Rosella Abeti

β-amyloid activates PARP causing astrocytic metabolic failure and neuronal death

Alzheimers disease is characterized by β-amyloid accumulation in the central nervous system. As β-amyloid is neurotoxic in culture, we have explored the mechanisms of toxicity in the search for therapeutic targets for Alzheimers disease and now identify a key role for poly(ADP-ribose) polymerase in β-amyloid-induced neuronal death. Exposure of hippocampal neuronal/glial co-cultures to β-amyloid peptides activates the glial nicotinamide adenine dinucleotide phosphate oxidase, followed by predominantly neuronal cell death. β-amyloid exposure caused the progressive loss of mitochondrial membrane potential in astrocytes, accompanied by transient mitochondrial depolarizations caused by reversible openings of the mitochondrial permeability transition pore. The transients were absent in cultures from cyclophilin D knockout mice, leaving the slow depolarization available for study in isolation. β-amyloid exposure decreased both nicotinamide adenine dinucleotide fluorescence and oxygen consumption, while provision of mitochondrial substrates reversed the depolarization, suggesting that substrate supply was limiting. Poly(ADP-ribose) polymerase is activated by oxidative stress and consumes nicotinamide adenine dinucleotide, decreasing substrate availability. β-amyloid exposure caused accumulation of the poly(ADP-ribose) polymerase product, poly-ADP-ribose polymers, in astrocytes. Inhibition of either poly(ADP-ribose) polymerase or of the nicotinamide adenine dinucleotide phosphate oxidase prevented the appearance of poly-ADP-ribose polymers and the mitochondrial depolarization. Exposure of co-cultures to β-amyloid for >8 h decreased nicotinamide adenine dinucleotide and mitochondrial membrane potential and increased cell death in neurons, all of which were prevented by poly(ADP-ribose) polymerase inhibitors. Poly-ADP-ribose polymers increased with age in the brains of the TASTPM Alzheimer mouse model. We conclude that β-amyloid-induced neuronal death is mediated by poly(ADP-ribose) polymerase in response to oxidative stress generated by the astrocytic nicotinamide adenine dinucleotide phosphate oxidase.

CLIC1 function is required for beta-amyloid-induced generation of reactive oxygen species by microglia.

The Alzheimers disease (AD) brain is characterized by plaques containing β-amyloid (Aβ) protein surrounded by astrocytes and reactive microglia. Activation of microglia by Aβ initiates production of reactive oxygen species (ROS) by the plasmalemmal NADPH oxidase; the resultant oxidative stress is thought to contribute to neurodegeneration in AD. We have previously shown that Aβ upregulates a chloride current mediated by the chloride intracellular channel 1 (CLIC1) protein in microglia. We now demonstrate that Aβ promotes the acute translocation of CLIC1 from the cytosol to the plasma membrane of microglia, where it mediates a chloride conductance. Both the Aβ induced Cl− conductance and ROS generation were prevented by pharmacological inhibition of CLIC1, by replacement of chloride with impermeant anions, by an anti-CLIC1 antibody and by suppression of CLIC1 expression using siRNA. Thus, the CLIC1-mediated Cl− conductance is required for Aβ-induced generation of neurotoxic ROS by microglia. Remarkably, CLIC1 activation is itself dependent on oxidation by ROS derived from the activated NADPH oxidase. We therefore propose that CLIC1 translocation from the cytosol to the plasma membrane, in response to redox modulation by NADPH oxidase-derived ROS, provides a feedforward mechanism that facilitates sustained microglial ROS generation by the NAPDH oxidase.

Activation of PARP by Oxidative Stress Induced by β-Amyloid: Implications for Alzheimer’s Disease

Alzheimer’s disease (AD) is a major neurodegenerative disease of old age, characterised by progressive cognitive impairment, dementia and atrophy of the central nervous system. The pathological hallmarks include the accumulation of the peptide β-amyloid (Aβ) which itself is toxic to neurons in culture. Recently, it has been discovered that Aβ activates the protein poly(ADP-ribosyl) polymerase-1 (PARP-1) specifically in astrocytes, leading indirectly to neuronal cell death. PARP-1 is a DNA repair enzyme, normally activated by single strand breaks associated with oxidative stress, which catalyses the formation of poly ADP-ribose polymers from nicotinamide adenine dinucleotide (NAD+). The pathological over activation of PARP-1 causes depletion of NAD+ and leads to cell death. Here we review the relationship between AD and PARP-1, and explore the role played by astrocytes in neuronal death. AD has so far proven refractory to any effective treatment. Identification of these pathways represents a step towards a greater understanding of the pathophysiology of this devastating disease with the potential to explore novel therapeutic targets.

Blockade of chloride intracellular ion channel 1 stimulates Aβ phagocytosis

In amyloid‐β (Aβ)‐stimulated microglial cells, blockade of chloride intracellular ion channel 1 (CLIC1) reverts the increase in tumor necrosis factor‐α and nitric oxide (NO) production and results in neuroprotection of cocultured neurons. This effect could be of therapeutic efficacy in Alzheimers disease (AD), where microglial activation may contribute to neurodegeneration, but it could reduce Aβ phagocytosis, which could facilitate amyloid plaque removal. Here, we analyzed the CLIC1 blockade effect on Aβ‐stimulated mononuclear phagocytosis. In the microglial cell line BV‐2, Aβ25–35 treatment enhanced fluorescent bead phagocytosis, which persisted also in the presence of IAA‐94, a CLIC1 channel blocker. The same result was obtained in rat primary microglia and in BV2 cells, where CLIC1 expression had been knocked down with a plasmid producing small interfering RNAs. To address specifically the issue of Aβ phagocytosis, we treated BV‐2 cells with biotinylated Aβ1–42 and measured intracellular amyloid by morphometric analysis. IAA‐94‐treated cells showed an increased Aβ phagocytosis after 24 hr and efficient degradation of ingested material after 72 hr. In addition, we tested Aβ1–42 phagocytosis in adult rat peritoneal macrophages. Also, these cells actively phagocytosed Aβ1–42 in the presence of IAA‐94. However, the increased expression of inducible NO synthase (iNOS), stimulated by Aβ, was reverted by IAA‐94. In parallel, a decrease in NO release was detected. These results suggest that blockade of CLIC1 stimulates Aβ phagocytosis in mononuclear phagocytes while inhibiting the induction of iNOS and further point to CLIC1 as a possible therapeutic target in AD.

Point mutations in the transmembrane region of the Clic1 ion channel selectively modify its biophysical properties

Chloride intracellular Channel 1 (CLIC1) is a metamorphic protein that changes from a soluble cytoplasmic protein into a transmembrane protein. Once inserted into membranes, CLIC1 multimerises and is able to form chloride selective ion channels. Whilst CLIC1 behaves as an ion channel both in cells and in artificial lipid bilayers, its structure in the soluble form has led to some uncertainty as to whether it really is an ion channel protein. CLIC1 has a single putative transmembrane region that contains only two charged residues: arginine 29 (Arg29) and lysine 37 (Lys37). As charged residues are likely to have a key role in ion channel function, we hypothesized that mutating them to neutral alanine to generate K37A and R29A CLIC1 would alter the electrophysiological characteristics of CLIC1. By using three different electrophysiological approaches: i) single channel Tip-Dip in artificial bilayers using soluble recombinant CLIC1, ii) cell-attached and iii) whole-cell patch clamp recordings in transiently transfected HEK cells, we determined that the K37A mutation altered the single-channel conductance while the R29A mutation affected the single-channel open probability in response to variation in membrane potential. Our results show that mutation of the two charged amino acids (K37 and R29) in the putative transmembrane region of CLIC1 alters the biophysical properties of the ion channel in both artificial bilayers and cells. Hence these charged residues are directly involved in regulating its ion channel activity. This strongly suggests that, despite its unusual structure, CLIC1 itself is able to form a chloride ion channel.

Glial cells are functionally impaired in juvenile neuronal ceroid lipofuscinosis and detrimental to neurons

The neuronal ceroid lipofuscinoses (NCLs or Batten disease) are a group of inherited, fatal neurodegenerative disorders of childhood. In these disorders, glial (microglial and astrocyte) activation typically occurs early in disease progression and predicts where neuron loss subsequently occurs. We have found that in the most common juvenile form of NCL (CLN3 disease or JNCL) this glial response is less pronounced in both mouse models and human autopsy material, with the morphological transformation of both astrocytes and microglia severely attenuated or delayed. To investigate their properties, we isolated glia and neurons from Cln3-deficient mice and studied their basic biology in culture. Upon stimulation, both Cln3-deficient astrocytes and microglia also showed an attenuated ability to transform morphologically, and an altered protein secretion profile. These defects were more pronounced in astrocytes, including the reduced secretion of a range of neuroprotective factors, mitogens, chemokines and cytokines, in addition to impaired calcium signalling and glutamate clearance. Cln3-deficient neurons also displayed an abnormal organization of their neurites. Most importantly, using a co-culture system, Cln3-deficient astrocytes and microglia had a negative impact on the survival and morphology of both Cln3-deficient and wildtype neurons, but these effects were largely reversed by growing mutant neurons with healthy glia. These data provide evidence that CLN3 disease astrocytes are functionally compromised. Together with microglia, they may play an active role in neuron loss in this disorder and can be considered as potential targets for therapeutic interventions.

Diabetes in Friedreich ataxia

© 2017 S. Karger AG, Basel. All rights reserved. Friedreich ataxia (FRDA) is the most common hereditary ataxia. It is a progressive autosomal recessive neurodegenerative disorder associated with an increased risk of impaired glucose tolerance and overt diabetes mellitus. FRDA is caused by a genetic mutation inserting a GAA (guanine-adenineadenine) repeat expansion within intron 1 of the FXN gene, resulting in its transcriptional silencing. The FXN gene encodes for frataxin, a protein ubiquitously expressed and located in the inner mitochondrial membrane. Frataxin is involved in the biogenesis of iron-sulphur clusters (ISCs). The progressive lack of frataxin leads to iron accumulation and decreased activity of those proteins that contain ISCs, such as complex I, II, and III of the mitochondrial electron transport chain, and aconitase, a crucial enzyme of the Krebs cycle (TCA). As a result, FRDA has been linked to mitochondrial dysfunction due to increased reactive oxidative species generation and decreased ATP production. Mitochondria of pancreatic β-cells are central to stimulus-secretion coupling, which is responsible for triggering and amplifying insulin secretion. Furthermore, the intrinsic pathway of apoptosis occurs in the mitochondria and has also been implicated in the pathogenesis of diabetes in FRDA. This chapter focuses on what is currently known about the pathophysiology of diabetes mellitus in the context of frataxin deficiency and its clinical management.

Novel Nrf2-Inducer Prevents Mitochondrial Defects and Oxidative Stress in Friedreich’s Ataxia Models

Friedreich’s Ataxia (FRDA) is an autosomal recessive neurodegenerative disorder, affecting dorsal root ganglia (DRG), cerebellar dentate nuclei and heart. It is caused by a GAA repeat expansion mutation within the frataxin gene (FXN). This impedes FXN transcription resulting in a progressive decrease of the mitochondrial protein, frataxin. Increased oxidative stress leading to a chronic depletion of endogenous antioxidants affects the survival of the cells and causes neurodegeneration. In particular, cerebellar granule neurons (CGNs) show a significant increase of reactive oxygen species (ROS), lipid peroxidation and lower level of reduced glutathione (GSH). In FRDA, one of the major pathways of oxidant scavengers, the Nrf2 antioxidant pathway, is defective. Previous studies on FRDA-like CGNs showed that the reduced level of frataxin and the oxidative stress induce mitochondrial impairments. By triggering the Nrf2 endogenous pathway pharmacologically we determined whether this could promote mitochondrial fitness and counteract oxidative stress. In this work, we sought to investigate the beneficial effect of a promising Nrf2-inducer, omaveloxolone (omav), in CGNs from two FRDA mouse models, KIKO and YG8R, and human fibroblasts from patients. We found that CGNs from both KIKO and YG8R presented Complex I deficiency and that omav was able to restore substrate availability and Complex I activity. This was also confirmed in human primary fibroblasts from FRDA patients. Although fibroblasts are not the major tissue affected, we found that they show significant differences recapitulating the disease; this is therefore an important tool to investigate patients’ pathophysiology. Interestingly, we found that patient fibroblasts had an increased level of endogenous lipid peroxidation and mitochondrial ROS (mROS), and lower GSH at rest. Omav was able to reverse this phenotype, protecting the cells against oxidative stress. By stimulating the cells with hydrogen peroxide (H2O2) and looking for potential mitochondrial pathophysiology, we found that fibroblasts could not maintain their mitochondrial membrane potential (ΔΨm). Remarkably, omav was protective to mitochondrial depolarization, promoting mitochondrial respiration and preventing cell death. Our results show that omav promotes Complex I activity and protect cells from oxidative stress. Omav could, therefore, be used as a novel therapeutic drug to ameliorate the pathophysiology of FRDA.

Calcium Deregulation: Novel Insights to Understand Friedreich’s Ataxia Pathophysiology

Friedreich’s Ataxia (FRDA) is a neurodegenerative disorder, characterized by degeneration of dorsal root ganglia, cerebellum and cardiomyopathy. Heart failure is one of the most common causes of death for FRDA patients. Deficiency of frataxin, a small mitochondrial protein, is responsible for all clinical and morphological manifestations of FRDA. The focus of our study was to investigate the unexplored Ca2+ homeostasis in cerebellar granule neurons (CGNs) and in cardiomyocytes of FRDA cellular models to understand the pathogenesis of degeneration. Ca2+ homeostasis in neurons and cardiomyocytes is not only crucial for the cellular wellbeing but more importantly to generate action potential in both neurons and cardiomyocytes. By challenging Ca2+ homeostasis in CGNs, and in adult and neonatal cardiomyocytes of FRDA models, we have assessed the impact of frataxin decrease on both neuronal and cardiac physiopathology. Interestingly, we have found that Ca2+ homeostasis is altered both cell types. CGNs showed a Ca2+ mishandling under depolarizing conditions and this was also reflected in the endoplasmic reticulum (ER) content. In cardiomyocytes we found that the sarcoplasmic reticulum (SR) Ca2+ content was pathologically reduced, and that mitochondrial Ca2+ uptake was impaired. This phenomenon is due to the excess of oxidative stress under FRDA like conditions and the consequent aberrant modulation of key players at the SR/ER and mitochondrial level that usually restore the Ca2+ homeostasis. Our findings demonstrate that in both neurons and cardiomyocytes the decreased Ca2+ level within the stores has a comparable detrimental impact in their physiology. In cardiomyocytes, we found that ryanodine receptors (RyRs) may be leaking and expel more Ca2+ out from the SR. At the same time mitochondrial uptake was altered and we found that Vitamin E can restore this defect. Moreover, Vitamin E protects from cell death induced by hypoxia-reperfusion injury, revealing novel properties of Vitamin E as potential therapeutic tool for FRDA cardiomyopathy.