Katja M. Kanninen

Endogenous TDP-43 localized to stress granules can subsequently form protein aggregates.

TDP-43 proteinopathies are characterized by loss of nuclear TDP-43 and accumulation of the protein in the cytosol as ubiquitinated protein aggregates. These protein aggregates may have an important role in subsequent neuronal degeneration in motor neuron disease, frontotemporal dementia and potentially other neurodegenerative diseases. Although the cellular mechanisms driving the abnormal accumulation of TDP-43 are not understood, recent studies have shown that an early change to TDP-43 metabolism in disease may be accumulation in cytosolic RNA stress granules (SGs). However, it is unclear whether the TDP-43 in these SGs progresses to become irreversible protein aggregates as observed in patients. We have shown recently that paraquat-treated cells are a useful model for examining TDP-43 SG localization. In this study, we used the paraquat model to examine if endogenous TDP-43 in SGs can progress to more stable protein aggregates. We found that after treatment of HeLa cells overnight with paraquat, TDP-43 co-localized to SGs together with the ubiquitous SG marker, human antigen R (HuR). However, after a further incubation in paraquat-free, conditioned medium for 6h, HuR-positive SGs were rarely detected yet TDP-43 positive aggregates remained present. The majority of these TDP-43 aggregates were positive for ubiquitin. Further evidence for persistence of TDP-43 aggregates was obtained by treating cultures with cycloheximide after paraquat treatment. Cycloheximide abolished nearly all cytosolic HuR aggregation (SGs) but large TDP-43-positive aggregates remained. Finally, we showed that addition of ERK and JNK inhibitors together with paraquat blocked TDP-43-positive SG formation, while treatment with inhibitors after 24h paraquat exposure failed to reverse the TDP-43 accumulation. This failure was most likely due to the addition of inhibitors after maximal activation of the kinases at 4h post-paraquat treatment. These findings provide strong evidence that once endogenous TDP-43 accumulates in SGs, it has the potential to progress to stable protein aggregates as observed in neurons in TDP-43 proteinopathies. This may provide a therapeutic opportunity to inhibit the transition of TDP-43 from SG protein to aggregate.

C-Jun N-terminal kinase controls TDP-43 accumulation in stress granules induced by oxidative stress

BackgroundTDP-43 proteinopathies are characterized by loss of nuclear TDP-43 expression and formation of C-terminal TDP-43 fragmentation and accumulation in the cytoplasm. Recent studies have shown that TDP-43 can accumulate in RNA stress granules (SGs) in response to cell stresses and this could be associated with subsequent formation of TDP-43 ubiquinated protein aggregates. However, the initial mechanisms controlling endogenous TDP-43 accumulation in SGs during chronic disease are not understood. In this study we investigated the mechanism of TDP-43 processing and accumulation in SGs in SH-SY5Y neuronal-like cells exposed to chronic oxidative stress. Cell cultures were treated overnight with the mitochondrial inhibitor paraquat and examined for TDP-43 and SG processing.ResultsWe found that mild stress induced by paraquat led to formation of TDP-43 and HuR-positive SGs, a proportion of which were ubiquitinated. The co-localization of TDP-43 with SGs could be fully prevented by inhibition of c-Jun N-terminal kinase (JNK). JNK inhibition did not prevent formation of HuR-positive SGs and did not prevent diffuse TDP-43 accumulation in the cytosol. In contrast, ERK or p38 inhibition prevented formation of both TDP-43 and HuR-positive SGs. JNK inhibition also inhibited TDP-43 SG localization in cells acutely treated with sodium arsenite and reduced the number of aggregates per cell in cultures transfected with C-terminal TDP-43 162-414 and 219-414 constructs.ConclusionsOur studies are the first to demonstrate a critical role for kinase control of TDP-43 accumulation in SGs and may have important implications for development of treatments for FTD and ALS, targeting cell signal pathway control of TDP-43 aggregation.

Oral Treatment with CuII(atsm) Increases Mutant SOD1 In Vivo but Protects Motor Neurons and Improves the Phenotype of a Transgenic Mouse Model of Amyotrophic Lateral Sclerosis

Mutations in the metallo-protein Cu/Zn-superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS) in humans and an expression level-dependent phenotype in transgenic rodents. We show that oral treatment with the therapeutic agent diacetyl-bis(4-methylthiosemicarbazonato)copperII [CuII(atsm)] increased the concentration of mutant SOD1 (SOD1G37R) in ALS model mice, but paradoxically improved locomotor function and survival of the mice. To determine why the mice with increased levels of mutant SOD1 had an improved phenotype, we analyzed tissues by mass spectrometry. These analyses revealed most SOD1 in the spinal cord tissue of the SOD1G37R mice was Cu deficient. Treating with CuII(atsm) decreased the pool of Cu-deficient SOD1 and increased the pool of fully metallated (holo) SOD1. Tracking isotopically enriched 65CuII(atsm) confirmed the increase in holo-SOD1 involved transfer of Cu from CuII(atsm) to SOD1, suggesting the improved locomotor function and survival of the CuII(atsm)-treated SOD1G37R mice involved, at least in part, the ability of the compound to improve the Cu content of the mutant SOD1. This was supported by improved survival of SOD1G37R mice that expressed the human gene for the Cu uptake protein CTR1. Improving the metal content of mutant SOD1 in vivo with CuII(atsm) did not decrease levels of misfolded SOD1. These outcomes indicate the metal content of SOD1 may be a greater determinant of the toxicity of the protein in mutant SOD1-associated forms of ALS than the mutations themselves. Improving the metal content of SOD1 therefore represents a valid therapeutic strategy for treating ALS caused by SOD1.

Targeting Glycogen Synthase Kinase-3β for Therapeutic Benefit against Oxidative Stress in Alzheimer's Disease: Involvement of the Nrf2-ARE Pathway

Specific regions of the Alzheimers disease (AD) brain are burdened with extracellular protein deposits, the accumulation of which is concomitant with a complex cascade of overlapping events. Many of these pathological processes produce oxidative stress. Under normal conditions, oxidative stress leads to the activation of defensive gene expression that promotes cell survival. At the forefront of defence is the nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor that regulates a broad spectrum of protective genes. Glycogen synthase kinase-3β (GSK-3β) regulates Nrf2, thus making this kinase a potential target for therapeutic intervention aiming to boost the protective activation of Nrf2. This paper aims to review the neuroprotective role of Nrf2 in AD, with special emphasis on the role of GSK-3β in the regulation of the Nrf2 pathway. We also examine the potential of inducing GSK-3β by small-molecule activators, dithiocarbamates, which potentially exert their beneficial therapeutic effects via the activation of the Nrf2 pathway.

Deregulation of subcellular biometal homeostasis through loss of the metal transporter, Zip7, in a childhood neurodegenerative disorder

BackgroundAberrant biometal metabolism is a key feature of neurodegenerative disorders including Alzheimer’s and Parkinson’s diseases. Metal modulating compounds are promising therapeutics for neurodegeneration, but their mechanism of action remains poorly understood. Neuronal ceroid lipofuscinoses (NCLs), caused by mutations in CLN genes, are fatal childhood neurodegenerative lysosomal storage diseases without a cure. We previously showed biometal accumulation in ovine and murine models of the CLN6 variant NCL, but the mechanism is unknown. This study extended the concept that alteration of biometal functions is involved in pathology in these disorders, and investigated molecular mechanisms underlying impaired biometal trafficking in CLN6 disease.ResultsWe observed significant region-specific biometal accumulation and deregulation of metal trafficking pathways prior to disease onset in CLN6 affected sheep. Substantial progressive loss of the ER/Golgi-resident Zn transporter, Zip7, which colocalized with the disease-associated protein, CLN6, may contribute to the subcellular deregulation of biometal homeostasis in NCLs. Importantly, the metal-complex, ZnII(atsm), induced Zip7 upregulation, promoted Zn redistribution and restored Zn-dependent functions in primary mouse Cln6 deficient neurons and astrocytes.ConclusionsThis study demonstrates the central role of the metal transporter, Zip7, in the aberrant biometal metabolism of CLN6 variants of NCL and further highlights the key contribution of deregulated biometal trafficking to the pathology of neurodegenerative diseases. Importantly, our results suggest that ZnII(atsm) may be a candidate for therapeutic trials for NCLs.

Increased zinc and manganese in parallel with neurodegeneration, synaptic protein changes and activation of Akt/GSK3 signaling in ovine CLN6 neuronal ceroid lipofuscinosis.

Mutations in the CLN6 gene cause a variant late infantile form of neuronal ceroid lipofuscinosis (NCL; Batten disease). CLN6 loss leads to disease clinically characterized by vision impairment, motor and cognitive dysfunction, and seizures. Accumulating evidence suggests that alterations in metal homeostasis and cellular signaling pathways are implicated in several neurodegenerative and developmental disorders, yet little is known about their role in the NCLs. To explore the disease mechanisms of CLN6 NCL, metal concentrations and expression of proteins implicated in cellular signaling pathways were assessed in brain tissue from South Hampshire and Merino CLN6 sheep. Analyses revealed increased zinc and manganese concentrations in affected sheep brain in those regions where neuroinflammation and neurodegeneration first occur. Synaptic proteins, the metal-binding protein metallothionein, and the Akt/GSK3 and ERK/MAPK cellular signaling pathways were also altered. These results demonstrate that altered metal concentrations, synaptic protein changes, and aberrant modulation of cellular signaling pathways are characteristic features in the CLN6 ovine form of NCL.

Altered biometal homeostasis is associated with CLN6 mRNA loss in mouse neuronal ceroid lipofuscinosis.

Summary Neuronal ceroid lipofuscinoses, the most common fatal childhood neurodegenerative illnesses, share many features with more prevalent neurodegenerative diseases. Neuronal ceroid lipofuscinoses are caused by mutations in CLN genes. CLN6 encodes a transmembrane endoplasmic reticulum protein with no known function. We characterized the behavioural phenotype of spontaneous mutant mice modeling CLN6 disease, and demonstrate progressive motor and visual decline and reduced lifespan in these mice, consistent with symptoms observed in neuronal ceroid lipofuscinosis patients. Alterations to biometal homeostasis are known to play a critical role in pathology in Alzheimers, Parkinsons, Huntingtons and motor neuron diseases. We have previously shown accumulation of the biometals, zinc, copper, manganese and cobalt, in CLN6 Merino and South Hampshire sheep at the age of symptom onset. Here we determine the physiological and disease-associated expression of CLN6, demonstrating regional CLN6 transcript loss, and concurrent accumulation of the same biometals in the CNS and the heart of presymptomatic CLN6 mice. Furthermore, increased expression of the ER/Golgi-localized cation transporter protein, Zip7, was detected in cerebellar Purkinje cells and whole brain fractions. Purkinje cells not only control motor function, an early symptomatic change in the CLN6 mice, but also display prominent neuropathological changes in mouse models and patients with different forms of neuronal ceroid lipofuscinoses. Whole brain fractionation analysis revealed biometal accumulation in fractions expressing markers for ER, Golgi, endosomes and lysosomes of CLN6 brains. These data are consistent with a link between CLN6 expression and biometal homeostasis in CLN6 disease, and provide further support for altered cation transporter regulation as a key factor in neurodegeneration.

Inhibition of TDP-43 Accumulation by Bis(thiosemicarbazonato)-Copper Complexes

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal, motor neuron disease with no effective long-term treatment options. Recently, TDP-43 has been identified as a key protein in the pathogenesis of some cases of ALS. Although the role of TDP-43 in motor neuron degeneration is not yet known, TDP-43 has been shown to accumulate in RNA stress granules (SGs) in cell models and in spinal cord tissue from ALS patients. The SG association may be an early pathological change to TDP-43 metabolism and as such a potential target for therapeutic intervention. Accumulation of TDP-43 in SGs induced by inhibition of mitochondrial activity can be inhibited by modulation of cellular kinase activity. We have also found that treatment of cells and animal models of neurodegeneration, including an ALS model, with bioavailable bis(thiosemicarbazonato)copperII complexes (CuII(btsc)s) can modulate kinase activity and induce neuroprotective effects. In this study we examined the effect of diacetylbis(-methylthiosemicarbazonato)copperII (CuII(atsm)) and glyoxalbis(-methylthiosemicarbazonato)copperII (CuII(gtsm)) on TDP-43-positive SGs induced in SH-SY5Y cells in culture. We found that the CuII(btsc)s blocked formation of TDP-43-and human antigen R (HuR)-positive SGs induced by paraquat. The CuII(btsc)s protected neurons from paraquat-mediated cell death. These effects were associated with inhibition of ERK phosphorylation. Co-treatment of cultures with either CuII(atsm) or an ERK inhibitor, PD98059 both prevented ERK activation and blocked formation of TDP-43-and HuR-positive SGs. CuII(atsm) treatment or ERK inhibition also prevented abnormal ubiquitin accumulation in paraquat-treated cells suggesting a link between prolonged ERK activation and abnormal ubiquitin metabolism in paraquat stress and inhibition by Cu. Moreover, CuII(atsm) reduced accumulation of C-terminal (219–414) TDP-43 in transfected SH-SY5Y cells. These results demonstrate that CuII(btsc) complexes could potentially be developed as a neuroprotective agent to modulate neuronal kinase function and inhibit TDP-43 aggregation. Further studies in TDP-43 animal models are warranted.

Biometals in rare neurodegenerative disorders of childhood

Copper, iron, and zinc are just three of the main biometals critical for correct functioning of the central nervous system (CNS). They have diverse roles in many functional processes including but not limited to enzyme catalysis, protein stabilization, and energy production. The range of metal concentrations within the body is tightly regulated and when the balance is perturbed, debilitating effects ensue. Homeostasis of brain biometals is mainly controlled by various metal transporters and metal sequestering proteins. The biological roles of biometals are vastly reviewed in the literature with a large focus on the connection to neurological conditions associated with ageing. Biometals are also implicated in a variety of debilitating inherited childhood disorders, some of which arise soon following birth or as the child progresses into early adulthood. This review acts to highlight what we know about biometals in childhood neurological disorders such as Wilsons disease (WD), Menkes disease (MD), neuronal ceroid lipofuscinoses (NCLs), and neurodegeneration with brain iron accumulation (NBIA). Also discussed are some of the animal models available to determine the pathological mechanisms in these childhood disorders, which we hope will aid in our understanding of the role of biometals in disease and in attaining possible therapeutics in the future.

A potential copper-regulatory role for cytosolic expression of the DNA repair protein XRCC5.

Copper (Cu) has a critical role in the generation of oxidative stress during neurodegeneration and cancer. Reactive oxygen species generated through abnormal elevation or deficiency of Cu can lead to lipid, protein, and DNA damage. Oxidation of DNA can induce strand breaks and is associated with altered cell fate including transformation or death. DNA repair is mediated through the action of the multimeric DNA-PK repair complex. The components of this complex are the Ku autoantigens, XRCC5 and XRCC6 (Ku80 and Ku70, respectively). How this repair complex responds to perturbed Cu homeostasis and Cu-mediated oxidative stress has not been investigated. We previously reported that XRCC5 expression is altered in response to cellular Cu levels, with low Cu inhibiting XRCC5 expression and high Cu levels enhancing expression. In this study we further investigated the interaction between XRCC5 and Cu. We report that cytosolic XRCC5 is increased in response to Cu, but not zinc, iron, or nickel, and the level of cytosolic XRCC5 correlates with protection against oxidative damage to DNA. These observations were made in both HeLa cells and fibroblasts. Cytosolic XRCC5 interacted with the Cu chaperone and detoxification protein human Atox1 homologue (HAH), and down regulation of XRCC5 expression using siRNA led to enhanced HAH expression when cells were exposed to Cu. XRCC5 could also be purified from cytosolic extracts using a Cu-loaded column. These findings provide further evidence that cytosolic XRCC5 has a key role in protection against DNA oxidation from Cu, through either direct sequestration or signaling through other Cu-detoxification molecules. Our findings have important implications for the development of therapeutic treatments targeting Cu in neurodegeneration and/or cancer.