, hippocampus and pre-frontal/frontal cortex after central administration of the neuropeptide PACAP

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that exerts pleiotropic functions, acting as a hypophysiotropic factor, a neurotrophic and a neuroprotective agent. The molecular pathways activated by PACAP to exert its physiological roles in brain are incompletely understood. In this study, adrenocorticotropic hormone (ACTH), prolactin, luteinising hormone (LH), follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), brain-derived neurotrophic factor and corticosterone blood levels were determined before and 20, 40, 60, and 120 min after PACAP intracerebroventricular administration. PACAP treatment increased ACTH, corticosterone, LH and FSH blood concentrations, while it decreased TSH levels. A proteomics investigation was carried out in hypothalamus, hippocampus and pre-frontal/frontal cortex (P/FC) using 2-dimensional gel electrophoresis at 120 min, the end-point suggested by studies on PACAP hypophysiotropic activities. Spots showing statistically significant alterations after PACAP treatment were identified by Matrix-assisted laser desorption/ionization-Time of flight mass spectrometry. Identified proteins were consistent with PACAP involvement in different molecular processes in brain. Altered expression levels were observed for proteins involved in cytoskeleton modulation and synaptic plasticity: actin in the hypothalamus; stathmin, dynamin, profilin and cofilin in hippocampus; synapsin in P/FC. Proteins involved in cellular differentiation were also modulated: glutathione-S-transferase \u3b1 and peroxiredoxin in hippocampus; nucleoside diphosphate kinase in P/FC. Alterations were detected in proteins involved in neuroprotection, neurodegeneration and apoptosis: ubiquitin carboxyl-terminal hydrolase isozyme L1 and heat shock protein 90-\u3b2 in hypothalamus; \u3b1-synuclein in hippocampus; glyceraldehyde-3-phosphate dehydrogenase and prohibitin in P/FC. This proteomics study identified new proteins involved in molecular mechanisms mediating PACAP functions in the central nervous system

Homer regulates calcium signalling in growth cone turning

BACKGROUND: Homer proteins are post-synaptic density proteins with known functions in receptor trafficking and calcium homeostasis. While they are key mediators of synaptic plasticity, they are also known to function in axon guidance, albeit by mechanisms that are yet to be elucidated. Homer proteins couple extracellular receptors – such as metabotropic glutamate receptors and the transient receptor potential canonical family of cation channels – to intracellular receptors such as inositol triphosphate and ryanodine receptors on intracellular calcium stores and, therefore, are well placed to regulate calcium dynamics within the neural growth cone. Here we used growth cones from dorsal root ganglia, a well established model in the field of axon guidance, and a growth cone turning assay to examine Homer1 function in axon guidance. RESULTS: Homer1 knockdown reversed growth cone turning from attraction to repulsion in response to the calcium-dependent guidance cues brain derived neurotrophic factor and netrin-1. Conversely, Homer1 knockdown had no effect on repulsion to the calcium-independent guidance cue Semaphorin-3A. This reversal of attractive turning suggested a requirement for Homer1 in a molecular switch. Pharmacological experiments confirmed that the operational state of a calcium-calmodulin dependent protein kinase II/calcineurin phosphatase molecular switch was dependent on Homer1 expression. Calcium imaging of motile growth cones revealed that Homer1 is required for guidance-cue-induced rise of cytosolic calcium and the attenuation of spontaneous cytosolic calcium transients. Homer1 knockdown-induced calcium transients and turning were inhibited by antagonists of store-operated channels. In addition, immunocytochemistry revealed the close association of Homer1 with the store-operated proteins TRPC1 and STIM1 within dorsal root ganglia growth cones. CONCLUSION: These experiments provide evidence that Homer1 is an essential component of the calcium signalling repertoire within motile growth cones, regulating guidance-cue-induced calcium release and maintaining basal cytosolic calcium

Prions Strongly Reduce NMDA Receptor S-Nitrosylation Levels at Pre-symptomatic and Terminal Stages of Prion Diseases

Prion diseases are fatal neurodegenerative disorders characterized by the cellular prion protein (PrPC) conversion into a misfolded and infectious isoform termed prion or PrPSc. The neuropathological mechanism underlying prion toxicity is still unclear, and the debate on prion protein gain- or loss-of-function is still open. PrPC participates to a plethora of physiological mechanisms. For instance, PrPC and copper cooperatively modulate N-methyl-D-aspartate receptor (NMDAR) activity by mediating S-nitrosylation, an inhibitory post-translational modification, hence protecting neurons from excitotoxicity. Here, NMDAR S-nitrosylation levels were biochemically investigated at pre- and post-symptomatic stages of mice intracerebrally inoculated with RML, 139A, and ME7 prion strains. Neuropathological aspects of prion disease were studied by histological analysis and proteinase K digestion. We report that hippocampal NMDAR S-nitrosylation is greatly reduced in all three prion strain infections in both pre-symptomatic and terminal stages of mouse disease. Indeed, we show that NMDAR S-nitrosylation dysregulation affecting prion-inoculated animals precedes the appearance of clinical signs of disease and visible neuropathological changes, such as PrPSc accumulation and deposition. The pre-symptomatic reduction of NMDAR S-nitrosylation in prion-infected mice may be a possible cause of neuronal death in prion pathology, and it might contribute to the pathology progression opening new therapeutic strategies against prion disorders