Evaluation of cyanotoxin L-BMAA effect on α-synuclein and TDP43 proteinopathy.

The complex interplay between genetic and environmental factors is considered the cause of neurodegenerative diseases including Parkinson's disease (PD) and Amyotrophic Lateral Sclerosis (ALS). Among the environmental factors, toxins produced by cyanobacteria have received much attention due to the significant increase in cyanobacteria growth worldwide. In particular, L-BMAA toxin, produced by diverse taxa of cyanobacteria, dinoflagellates and diatoms, has been extensively correlated to neurodegeneration. The molecular mechanism of L-BMAA neurotoxicity is still cryptic and far from being understood. In this research article, we have investigated the molecular pathways altered by L-BMAA exposure in cell systems, highlighting a significant increase in specific stress pathways and an impairment in autophagic processes. Interestingly, these changes lead to the accumulation of both α-synuclein and TDP43, which are correlated with PD and ALS proteinopathy, respectively. Finally, we were able to demonstrate specific alterations of TDP43 WT or pathological mutants with respect to protein accumulation, aggregation and cytoplasmic translocation, some of the typical features of both sporadic and familial ALS.

Inhibition of the Exocyst Complex Attenuates the LRRK2 Pathological Effects.

Pathological mutations in leucine-rich repeat kinase 2 (LRRK2) gene are the major genetic cause of Parkinson's disease (PD). Multiple lines of evidence link LRRK2 to the control of vesicle dynamics through phosphorylation of a subset of RAB proteins. However, the molecular mechanisms underlying these processes are not fully elucidated. We have previously demonstrated that LRRK2 increases the exocyst complex assembly by Sec8 interaction, one of the eight members of the exocyst complex, and that Sec8 over-expression mitigates the LRRK2 pathological effect in PC12 cells. Here, we extend this analysis using LRRK2 drosophila models and show that the LRRK2-dependent exocyst complex assembly increase is downstream of RAB phosphorylation. Moreover, exocyst complex inhibition rescues mutant LRRK2 pathogenic phenotype in cellular and drosophila models. Finally, prolonged exocyst inhibition leads to a significant reduction in the LRRK2 protein level, overall supporting the role of the exocyst complex in the LRRK2 pathway. Taken together, our study suggests that modulation of the exocyst complex may represent a novel therapeutic target for PD.

The E3 ligase TRIM1 ubiquitinates LRRK2 and controls its localization, degradation, and toxicity.

Missense mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease (PD); however, pathways regulating LRRK2 subcellular localization, function, and turnover are not fully defined. We performed quantitative mass spectrometry-based interactome studies to identify 48 novel LRRK2 interactors, including the microtubule-associated E3 ubiquitin ligase TRIM1 (tripartite motif family 1). TRIM1 recruits LRRK2 to the microtubule cytoskeleton for ubiquitination and proteasomal degradation by binding LRRK2911-919, a nine amino acid segment within a flexible interdomain region (LRRK2853-981), which we designate the "regulatory loop" (RL). Phosphorylation of LRRK2 Ser910/Ser935 within LRRK2 RL influences LRRK2's association with cytoplasmic 14-3-3 versus microtubule-bound TRIM1. Association with TRIM1 modulates LRRK2's interaction with Rab29 and prevents upregulation of LRRK2 kinase activity by Rab29 in an E3-ligase-dependent manner. Finally, TRIM1 rescues neurite outgrowth deficits caused by PD-driving mutant LRRK2 G2019S. Our data suggest that TRIM1 is a critical regulator of LRRK2, controlling its degradation, localization, binding partners, kinase activity, and cytotoxicity.

Cyanobacteria, Cyanotoxins, and Neurodegenerative Diseases: Dangerous Liaisons.

The prevalence of neurodegenerative disease (ND) is increasing, partly owing to extensions in lifespan, with a larger percentage of members living to an older age, but the ND aetiology and pathogenesis are not fully understood, and effective treatments are still lacking. Neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis are generally thought to progress as a consequence of genetic susceptibility and environmental influences. Up to now, several environmental triggers have been associated with NDs, and recent studies suggest that some cyanotoxins, produced by cyanobacteria and acting through a variety of molecular mechanisms, are highly neurotoxic, although their roles in neuropathy and particularly in NDs are still controversial. In this review, we summarize the most relevant and recent evidence that points at cyanotoxins as environmental triggers in NDs development.

Parkinson's Disease-Related Genes and Lipid Alteration.

Parkinson's disease (PD) is a complex and progressive neurodegenerative disorder with a prevalence of approximately 0.5-1% among those aged 65-70 years. Although most of its clinical manifestations are due to a loss of dopaminergic neurons, the PD etiology is largely unknown. PD is caused by a combination of genetic and environmental factors, and the exact interplay between genes and the environment is still debated. Several biological processes have been implicated in PD, including mitochondrial or lysosomal dysfunctions, alteration in protein clearance, and neuroinflammation, but a common molecular mechanism connecting the different cellular alterations remains incompletely understood. Accumulating evidence underlines a significant role of lipids in the pathological pathways leading to PD. Beside the well-described lipid alteration in idiopathic PD, this review summarizes the several lipid alterations observed in experimental models expressing PD-related genes and suggests a possible scenario in relationship to the molecular mechanisms of neuronal toxicity. PD could be considered a lipid-induced proteinopathy, where alteration in lipid composition or metabolism could induce protein alteration-for instance, alpha-synuclein accumulation-and finally neuronal death.

LRRK2 Modulates the Exocyst Complex Assembly by Interacting with Sec8.

Mutations in LRRK2 play a critical role in both familial and sporadic Parkinson's disease (PD). Up to date, the role of LRRK2 in PD onset and progression remains largely unknown. However, experimental evidence highlights a critical role of LRRK2 in the control of vesicle trafficking, likely by Rab phosphorylation, that in turn may regulate different aspects of neuronal physiology. Here we show that LRRK2 interacts with Sec8, one of eight subunits of the exocyst complex. The exocyst complex is an evolutionarily conserved multisubunit protein complex mainly involved in tethering secretory vesicles to the plasma membrane and implicated in the regulation of multiple biological processes modulated by vesicle trafficking. Interestingly, Rabs and exocyst complex belong to the same protein network. Our experimental evidence indicates that LRRK2 kinase activity or the presence of the LRRK2 kinase domain regulate the assembly of exocyst subunits and that the over-expression of Sec8 significantly rescues the LRRK2 G2019S mutant pathological effect. Our findings strongly suggest an interesting molecular mechanism by which LRRK2 could modulate vesicle trafficking and may have important implications to decode the complex role that LRRK2 plays in neuronal physiology.

HDAC1 inhibition ameliorates TDP-43-induced cell death in vitro and in vivo.

TDP-43 pathology is a disease hallmark that characterizes both amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP). TDP-43 undergoes several posttranslational modifications that can change its biological activities and its aggregative propensity, which is a common hallmark of different neurodegenerative conditions. New evidence is provided by the current study pointing at TDP-43 acetylation in ALS cellular models. Using both in vitro and in vivo approaches, we demonstrate that TDP-43 interacts with histone deacetylase 1 (HDAC1) via RRM1 and RRM2 domains, that are known to contain the two major TDP-43 acetylation sites, K142 and K192. Moreover, we show that TDP-43 is a direct transcriptional activator of CHOP promoter and this activity is regulated by acetylation. Finally and most importantly, we observe both in cell culture and in Drosophila that a HDCA1 reduced level (genomic inactivation or siRNA) or treatment with pan-HDAC inhibitors exert a protective role against WT or pathological mutant TDP-43 toxicity, suggesting TDP-43 acetylation as a new potential therapeutic target. HDAC inhibition efficacy in neurodegeneration has long been debated, but future investigations are warranted in this area. Selection of more specific HDAC inhibitors is still a promising option for neuronal protection especially as HDAC1 appears as a downstream target of both TDP- 43 and FUS, another ALS-related gene.

Levetiracetam treatment ameliorates LRRK2 pathological mutant phenotype.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson's disease (PD). The LRRK2 physiological and pathological function is still debated. However, different experimental evidence based on LRRK2 cellular localization and LRRK2 protein interactors suggests that LRRK2 may be part and regulate a protein network modulating vesicle dynamics/trafficking. Interestingly, the synaptic vesicle protein SV2A is part of this protein complex. Importantly, SV2A is the binding site of the levetiracetam (LEV), a compound largely used in human therapy for epilepsy treatment. The binding of LEV to SV2A reduces the neuronal firing by the modulation of vesicle trafficking although by an unclear molecular mechanism. In this short communication, we have analysed the interaction between the LRRK2 and SV2A pathways by LEV treatment. Interestingly, LEV significantly counteracts the effect of LRRK2 G2019S pathological mutant expression in three different cellular experimental models. Our data strongly suggest that LEV treatment may have a neuroprotective effect on LRRK2 pathological mutant toxicity and that LEV repositioning could be a viable compound for PD treatment.

Epigenetic Changes Associated with the Expression of Amyotrophic Lateral Sclerosis (ALS) Causing Genes.

Neurodegenerative disorders, including Amyotrophic Lateral Sclerosis (ALS), have been associated to alterations in chromatin structure resulting in long-lasting changes in gene expression. ALS is predominantly a sporadic disease and environmental triggers may be involved in its onset. In this respect, alterations in the epigenome can provide the key to transform the genetic information into phenotype. In this paper, we demonstrate that two modifications associated with transcriptional activation, namely dimethylation of lysine 4 on H3 tail (H3K4me2) and phospho-acetylation of serine 10 and lysine 14 on H3 tail (H3K14ac-S10ph), and two modifications associated to transcriptional repression, namely trimethylation of lysine 9 on H3 tail (H3K9me3) and DNA methylation are selectively altered in cellular and animal model of ALS. These results reinforce the idea that epigenetic therapy may represent a potential and attractive approach for ALS treatment.

Role of LRRK2 in the regulation of dopamine receptor trafficking.

Mutations in LRRK2 play a critical role in both familial and sporadic Parkinson's disease (PD). Up to date, the role of LRRK2 in PD onset and progression remains largely unknown. However, experimental evidence highlights a critical role of LRRK2 in the control of vesicle trafficking that in turn may regulate different aspects of neuronal physiology. We have analyzed the role of LRRK2 in regulating dopamine receptor D1 (DRD1) and D2 (DRD2) trafficking. DRD1 and DRD2 are the most abundant dopamine receptors in the brain. They differ in structural, pharmacological and biochemical properties, as well as in localization and internalization mechanisms. Our results indicate that disease-associated mutant G2019S LRRK2 impairs DRD1 internalization, leading to an alteration in signal transduction. Moreover, the mutant forms of LRRK2 affect receptor turnover by decreasing the rate of DRD2 trafficking from the Golgi complex to the cell membrane. Collectively, our findings are consistent with the conclusion that LRRK2 influences the motility of neuronal vesicles and the neuronal receptor trafficking. These findings have important implications for the complex role that LRRK2 plays in neuronal physiology and the possible pathological mechanisms that may lead to neuronal death in PD.

Redox-sensitive GFP to monitor oxidative stress in neurodegenerative diseases.

Redox processes are key events in the degenerative cascade of many adult-onset neurodegenerative diseases (NDs), but the biological relevance of a single redox change is often dependent on the redox couple involved and on its subcellular origin. The biosensors based on engineered fluorescent proteins (redox-sensitive GFP [roGFP]) offer a unique opportunity to monitor redox changes in both physiological and pathological contexts in living animals and plants. Here, we review the use of roGFPs to monitor oxidative stress in different three adult-onset NDs: Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). Despite the many differences spanning from incidence to onset, the hypotheses on biological processes underlying both sporadic and familiar ND forms in humans outline a model in which noncompeting mechanisms are likely to converge in various unsuccessful patterns to mediate the selective degeneration of a specific neuronal population. roGFPs, targeted to different cell compartments, are successfully used as specific markers of cell toxicity, induced by expression of causative genes linked to a determined ND. We also report the use of roGFP to monitor oxidative stress induced by the expression of the ALS-causative gene SOD1.

Single-step green synthesis and characterization of gold-conjugated polyphenol nanoparticles with antioxidant and biological activities.

BACKGROUND: Gold nanoparticles (GNPs) are likely to provide an attractive platform for combining a variety of biophysicochemical properties into a unified nanodevice with great therapeutic potential. In this study we investigated the capabilities of three different natural polyphenols, epigallocatechin-3-gallate (EGCG), resveratrol (RSV), and fisetin (FS), to allow synergistic chemical reduction of gold salts to GNPs and stabilization in a single-step green process. Moreover, antioxidant properties of the nanosystems, as well as preliminary antiproliferative activity and apoptotic process investigation of model EGCG-GNPs on stable clones of neuroblastoma SH-SY5Y cells expressing CFP-DEVD-YFP reporter, were examined. METHODS: The GNPs were characterized by physicochemical techniques, polyphenol content, and in vitro stability. The antioxidant activity of the GNPs was also determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) cation (ABTS) radical-scavenging assays. Stable clones of neuronal SH-SY5Y-CFP-DEVD-YFP were generated and characterized, and cell viability after treatment with EGCG-GNPs was assessed after 72 hours through a 3(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. Activation of the apoptotic pathways was also investigated by Western blot analysis. RESULTS: With a diameter in the size range of 10-25 nm, the obtained nanoparticles (NPs) were found to contain 2.71%, 3.23%, and 5.47% of EGCG, RSV, and FS, respectively. Nanoprototypes exhibited remarkable in vitro stability in various media, suggesting that NP surface coating with phytochemicals prevents aggregation in different simulated physiological conditions. The scavenging activities for DPPH and ABTS were highly correlated with EGCG, RSV, and FS content. Moreover, high correlation coefficients between the ABTS and DPPH values were found for the prepared nanosystems. EGCG-GNPs induce a dose-dependent reduction on SH-SY5Y-CFP-DEVD-YFP cell viability that is likely to involve the activation of the apoptotic pathways, similarly to free EGCG, as suggested by the processing of the CFP-DEVD-YFP reporter. CONCLUSION: These results prompted us to propose the ecofriendly synthesized EGCG-, RSV-, and FS-based nanogold conjugates as suitable carriers for bioactive polyphenols to be used for the treatment of disorders associated with oxidative stress, including neurodegenerative disorders, cardiovascular disease, and cancer.

Differential protein-protein interactions of LRRK1 and LRRK2 indicate roles in distinct cellular signaling pathways.

Genetic studies show that LRRK2, and not its closest paralogue LRRK1, is linked to Parkinson's disease. To gain insight into the molecular and cellular basis of this discrepancy, we searched for LRRK1- and LRRK2-specific cellular processes by identifying their distinct interacting proteins. A protein microarray-based interaction screen was performed with recombinant 3xFlag-LRRK1 and 3xFlag-LRRK2 and, in parallel, co-immunoprecipitation followed by mass spectrometry was performed from SH-SY5Y neuroblastoma cell lines stably expressing 3xFlag-LRRK1 or 3xFlag-LRRK2. We identified a set of LRRK1- and LRRK2-specific as well as common interactors. One of our most prominent findings was that both screens pointed to epidermal growth factor receptor (EGF-R) as a LRRK1-specific interactor, while 14-3-3 proteins were LRRK2-specific. This is consistent with phosphosite mapping of LRRK1, revealing phosphosites outside of 14-3-3 consensus binding motifs. To assess the functional relevance of these interactions, SH-SY5Y-LRRK1 and -LRRK2 cell lines were treated with LRRK2 kinase inhibitors that disrupt 14-3-3 binding, or with EGF, an EGF-R agonist. Redistribution of LRRK2, not LRRK1, from diffuse cytoplasmic to filamentous aggregates was observed after inhibitor treatment. Similarly, EGF induced translocation of LRRK1, but not of LRRK2, to endosomes. Our study confirms that LRRK1 and LRRK2 can carry out distinct functions by interacting with different cellular proteins. LRRK1 and LRRK2 (leucine-rich repeat kinase) interaction partners were identified by two different protein-protein interaction screens. These confirmed epidermal growth factor receptor (EGR-R) as a LRRK1-specific interactor, while 14-3-3 proteins were LRRK2-specific. Functional analysis of these interactions and the pathways they mediate shows that LRRK1 and LRRK2 signaling do not intersect, reflective of the differential role of both LRRKs in Parkinson's disease.

LRRK2 affects vesicle trafficking, neurotransmitter extracellular level and membrane receptor localization.

The leucine-rich repeat kinase 2 (LRRK2) gene was found to play a role in the pathogenesis of both familial and sporadic Parkinson's disease (PD). LRRK2 encodes a large multi-domain protein that is expressed in different tissues. To date, the physiological and pathological functions of LRRK2 are not clearly defined. In this study we have explored the role of LRRK2 in controlling vesicle trafficking in different cellular or animal models and using various readouts. In neuronal cells, the presence of LRRK2(G2019S) pathological mutant determines increased extracellular dopamine levels either under basal conditions or upon nicotine stimulation. Moreover, mutant LRRK2 affects the levels of dopamine receptor D1 on the membrane surface in neuronal cells or animal models. Ultrastructural analysis of PC12-derived cells expressing mutant LRRK2(G2019S) shows an altered intracellular vesicle distribution. Taken together, our results point to the key role of LRRK2 to control vesicle trafficking in neuronal cells.

LRRK2 and vesicle trafficking.

Mutations in LRRK2 (leucine-rich repeat kinase 2) (also known as PARK8 or dardarin) are responsible for the autosomal-dominant form of PD (Parkinson's disease). LRRK2 mutations were found in approximately 3-5% of familial and 1-3% of sporadic PD cases with the highest prevalence (up to 40%) in North Africans and Ashkenazi Jews. To date, mutations in LRRK2 are a major genetic risk factor for familial and sporadic PD. Despite the fact that 8 years have passed from the establishment of the first link between PD and dardarin in 2004, the pathophysiological role of LRRK2 in PD onset and progression is far from clearly defined. Also the generation of different LRRK2 transgenic or knockout animals has not provided new hints on the function of LRRK2 in the brain. The present paper reviews recent evidence regarding a potential role of LRRK2 in the regulation of membrane trafficking from vesicle generation to the movement along cytoskeleton and finally to vesicle fusion with cell membrane.

Bcl2-A1 interacts with pro-caspase-3: implications for amyotrophic lateral sclerosis.

Expression of mutant SOD1 typical of familial amyotrophic lateral sclerosis (ALS) induces the expression of Bcl2-A1, a member of the Bcl2 family of proteins, specifically in motor neurons of transgenic mice. In this work, we have used immortalized motor neurons (NSC-34) and transgenic mice expressing mutant SOD1 to unravel the molecular mechanisms and the biological meaning of this up-regulation. We report that up-regulation of Bcl2-A1 by mutant SOD1 is mediated by activation of the redox sensitive transcription factor AP1 and that Bcl2-A1 interacts with pro-caspase-3 via its C-terminal helix α9. Furthermore, Bcl2-A1 inhibits pro-caspase-3 activation in immortalized motor neurons expressing mutant SOD1 and thus induction of Bcl2-A1 in ALS mice represents a pro-survival strategy aimed at counteracting the toxic effects of mutant SOD1. These data provide significant new insights on how molecular signaling, driven by expression of the ALS-causative gene SOD1, affects regulation of apoptosis in motor neurons and thus may have implications for ALS therapy, where prevention of motor neuronal cell death is one of the major aims.

Prune melanoidins protect against oxidative stress and endothelial cell death.

The health-promoting effects of fruit and vegetable consumption are thought to be due to phytochemicals contained in fresh plant material. Whether processed plant foods provide the same benefits as unprocessed ones is an open question. Melanoidins from heat-processed plums (prunes) were isolated and their presence confirmed by hydroxymethylfurfural content and browning index. Oxidative-induced endothelial cell (EC) damage is the trigger for the development of cardiovascular diseases (CVD); therefore the potential protective effect of prune melanoidins on hydrogen peroxide-induced oxidative cell damage was investigated on human endothelial ECV304 cells. Cytoplasmic and mitochondrial redox status was assessed by using the novel, redox-sensitive, ratiometric fluorescent protein sensor (roGFP), while mitochondrial membrane potential (MMP) was investigated with the fluorescent dye, JC-1. Treatment of ECV304 cells with hydrogen peroxide dose-dependently induced both mitochondrial and cytoplasmic oxidation, in addition to MMP dissipation, with ensuing cell death. Pretreatment of ECV304 with prune melanoidins, significantly counteracted and ultimately abolished hydrogen peroxide elicited phenomena, clearly indicating that these polymers protect human EC against oxidative stress.

Astroglial inhibition of NF-κB does not ameliorate disease onset and progression in a mouse model for amyotrophic lateral sclerosis (ALS).

Motor neuron death in amyotrophic lateral sclerosis (ALS) is considered a "non-cell autonomous" process, with astrocytes playing a critical role in disease progression. Glial cells are activated early in transgenic mice expressing mutant SOD1, suggesting that neuroinflammation has a relevant role in the cascade of events that trigger the death of motor neurons. An inflammatory cascade including COX2 expression, secretion of cytokines and release of NO from astrocytes may descend from activation of a NF-κB-mediated pathway observed in astrocytes from ALS patients and in experimental models. We have attempted rescue of transgenic mutant SOD1 mice through the inhibition of the NF-κB pathway selectively in astrocytes. Here we show that despite efficient inhibition of this major pathway, double transgenic mice expressing the mutant SOD1(G93A) ubiquitously and the dominant negative form of IκBα (IκBαAA) in astrocytes under control of the GFAP promoter show no benefit in terms of onset and progression of disease. Our data indicate that motor neuron death in ALS cannot be prevented by inhibition of a single inflammatory pathway because alternative pathways are activated in the presence of a persistent toxic stimulus.

Oligomerization of mutant SOD1 in mitochondria of motoneuronal cells drives mitochondrial damage and cell toxicity.

Increasing evidence indicates that the accumulation and aggregation of mutant Cu,Zn superoxide dismutase (mutSOD1) in spinal cord mitochondria is implicated in the pathogenesis of familial amyotrophic lateral sclerosis (FALS). Although the mechanisms underlying this effect are only partially understood, a deficit in the import mechanism of mutSOD1 and/or in its folding and maturation inside mitochondria is likely involved. To investigate this issue, we overexpressed mitochondria-targeted wild-type and mutSOD1s in neuronal cell lines. Mitochondria-targeted G93A mutSOD1 induces a significant impairment of mitochondrial morphology and metabolism, resulting in caspase-3 activation and cell death. These effects are paralleled by the formation of disulfide-linked, insoluble oligomers of mutSOD1 inside mitochondria. Overexpression of the copper chaperone for SOD1 (CCS) improves the solubility of cytosolic mutSOD1s, but has no effect or even worsens the insolubility of mitochondria-targeted G93A mutSOD1, indicating that CCS may increase the availability of an aggregating form of mutSOD1. Interestingly, prevention of the formation of such aggregates by removal of disulfide-bonded cysteines counteracts the effects produced by mutSOD1 accumulated inside mitochondria. Overall, our results demonstrate for the first time that aggregation of mutSOD1s into mitochondria is important for mutSOD1 to induce damage, although other forms of misfolded SOD1s might be involved.