Bioconjugates of curcumin display improved protection against glutathione depletion mediated oxidative stress in a dopaminergic neuronal cell line: Implications for Parkinson’s disease

Oxidative stress is implicated in mitochondrial dysfunction associated with neurodegeneration in Parkinson’s disease (PD). Depletion of the cellular antioxidant glutathione (GSH) resulting in oxidative stress is considered as an early event in neurodegeneration. We previously showed that curcumin, a dietary polyphenol from turmeric induced GSH synthesis in experimental models and protected against oxidative stress. Here we tested the effect of three bioconjugates of curcumin (involving diesters of demethylenated piperic acid, valine and glutamic acid) against GSH depletion mediated oxidative stress in dopaminergic neuronal cells and found that the glutamic acid derivative displayed improved neuroprotection compared to curcumin.     

Molecular mechanisms of pesticide-induced neurotoxicity: Relevance to Parkinson's disease

Pesticides are widely used in agricultural and other settings, resulting in continued human exposure. Pesticide toxicity has been clearly demonstrated to alter a variety of neurological functions. Particularly, there is strong evidence suggesting that pesticide exposure predisposes to neurodegenerative diseases. Epidemiological data have suggested a relationship between pesticide exposure and brain neurodegeneration. However, an increasing debate has aroused regarding this issue. Paraquat is a highly toxic quaternary nitrogen herbicide which has been largely studied as a model for Parkinson's disease providing valuable insight into the molecular mechanisms involved in the toxic effects of pesticides and their role in the progression of neurodegenerative diseases. In this work, we review the molecular mechanisms involved in the neurotoxic action of pesticides, with emphasis on the mechanisms associated with the induction of neuronal cell death by paraquat as a model for Parkinsonian neurodegeneration.     

The neurotoxicity of iron,copper and manganese in Parkinson's and Wilson's diseases

Impaired cellular homeostasis of metals, particularly of Cu, Fe and Mn may trigger neurodegeneration through various mechanisms, notably induction of oxidative stress, promotion of α-synuclein aggregation and fibril formation, activation of microglial cells leading to inflammation and impaired production of metalloproteins. In this article we review available studies concerning Fe, Cu and Mn in Parkinson's disease and Wilson's disease. In Parkinson's disease local dysregulation of iron metabolism in the substantia nigra (SN) seems to be related to neurodegeneration with an increase in SN iron concentration, accompanied by decreased SN Cu and ceruloplasmin concentrations and increased free Cu concentrations and decreased ferroxidase activity in the cerebrospinal fluid. Available data in Wilson's disease suggest that substantial increases in CNS Cu concentrations persist for a long time during chelating treatment and that local accumulation of Fe in certain brain nuclei may occur during the course of the disease. Consequences for chelating treatment strategies are discussed.     

Disease-associated protein seeding suggests a dissociation between misfolded protein accumulation and neurodegeneration in prion disease

Chronic neurodegenerative diseases, such as prion diseases or Alzheimer's disease, are associated with progressive accumulation of host proteins which misfold and aggregate. Neurodegeneration is restricted to specific neuronal populations which show clear accumulation of misfolded proteins, whilst neighbouring neurons remain unaffected. Such data raise interesting questions about the vulnerability of specific neuronal populations to neurodegeneration and much research has concentrated only on the mechanisms of neurodegeneration in afflicted neuronal populations. An alternative, undervalued and almost completely unstudied question however is how and why neuronal populations are resilient to neurodegeneration. One potential answer is unaffected regions do not accumulate misfolded proteins, thus mechanisms of neurodegeneration do not become activated. In this perspectives, we discuss novel data from our laboratories which demonstrate that misfolded proteins do accumulate in regions of the brain which do not show evidence of neurodegeneration and further evidence that microglial responses may define the severity of neurodegeneration.     

The role of lysosomal cathepsins in neurodegeneration: Mechanistic insights,diagnostic potential and therapeutic approaches

Lysosomes are ubiquitous organelles with a fundamental role in maintaining cellular homeostasis by mediating degradation and recycling processes. Cathepsins are the most abundant lysosomal hydrolyses and are responsible for the bulk degradation of various substrates. A correct autophagic function is essential for neuronal survival, as most neurons are post-mitotic and thus susceptible to accumulate cellular components. Increasing evidence suggests a crucial role of the lysosome in neurodegeneration as a key regulator of aggregation-prone and disease-associated proteins, such as α-synuclein, β-amyloid and huntingtin. Particularly, alterations in lysosomal cathepsins CTSD, CTSB and CTSL can contribute to the pathogenesis of neurodegenerative diseases as seen for neuronal ceroid lipofuscinosis, synucleinopathies (Parkinson's disease, Dementia with Lewy Body and Multiple System Atrophy) as well as Alzheimer's and Huntington's disease. In this review, we provide an overview of recent evidence implicating CTSD, CTSB and CTSL in neurodegeneration, with a special focus on the role of these enzymes in α-synuclein metabolism. In addition, we summarize the potential role of lysosomal cathepsins as clinical biomarkers in neurodegenerative diseases and discuss potential therapeutic approaches by targeting lysosomal function.     

Neurodegeneration in Parkinson's Disease: Interactions of Oxidative Stress,Tryptophan Catabolites and Depression with Mitochondria and Sirtuins

The biological underpinnings to the etiology and course of neurodegeneration in Parkinson's disease are an area of extensive research that has yet to produce an early biological marker or disease-slowing or preventative treatment. Recent conceptualizations of Parkinson's disease have integrated immuno-inflammation and oxidative and nitrosative stress occurring in depression, somatization and peripheral inflammation into the course of Parkinson's disease. We review the data showing the importance of immuno-inflammatory processes and oxidative and nitrosative stress in such classically conceived ‘comorbidities’, suggesting that lifetime, prodromal and concurrent depression and somatization may be intricately involved in the etiology and course of Parkinson's disease, rather than psychiatric comorbidities. This produces a longer term developmental perspective of Parkinson's disease, which incorporates tryptophan catabolites (TRYCATs), lipid peroxidation, sirtuins, cyclic adenosine monophosphate, aryl hydrocarbon receptor, and circadian genes. This integrates wider bodies of data pertaining to neuronal loss in Parkinson's disease, emphasizing how these interact with susceptibility genes to drive changes in mitochondria, blood–brain barrier permeability and intercellular signalling. We review this data here in the context of neurodegeneration in Parkinson's disease and to the future directions indicated for slowing disease progression.     

DJ-1 deficiency in astrocytes selectively enhances mitochondrial Complex I inhibitor-induced neurotoxicity

Parkinson's disease (PD) brains show evidence of mitochondrial respiratory Complex I deficiency, oxidative stress, and neuronal death. Complex I-inhibiting neurotoxins, such as the pesticide rotenone, cause neuronal death and parkinsonism in animal models. We have previously shown that DJ-1 over-expression in astrocytes augments their capacity to protect neurons against rotenone, that DJ-1 knock-down impairs astrocyte-mediated neuroprotection against rotenone, and that each process involves astrocyte-released factors. To further investigate the mechanism behind these findings, we developed a high-throughput, plate-based bioassay that can be used to assess how genetic manipulations in astrocytes affect their ability to protect co-cultured neurons. We used this bioassay to show that DJ-1 deficiency-induced impairments in astrocyte-mediated neuroprotection occur solely in the presence of pesticides that inhibit Complex I (rotenone, pyridaben, fenazaquin, and fenpyroximate); not with agents that inhibit Complexes II-V, that primarily induce oxidative stress, or that inhibit the proteasome. This is a potentially PD-relevant finding because pesticide exposure is epidemiologically-linked with an increased risk for PD. Further investigations into our model suggested that astrocytic GSH and heme oxygenase-1 antioxidant systems are not central to the neuroprotective mechanism.     

Dopaminergic neurotoxicity of S‐ethyl N,N‐dipropylthiocarbamate (EPTC), molinate,and S‐methyl‐N,N‐diethylthiocarbamate (MeDETC) in Caenorhabditis elegans

Epidemiological studies corroborate a correlation between pesticide use and Parkinson's disease (PD). Thiocarbamate and dithiocarbamate pesticides are widely used and produce neurotoxicity in the peripheral nervous system. Recent evidence from rodent studies suggests that these compounds also cause dopaminergic (DAergic) dysfunction and altered protein processing, two hallmarks of PD. However, DAergic neurotoxicity has yet to be documented. We assessed DAergic dysfunction in Caenorhabditis elegans (C. elegans) to investigate the ability of thiocarbamate pesticides to induce DAergic neurodegeneration. Acute treatment with either S‐ethyl N,N‐dipropylthiocarbamate (EPTC), molinate, or a common reactive intermediate of dithiocarbamate and thiocarbamate metabolism, S‐methyl‐N,N‐diethylthiocarbamate (MeDETC), to gradual loss of DAergic cell morphology and structure over the course of 6 days in worms expressing green fluorescent protein (GFP) under a DAergic cell specific promoter. HPLC analysis revealed decreased DA content in the worms immediately following exposure to MeDETC, EPTC, and molinate. In addition, worms treated with the three test compounds showed a drastic loss of DAergic‐dependent behavior over a time course similar to changes in DAergic cell morphology. Alterations in the DAergic system were specific, as loss of cell structure and neurotransmitter content was not observed in cholinergic, glutamatergic, or GABAergic systems. Overall, our data suggest that thiocarbamate pesticides promote neurodegeneration and DAergic cell dysfunction in C. elegans, and may be an environmental risk factor for PD.     

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)copper(II) complexes (Cu(II)(btsc)s) can modulate kinase activity and induce neuroprotective effects. In this study we examined the effect of diacetylbis(-methylthiosemicarbazonato)copper(II) (Cu(II)(atsm)) and glyoxalbis(-methylthiosemicarbazonato)copper(II) (Cu(II)(gtsm)) on TDP-43-positive SGs induced in SH-SY5Y cells in culture. We found that the Cu(II)(btsc)s blocked formation of TDP-43-and human antigen R (HuR)-positive SGs induced by paraquat. The Cu(II)(btsc)s protected neurons from paraquat-mediated cell death. These effects were associated with inhibition of ERK phosphorylation. Co-treatment of cultures with either Cu(II)(atsm) or an ERK inhibitor, PD98059 both prevented ERK activation and blocked formation of TDP-43-and HuR-positive SGs. Cu(II)(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, Cu(II)(atsm) reduced accumulation of C-terminal (219-414) TDP-43 in transfected SH-SY5Y cells. These results demonstrate that Cu(II)(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.     

Neuroprotective potential of choline alfoscerate against β‐amyloid injury: Involvement of neurotrophic signals

Alzheimer's disease represents the most prevalent neurodegeneration worldwide, clinically characterized by cognitive and memory impairment. New therapeutic approaches are extremely important to counteract this disorder. This research is focused on the potential use of choline alfoscerate in preventing neuronal death using in vitro models of Alzheimer's disease, representing the early stage of the disease, treated before or after the insult with glycerylphosphorylcholine. On the light of the results collected, we can postulate that choline alfoscerate, by the activation of the neurotrophin survival pathway, was able to counteract the detrimental effect of β‐amyloid in both in vitro models, reducing apoptotic cell death and preserving the neuronal morphology.     

alpha-synuclein and Parkinson's disease: the first roadblock

alpha-synuclein gene mutations are major underlying genetic defects known in familial juvenile onset Parkinson's disease (PD), and alpha-synuclein is a major constituent of Lewy Bodies, the pathological hallmark of PD. The normal cellular function of alpha-synuclein has been elusive, and its exact etiological mechanism in causing dopaminergic neuronal death in PD is also not clearly understood. Very recent reports now indicate that mutant or simply over-expressed alpha- synuclein could cause damage by interfering with particular steps of neuronal membrane traffic. alpha-synuclein selectively blocks endoplamic reticulum-to-Golgi transport, thus causing ER stress. A screen in a yeast revealed that alpha- synuclein toxicity could be suppressed by over-expression of the small GTPase Ypt1/Rab1, and that over-expression of the latter rescues neuron loss in invertebrate and mammalian models of alpha-synuclein-induced neurodegeneration. alpha-synuclein may also serve a chaperone function for the proper folding of synaptic SNAREs that are important for neurotransmitter release. We discuss these recent results and the emerging pathophysiological interaction of alpha-synuclein with components of neuronal membrane traffic.     

Proteomics in human Parkinson's disease research

During the last decades, considerable advances in the understanding of specific mechanisms underlying neurodegeneration in Parkinson's disease have been achieved, yet neither definite etiology nor unifying sequence of molecular events has been formally established. Current unmet needs in Parkinson's disease research include exploring new hypotheses regarding disease susceptibility, occurrence and progression, identifying reliable diagnostic, prognostic and therapeutic biomarkers, and translating basic research into appropriate disease-modifying strategies. The most popular view proposes that Parkinson's disease results from the complex interplay between genetic and environmental factors and mechanisms believed to be at work include oxidative stress, mitochondrial dysfunction, excitotoxicity, iron deposition and inflammation. More recently, a plethora of data has accumulated pinpointing an abnormal processing of the neuronal protein α-synuclein as a pivotal mechanism leading to aggregation, inclusions formation and degeneration. This protein-oriented scenario logically opens the door to the application of proteomic strategies to this field of research. We here review the current literature on proteomics applied to Parkinson's disease research, with particular emphasis on pathogenesis of sporadic Parkinson's disease in humans. We propose the view that Parkinson's disease may be an acquired or genetically-determined brain proteinopathy involving an abnormal processing of several, rather than individual neuronal proteins, and discuss some pre-analytical and analytical developments in proteomics that may help in verifying this concept.     

The early cellular pathology of Huntington’s disease

Huntington's disease (HD) is an inherited neurodegenerative disorder that affects about one in 10,000 individuals in North America. The genetic defect responsible for the disease is an expansion of a CAG repeat that encodes a polyglutamine tract in the expressed protein, huntingtin. The disease is characterized by involuntary movements, cognitive impairment, and emotional disturbance. Despite the widespread expression of huntingtin, the brains of HD patients show selective neuronal loss in the striatum and the deep layers of the cerebral cortex. Recent studies have shown that polyglutamine expansion causes huntingtin to aggregate, to accumulate in the nucleus, and to interact abnormally with other proteins. Several cellular and animal models for HD have revealed that intranuclear accumulation of mutant huntingtin and the formation of neuropil aggregates precede neurological symptoms and neurodegeneration. Intranuclear huntingtin may affect nuclear function and the expression of genes important for neuronal function, whereas neuropil aggregates may interfere with neuritic transport and function. These early pathological events, which occur in the absence of neurodegeneration, may contribute to the neurological symptoms of HD and ultimately lead to neuronal cell death.     

Harnessing the power of yeast to unravel the molecular basis of neurodegeneration

Several neurodegenerative diseases, such as Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), or prion diseases, are known for their intimate association with protein misfolding and aggregation. These disorders are characterized by the loss of specific neuronal populations in the brain and are highly associated with aging, suggesting a decline in proteostasis capacity may contribute to pathogenesis. Nevertheless, the precise molecular mechanisms that lead to the selective demise of neurons remain poorly understood. As a consequence, appropriate therapeutic approaches and effective treatments are largely lacking. The development of cellular and animal models that faithfully reproduce central aspects of neurodegeneration has been crucial for advancing our understanding of these diseases. Approaches involving the sequential use of different model systems, starting with simpler cellular models and ending with validation in more complex animal models, resulted in the discovery of promising therapeutic targets and small molecules with therapeutic potential. Within this framework, the simple and well‐characterized eukaryote Saccharomyces cerevisiae, also known as budding yeast, is being increasingly used to study the molecular basis of several neurodegenerative disorders. Yeast provides an unprecedented toolbox for the dissection of complex biological processes and pathways. Here, we summarize how yeast models are adding to our current understanding of several neurodegenerative disorders.     

The downward spiral of tau and autolysosomes

A growing body of research has connected autophagy to neurodegenerative diseases such as Alzheimer disease (AD). In autopsied AD brain, large multivesicular bodies accumulate in neurons. Knockout mice deficient for key autophagy genes demonstrate age-dependent neurodegeneration. Most neurodegenerative diseases are characterized by accumulation of insoluble protein species; the type of protein and the location of aggregates within the nervous system help to define the type of disorder. It has been hypothesized that the inability to degrade such aggregates is a major causative factor in neuronal dysfunction and eventual neuronal death. As neurons are postmitotic and thus cannot regenerate themselves, mechanisms of protein clearance have received much attention in the field. The function of the ubiquitin-proteasome system (UPS) is impaired in models of neurodegeneration, and overexpression of chaperone proteins, such as those in the HSP70 family, leads to beneficial effects in many models of proteinopathies. Recently, studies of the effects of autophagy as a clearance mechanism have uncovered compelling evidence that inducing autophagy can alleviate many pathogenic and behavioral symptoms in animal and cellular models of neurodegeneration.     

The downward spiral of tau and autolysosomes: A new hypothesis in neurodegeneration

A growing body of research has connected autophagy to neurodegenerative diseases such as Alzheimer disease (AD). In autopsied AD brain, large multivesicular bodies accumulate in neurons. Knockout mice deficient for key autophagy genes demonstrate age-dependent neurodegeneration. Most neurodegenerative diseases are characterized by accumulation of insoluble protein species; the type of protein and the location of aggregates within the nervous system help to define the type of disorder. It has been hypothesized that the inability to degrade such aggregates is a major causative factor in neuronal dysfunction and eventual neuronal death. As neurons are postmitotic and thus cannot regenerate themselves, mechanisms of protein clearance have received much attention in the field. The function of the ubiquitin-proteasome system (UPS) is impaired in models of neurodegeneration, and overexpression of chaperone proteins, such as those in the HSP70 family, leads to beneficial effects in many models of proteinopathies. Recently, studies of the effects of autophagy as a clearance mechanism have uncovered compelling evidence that inducing autophagy can alleviate many pathogenic and behavioral symptoms in animal and cellular models of neurodegeneration.     

A single amino acid substitution in a proteasome subunit triggers aggregation of ubiquitinated proteins in stressed neuronal cells

Accumulation of ubiquitinated proteins in inclusions is common to various neurodegenerative disorders such as Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis, although it occurs in selective neurons in each disease. The mechanisms generating such abnormal aggregates and their role in neurodegeneration remain unclear. Inclusions appear in familial and non-familial cases of neurodegenerative disorders, suggesting that factors other than particular mutations contribute to protein accumulation and aggregation. Proteasome impairment triggered by aging or conditions such as oxidative stress may contribute to protein accumulation and aggregation in neurodegeneration. To test this hypothesis in mouse neuronal cells, we overexpressed a 20S proteasome beta5 subunit with an active site mutation. The N-terminal threonine to alanine substitution resulted in impairment of the chymotrypsin-like activity, which is a rate-limiting step in protein degradation by the proteasome. The Thr1Ala mutation was not lethal under homeostatic conditions. However, this single amino acid substitution significantly hypersensitized the cells to oxidative stress, triggering not only the accumulation and aggregation of ubiquitinated proteins, including synuclein, but also cell death. Our results demonstrate that this genetic manipulation of proteasome activity involving a single amino acid substitution causes the formation of protein aggregates in stressed neuronal cells independently of the occurrence of mutations in other cellular proteins. These results support the notion that proteasome disruption may be central to the development of familial as well as sporadic cases of neurodegeneration.