Stress response mechanisms in protein misfolding diseases: Profiling a cellular model of Huntington's disease.

Stress response pathways like the integrated stress response (ISR), the mitochondrial unfolded protein response (UPRmt) and the heat shock response (HSR) have emerged as part of the pathophysiology of neurodegenerative diseases, including Huntington's disease (HD) - a currently incurable disease caused by the production of mutant huntingtin (mut-Htt). Previous data from HD patients suggest that ISR is activated while UPRmt and HSR are impaired in HD. The study of these stress response pathways as potential therapeutic targets in HD requires cellular models that mimic the activation status found in HD patients of such pathways. PC12 cells with inducible expression of the N-terminal fragment of mut-Htt are among the most used cell lines to model HD, however the activation of stress responses remains unclear in this model. The goal of this study is to characterize the activation of ISR, UPRmt and HSR in this HD cell model and evaluate if it mimics the activation status found in HD patients. We show that PC12 HD cell model presents reduced levels of Hsp90 and mitochondrial chaperones, suggesting an impaired activation or function of HSR and UPRmt. This HD model also presents increased levels of phosphorylated eIF2α, the master regulator of the ISR, but overall similar levels of ATF4 and decreased levels of CHOP - transcription factors downstream to eIF2α - in comparison to control, suggesting an initial activation of ISR. These results show that this model mimics the ISR activation and the impaired UPRmt and HSR found in HD patients. This work suggests that the PC12 N-terminal HD model is suitable for studying the role of stress response pathways in the pathophysiology of HD and for exploratory studies investigating the therapeutic potential of drugs targeting stress responses.

Does the antidepressant sertraline show chronic effects on aquatic invertebrates at environmentally relevant concentrations? A case study with the keystone amphipod, Gammarus locusta.

The increasing use of Sertraline (SER) as antidepressant and its consequent presence in the aquatic environment is raising concern about the chronic effects of this pharmaceutical to aquatic organisms. As the current concentrations of SER in surface waters are typically in the low ng/L range, acute toxicity is unlikely to occur. However, prolonged exposure to low concentrations of SER may lead to sub-lethal effects in aquatic organisms, including alterations in important physiological functions like growth, reproduction, behaviour, and also in key biochemical processes, such as those associated with neurotransmission and redox balance. To test this hypothesis, we selected the amphipod Gammarus locusta, a keystone species used in ecotoxicological hazard assessment. In the present study, juveniles' G. locusta from a permanent laboratory culture were chronically exposed to low concentrations of SER (8-1000 ng/L) in a bioassay that lasted for 48 days, allowing for a life-cycle study including effects on reproduction. At the lowest SER concentrations with environmental relevance (8, 40 and 200 ng/L) we detected no significant changes in key ecological endpoints such as survival, growth, reproduction and movement behaviour, or in any of the biochemical markers analysed. However, at 1000 ng/L SER (a concentration one order of magnitude higher than the levels reported in aquatic environments) females showed a significant increase in movement versus control, whereas no activity changes were observed in males. Overall, these findings indicate that G. locusta females are potentially more susceptible to the chronic effects of SER. Moreover, the current environmental SER concentrations are unlikely to affect amphipod's ecological endpoints because only SER concentrations higher than the levels reported in aquatic environments produced effects on the behaviour of G. locusta females. However, the increasing consumption of SER, highlights the importance of monitoring its chronic risk to the aquatic wildlife.

Chronic effects of triclocarban in the amphipod Gammarus locusta: Behavioural and biochemical impairment.

Triclocarban (TCC), a common antimicrobial agent widely used in many household and personal care products, has been widely detected in aquatic ecosystems worldwide. Due to its high lipophilicity and persistence in the aquatic ecosystems, TCC is of emerging environmental concern. Despite the frequently reported detection of TCC in the environment and significant uncertainties about its long term effects on aquatic ecosystems, few studies have addressed the chronic effects of TCC in aquatic organisms at ecologically relevant concentrations. Therefore, we aimed at testing a broad range of biological responses in the amphipod Gammarus locusta following a chronic (60 days) exposure to environmentally relevant concentrations of TCC (100, 500 and 2500ng/L). This work integrated biochemical markers of oxidative stress (catalase (CAT), glutathione-s-transferase (GST) and lipid peroxidation (LPO)) and neurotransmission (acetylcholinesterase (AChE)) with several key ecological endpoints, i.e. behaviour, survival, individual growth and reproduction. Significant alterations were observed in all biochemical markers. While AChE showed a dose-response curve (with a significant increased activity at a TCC concentration of 2500ng/L), oxidative stress markers did not follow a dose-response curve, with significant increase at 100 and/or 500ng/L and a decreased activity in the highest concentration (2500ng/L). The same effect was observed in the females' behavioural response, whereas males' behaviour was not affected by TCC exposure. The present study represents a first approach to characterize the hazard of TCC to crustaceans.

HDAC6 inhibition induces mitochondrial fusion, autophagic flux and reduces diffuse mutant huntingtin in striatal neurons.

Striatal neurons are vulnerable to Huntington's disease (HD). Decreased levels of acetylated alpha-tubulin and impaired mitochondrial dynamics, such as reduced motility and excessive fission, are associated with HD; however, it remains unclear whether and how these factors might contribute to the preferential degeneration of striatal neurons. Inhibition of the alpha-tubulin deacetylase HDAC6 has been proposed as a therapeutic strategy for HD, but remains controversial - studies in neurons show improved intracellular transport, whereas studies in cell-lines suggest it may impair autophagosome-lysosome fusion, and reduce clearance of mutant huntingtin (mHtt) and damaged mitochondria (mitophagy). Using primary cultures of rat striatal and cortical neurons, we show that mitochondria are intrinsically less motile and more balanced towards fission in striatal than in cortical neurons. Pharmacological inhibition of the HDAC6 deacetylase activity with tubastatin A (TBA) increased acetylated alpha-tubulin levels, and induced mitochondrial motility and fusion in striatal neurons to levels observed in cortical neurons. Importantly, TBA did not block neuronal autophagosome-lysosome fusion, and did not change mitochondrial DNA levels, suggesting no impairment in autophagy or mitochondrial clearance. Instead, TBA increased autophagic flux and reduced diffuse mHtt in striatal neurons, possibly by promoting transport of initiation factors to sites of autophagosomal biogenesis. This study identifies the pharmacological inhibition of HDAC6 deacetylase activity as a potential strategy to reduce the vulnerability of striatal neurons to HD.

Mitochondrial dynamics and quality control in Huntington's disease.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by polyglutamine expansion mutations in the huntingtin protein. Despite its ubiquitous distribution, expression of mutant huntingtin (mHtt) is particularly detrimental to medium spiny neurons within the striatum. Mitochondrial dysfunction has been associated with HD pathogenesis. Here we review the current evidence for mHtt-induced abnormalities in mitochondrial dynamics and quality control, with a particular focus on brain and neuronal data pertaining to striatal vulnerability. We address mHtt effects on mitochondrial biogenesis, protein import, complex assembly, fission and fusion, mitochondrial transport, and on the degradation of damaged mitochondria via autophagy (mitophagy). For an integrated perspective on potentially converging pathogenic mechanisms, we also address impaired autophagosomal transport and abnormal mHtt proteostasis in HD.

Pharmacological modulation of HDAC1 and HDAC6 in vivo in a zebrafish model: Therapeutic implications for Parkinson's disease.

Histone deacetylases (HDACs) are key epigenetic enzymes and emerging drug targets in cancer and neurodegeneration. Pan-HDAC inhibitors provided neuroprotection in Parkinson's Disease (PD) models, however, the HDAC isoforms with highest neuroprotective potential remain unknown. Zebrafish larvae (powerful pharmacological testing tools bridging cellular and in vivo studies) have thus far been used in PD modelling with limited phenotypic characterization. Here we characterize the behavioural and metabolic phenotypes of a zebrafish PD model induced with MPP(+), assess the feasibility of targeting zebrafish HDAC1 and HDAC6 isoforms, and test the in vivo effects of their selective inhibitors MS-275 and tubastatin A, respectively. MPP(+) induced a concentration-dependent decrease in metabolic activity and sensorimotor reflexes, and induced locomotor impairments rescuable by the dopaminergic agonist apomorphine. Zebrafish HDAC1 and HDAC6 isoforms show high sequence identity with mammalian homologues at the deacetylase active sites, and pharmacological inhibition increased acetylation of their respective histone and tubulin targets. MS-275 and tubastatin rescued the MPP(+)-induced decrease in diencephalic tyrosine hydroxylase immunofluorescence and in whole-larvae metabolic activity, without modifying mitochondrial complex activity or biogenesis. MS-275 or tubastatin alone modulated spontaneous locomotion. When combined with MPP(+), however, neither MS-275 nor tubastatin rescued locomotor impairments, although tubastatin did ameliorate the head-reflex impairment. This study demonstrates the feasibility of pharmacologically targeting the zebrafish HDAC1 and HDAC6 isoforms, and indicates that their inhibition can rescue cellular metabolism in a PD model. Absence of improvement in locomotion, however, suggests that monotherapy with either HDAC1 or HDAC6 inhibitors is unlikely to provide strong benefits in PD. This study highlights parameters dependent on the integrity of zebrafish neuronal circuits as a valuable complement to cell-based studies. Also, the demonstrated feasibility of pharmacologically targeting HDAC1 and HDAC6 in this organism paves the way for future studies investigating HDAC inhibitors in other diseases modelled in zebrafish.

Mutation of the human mitochondrial phenylalanine-tRNA synthetase causes infantile-onset epilepsy and cytochrome c oxidase deficiency.

Mitochondrial aminoacyl-tRNA synthetases (aaRSs) are essential enzymes in protein synthesis since they charge tRNAs with their cognate amino acids. Mutations in the genes encoding mitochondrial aaRSs have been associated with a wide spectrum of human mitochondrial diseases. Here we report the identification of pathogenic mutations (a partial genomic deletion and a highly conserved p. Asp325Tyr missense variant) in FARS2, the gene encoding mitochondrial phenylalanyl-tRNA synthetase, in a patient with early-onset epilepsy and isolated complex IV deficiency in muscle. The biochemical defect was expressed in myoblasts but not in fibroblasts and associated with decreased steady state levels of COXI and COXII protein and reduced steady state levels of the mt-tRNA(Phe) transcript. Functional analysis of the recombinant mutant p. Asp325Tyr FARS2 protein showed an inability to bind ATP and consequently undetectable aminoacylation activity using either bacterial tRNA or human mt-tRNA(Phe) as substrates. Lentiviral transduction of cells with wildtype FARS2 restored complex IV protein levels, confirming that the p.Asp325Tyr mutation is pathogenic, causing respiratory chain deficiency and neurological deficits on account of defective aminoacylation of mt-tRNA(Phe).

Lysine deacetylases and mitochondrial dynamics in neurodegeneration.

Lysine acetylation is a key post-translational modification known to regulate gene transcription, signal transduction, cellular transport and metabolism. Lysine deacetylases (KDACs), including classical KDACs (a.k.a. histone deacetylases; HDACs) and sirtuins (SIRTs), are emerging therapeutic targets in neurodegeneration. Given the strong link between abnormal mitochondrial dynamics and neurodegenerative disorders (e.g. in Alzheimer, Parkinson and Huntington diseases), here we examine the evidence for KDAC-mediated regulation of mitochondrial biogenesis, fission-fusion, movement and mitophagy. Mitochondrial biogenesis regulation was reported for SIRT1, SIRT3, and class IIa KDACs, mainly via PGC-1alpha modulation. SIRT1 or SIRT3 overexpression rescued mitochondrial density and fission-fusion balance in neurodegeneration models. Mitochondrial fission decreased with pan-classical-KDAC inhibitors and increased with nicotinamide (pan-sirtuin-inhibitor/activator depending on concentration and NAD(+) conversion). Mitochondrial movement increased with HDAC6 inhibition, but this is not yet reported for the other tubulin deacetylase SIRT2. Inhibition of HDAC6 or SIRT2 was reported neuroprotective. Mitophagy is assisted by the HDAC6 ubiquitin-binding and autophagosome-lysosome fusion promoting activities, and was also associated with SIRT1 activation. In summary, KDACs can potentially modulate multiple components of mitochondrial dynamics, however, several key points require clarification. The SIRT1-biogenesis connection relies heavily in controversial caloric restriction (CR) regimes or CR-mimetic drugs, and appears cell-type dependent, recommending caution before linking SIRT1 activation with general neuroprotection. Future studies should clarify mitochondrial fission-fusion regulation by KDACs, and the interplay between HDAC6 and SIRT1 in mitophagy. Also, further studies are required to ascertain whether HDAC6 inhibition to enhance mitochondrial trafficking does not compromise autophagy or clearance of misfolded proteins in neurodegenerative disorders.

Edaravone counteracts redox and metabolic disruptions in an emerging zebrafish model of sporadic ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease in which the death of motor neurons leads to loss of muscle function. Additionally, cognitive and circadian disruptions are common in ALS patients, contributing to disease progression and burden. Most ALS cases are sporadic, and environmental exposures contribute to their aetiology. However, animal models of these sporadic ALS cases are scarce. The small vertebrate zebrafish is a leading organism to model neurodegenerative diseases; previous studies have proposed bisphenol A (BPA) or ß-methylamino-l-alanine (BMAA) exposure to model sporadic ALS in zebrafish, damaging motor neurons and altering motor responses. Here we characterise the face and predictive validity of sporadic ALS models, showing their potential for the mechanistic study of ALS drugs. We phenotypically characterise the BPA and BMAA-induced models, going beyond motor activity and motor axon morphology, to include circadian, redox, proteostasis, and metabolomic phenotypes, and assessing their predictive validity for ALS modelling. BPA or BMAA exposure induced concentration-dependent activity impairments. Also, exposure to BPA but not BMAA induced motor axonopathy and circadian alterations in zebrafish larvae. Our further study of the BPA model revealed loss of habituation to repetitive startles, increased oxidative damage, endoplasmic reticulum (ER) stress, and metabolome abnormalities. The BPA-induced model shows predictive validity, since the approved ALS drug edaravone counteracted BPA-induced motor phenotypes, ER stress, and metabolic disruptions. Overall, BPA exposure is a promising model of ALS-related redox and ER imbalances, contributing to fulfil an unmet need for validated sporadic ALS models.

PERK inhibition in zebrafish mimics human Wolcott-Rallison syndrome phenotypes.

Wolcott-Rallison Syndrome (WRS) is the most common cause of permanent neonatal diabetes mellitus among consanguineous families. The diabetes associated with WRS is non-autoimmune, insulin-requiring and associated with skeletal dysplasia and growth retardation. The therapeutic options for WRS patients rely on permanent insulin pumping or on invasive transplants of liver and pancreas. WRS has a well identified genetic cause: loss-of-function mutations in the gene coding for an endoplasmic reticulum kinase named PERK (protein kinase R-like ER kinase). Currently, WRS research is facilitated by cellular and rodent models with PERK ablation. While these models have unique strengths, cellular models incompletely replicate the organ/system-level complexity of WRS, and rodents have limited scalability for efficiently screening potential therapeutics. To address these challenges, we developed a new in vivo model of WRS by pharmacologically inhibiting PERK in zebrafish. This small vertebrate displays high fecundity, rapid development of organ systems and is amenable to highly efficient in vivo drug testing. PERK inhibition in zebrafish produced typical WRS phenotypes such as glucose dysregulation, skeletal defects, and impaired development. PERK inhibition in zebrafish also produced broad-spectrum WRS phenotypes such as impaired neuromuscular function, compromised cardiac function and muscular integrity. These results show that zebrafish holds potential as a versatile model to study WRS mechanisms and contribute to the identification of promising therapeutic options for WRS.

Swimming against ALS: How to model disease in zebrafish for pathophysiological and behavioral studies.

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease that leads to progressive disability and motor impairment. Existing therapies provide modest improvements in patient survival, raising a need for new treatments for ALS. Zebrafish is a promising model animal for translational and fundamental research in ALS - it is an experimentally tractable vertebrate, with high homology to humans and an ample experimental toolbox. These advantages allow high-throughput study of behavioral and pathophysiological phenotypes. The last decade saw an increased interest in modelling ALS in zebrafish, leading to the current abundance and variety of available methods and models. Additionally, the rise of gene editing techniques and toxin combination studies has created novel opportunities for ALS studies in zebrafish. In this review, we address the relevance of zebrafish as a model animal for ALS studies, the strategies for model induction and key phenotypical evaluation. Furthermore, we discuss established and emerging zebrafish models of ALS, analyzing their validity, including their potential for drug testing, and highlighting research opportunities in this area.

The PERKs of mitochondria protection during stress: insights for PERK modulation in neurodegenerative and metabolic diseases.

Protein kinase RNA-like ER kinase (PERK) is an endoplasmic reticulum (ER) stress sensor that responds to the accumulation of misfolded proteins. Once activated, PERK initiates signalling pathways that halt general protein production, increase the efficiency of ER quality control, and maintain redox homeostasis. PERK activation also protects mitochondrial homeostasis during stress. The location of PERK at the contact sites between the ER and the mitochondria creates a PERK-mitochondria axis that allows PERK to detect stress in both organelles, adapt their functions and prevent apoptosis. During ER stress, PERK activation triggers mitochondrial hyperfusion, preventing premature apoptotic fragmentation of the mitochondria. PERK activation also increases the formation of mitochondrial cristae and the assembly of respiratory supercomplexes, enhancing cellular ATP-generating capacity. PERK strengthens mitochondrial quality control during stress by promoting the expression of mitochondrial chaperones and proteases and by increasing mitochondrial biogenesis and mitophagy, resulting in renewal of the mitochondrial network. But how does PERK mediate all these changes in mitochondrial homeostasis? In addition to the classic PERK-eukaryotic translation initiation factor 2α (eIF2α)-activating transcription factor 4 (ATF4) pathway, PERK can activate other protective pathways - PERK-O-linked N-acetyl-glucosamine transferase (OGT), PERK-transcription factor EB (TFEB), and PERK-nuclear factor erythroid 2-related factor 2 (NRF2) - contributing to broader regulation of mitochondrial dynamics, metabolism, and quality control. The pharmacological activation of PERK is protective in models of neurodegenerative and metabolic diseases, such as Huntington's disease, progressive supranuclear palsy and obesity, while the inhibition of PERK was protective in models of Parkinson's and prion diseases and diabetes. In this review, we address the molecular mechanisms by which PERK regulates mitochondrial dynamics, metabolism and quality control, and discuss the therapeutic potential of targeting PERK in neurodegenerative and metabolic diseases.

Disruptions of circadian rhythms, sleep, and stress responses in zebrafish: New infrared-based activity monitoring assays for toxicity assessment.

Behavioural disruptions are sensitive indicators of alterations to normal animal physiology and can be used for toxicity assessment. The small vertebrate zebrafish is a leading model organism for toxicological studies. The ability to continuously monitor the toxicity of drugs, pollutants, or environmental changes over several days in zebrafish can have high practical application. Although video-recordings can be used to monitor short-term zebrafish behaviour, it is challenging to videorecord prolonged experiments (e.g. circadian behaviour over several days) because of the darkness periods (nights) and the heavy data storage and image processing requirements. Alternatively, infrared-based activity monitors, widely used in invertebrate models such as drosophila, generate simple and low-storage data and could optimize large-scale prolonged behavioural experiments in zebrafish, thus favouring the implementation of high-throughput testing strategies. Here, we validate the use of a Locomotor Activity Monitor (LAM) to study the behaviour of zebrafish larvae, and we characterize the behavioural phenotypes induced by abnormal light conditions and by the Parkinsonian toxin MPP+. When zebrafish were deprived from daily light-cycle synchronization, the LAM detected various circadian disruptions, such as increased activity period, phase shifts, and decreased inter-daily stability. Zebrafish exposed to MPP+ (10, 100, 500 µM) showed a concentration-dependent decrease in activity, sleep disruptions, impaired habituation to repetitive startles (visual-motor responses), and a slower recovery to normal activity after the startle-associated stress. These phenotypes evidence the feasibility of using infrared-based LAM to assess multi-parameter behavioural disruptions in zebrafish. The procedures in this study have wide applicability and may yield standard methods for toxicity testing.

Automated analysis of activity, sleep, and rhythmic behaviour in various animal species with the Rtivity software.

Behavioural studies provide insights into normal and disrupted biological mechanisms. In many research areas, a growing spectrum of animal models-particularly small organisms-is used for high-throughput studies with infrared-based activity monitors, generating counts per time data. The freely available software to analyse such data, however, are primarily optimized for drosophila and circadian analysis. Researchers investigating other species or non-circadian behaviour would thus benefit from a more versatile software. Here we report the development of a free and open-source software-Rtivity-allowing customisation of species-specific parameters, and offering a versatile analysis of behavioural patterns, biological rhythms, stimulus responses, and survival. Rtivity is based on the R language and uses Shiny and the recently developed Rethomics package for a user-friendly graphical interface without requiring coding skills. Rtivity automatically assesses survival, computes various activity, sleep, and rhythmicity parameters, and performs fractal analysis of activity fluctuations. Rtivity generates multiple informative graphs, and exports structured data for efficient interoperability with common statistical software. In summary, Rtivity facilitates and enhances the versatility of the behavioural analysis of diverse animal species (e.g. drosophila, zebrafish, daphnia, ants). It is thus suitable for a broad range of researchers from multidisciplinary fields such as ecology, neurobiology, toxicology, and pharmacology.

Allosteric activation of Hsp70 reduces mutant huntingtin levels, the clustering of N-terminal fragments, and their nuclear accumulation.

AIMS: Huntington's disease (HD) is caused by a mutant huntingtin protein that misfolds, yields toxic N-terminal fragments, aggregates, and disrupts proteostasis. The Hsp70 chaperone is a potential therapeutic target as it prevents proteotoxicity by favouring protein folding, disaggregation, or degradation. We tested the hypothesis that allosteric Hsp70 activation with a pharmacological mimetic of the Hsp70 co-chaperone Hip, YM-1, could modulate huntingtin proteostasis. MAIN METHODS: We used HD cell models expressing either N-terminal or full-length huntingtin. Using single-cell analysis we studied huntingtin aggregation in different cellular compartments by fluorescence microscopy. Protein interaction was evaluated by immunoprecipitation, while protein levels were quantified by immunofluorescence and western-blot. KEY FINDINGS: N-terminal huntingtin interacted with Hsp70 and increased its levels. Treatment with YM-1 reduced N-terminal huntingtin clustering and nuclear aggregation. Full-length mutant huntingtin also interacted with Hsp70, and treatment with YM-1 reduced huntingtin levels when combined with Hsp70 induction by heat shock. Mechanistically, YM-1 increases the Hsp70 affinity for substrates, promoting their proteasomal degradation. Consistently, YM-1 reduced the levels of ubiquitinated proteins. Interestingly, YM-1 accumulated in mitochondria, interfered with its Hsp70 isoform involved in protein import, and increased NRF1 levels, a regulator of proteasome genes. We thus suggest that YM-1 may trigger the coordination of mitochondrial and cytosolic proteostasis, enhancing protein degradation. SIGNIFICANCE: Our findings show that the strategy of allosteric Hsp70 activation holds potential for HD. While drug efficacy may be limited to tissues with elevated Hsp70, combined therapies with Hsp70 elevating strategies could harness the full potential of allosteric Hsp70 activators for HD.

Adenosine A2A receptor blockade prevents synaptotoxicity and memory dysfunction caused by beta-amyloid peptides via p38 mitogen-activated protein kinase pathway.

Alzheimer's disease (AD) is characterized by memory impairment, neurochemically by accumulation of beta-amyloid peptide (namely Abeta(1-42)) and morphologically by an initial loss of nerve terminals. Caffeine consumption prevents memory dysfunction in different models, which is mimicked by antagonists of adenosine A(2A) receptors (A(2A)Rs), which are located in synapses. Thus, we now tested whether A(2A)R blockade prevents the early Abeta(1-42)-induced synaptotoxicity and memory dysfunction and what are the underlying signaling pathways. The intracerebral administration of soluble Abeta(1-42) (2 nmol) in rats or mice caused, 2 weeks later, memory impairment (decreased performance in the Y-maze and object recognition tests) and a loss of nerve terminal markers (synaptophysin, SNAP-25) without overt neuronal loss, astrogliosis, or microgliosis. These were prevented by pharmacological blockade [5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH58261); 0.05 mg . kg(-1) . d(-1), i.p.; for 15 d] in rats, and genetic inactivation of A(2A)Rs in mice. Moreover, these were synaptic events since purified nerve terminals acutely exposed to Abeta(1-42) (500 nm) displayed mitochondrial dysfunction, which was prevented by A(2A)R blockade. SCH58261 (50 nm) also prevented the initial synaptotoxicity (loss of MAP-2, synaptophysin, and SNAP-25 immunoreactivity) and subsequent loss of viability of cultured hippocampal neurons exposed to Abeta(1-42) (500 nm). This A(2A)R-mediated control of neurotoxicity involved the control of Abeta(1-42)-induced p38 phosphorylation and was independent from cAMP/PKA (protein kinase A) pathway. Together, these results show that A(2A)Rs play a crucial role in the development of Abeta-induced synaptotoxicity leading to memory dysfunction through a p38 MAPK (mitogen-activated protein kinase)-dependent pathway and provide a molecular basis for the benefits of caffeine consumption in AD.

Nature and cause of mitochondrial dysfunction in Huntington's disease: focusing on huntingtin and the striatum.

Polyglutamine expansion mutation in huntingtin causes Huntington's disease (HD). How mutant huntingtin (mHtt) preferentially kills striatal neurons remains unknown. The link between mitochondrial dysfunction and HD pathogenesis stemmed from postmortem brain data and mitochondrial toxin models. Current evidence from genetic models, containing mHtt, supports mitochondrial dysfunction with yet uncertain nature and cause. Because mitochondria composition and function varies across tissues and cell-types, mitochondrial dysfunction in HD vulnerable striatal neurons may have distinctive features. This review focuses on mHtt and the striatum, integrating experimental evidence from patients, mice, primary cultures and striatal cell-lines. I address the nature (specific deficits) and cause (mechanisms linked to mHtt) of HD mitochondrial dysfunction, considering limitations of isolated vs. in situ mitochondria approaches, and the complications introduced by glia and glycolysis in brain and cell-culture studies. Current evidence relegates respiratory chain impairment to a late secondary event. Upstream events include defective mitochondrial calcium handling, ATP production and trafficking. Also, transcription abnormalities affecting mitochondria composition, reduced mitochondria trafficking to synapses, and direct interference with mitochondrial structures enriched in striatal neurons, are possible mechanisms by which mHtt amplifies striatal vulnerability. Insights from common neurodegenerative disorders with selective vulnerability and mitochondrial dysfunction (Alzheimer's and Parkinson's diseases) are also addressed.

Mitochondrial bioenergetics and dynamics in Huntington's disease: tripartite synapses and selective striatal degeneration.

Preferential striatal neurodegeneration is a hallmark of Huntington's disease (HD) pathogenesis, which has been associated with mitochondrial dysfunction. Evidence from genetic HD models suggest that mutant huntingtin (mHtt) compromises mitochondrial bioenergetics and dynamics, preventing efficient calcium handling and ATP generation in neuronal networks. Striatal neurons receive abundant glutamatergic input from the cortex, forming tripartite synapses with astrocytic partners. These are involved in bidirectional communication, play neuroprotective roles, and emerging evidence suggests that astrocyte dysfunction supports non-cell autonomous neurodegeneration. In addition to mHtt effects, inherent mitochondria vulnerability within striatal neurons and astrocytes may contribute for preferential neurodegeneration in HD. Dysfunctional astrocytic mitochondria in cortico-striatal tripartite synapses might be particularly relevant in the pathogenesis of juvenile/infantile HD, frequently associated with seizures and abnormally large mHtt polyglutamine expansions. This review discusses our work, primarily addressing in situ mitochondrial function in neurons and astrocytes, in the context of related work within the HD-mitochondria field.

Guanylate cyclase regulates ileal longitudinal muscle contractions induced by neurogenic nitrergic activity in the rat.

1. Nitrergic neurons regulate gastrointestinal (GI) activity and their dysfunction has been associated with various GI diseases. Nitric oxide (NO) typically relaxes GI smooth muscle, but nitrergic contractions also occur. Although guanylate cyclase is well established as mediating nitrergic GI relaxation, its role in contraction remains uncertain. 2. We used electrical field stimulation (EFS; 0.3 msec pulses, three trains of 1.2 s width, 2 Hz, at 30 s intervals) to evoke biphasic contraction-relaxation responses in rat ileum strips (longitudinal muscle-myenteric plexus preparations), mediated by the endogenous nitrergic transmitter, under non-adrenergic, non-cholinergic (NANC) conditions (1 micromol/L atropine and 4 micromol/L guanethidine). 3. All EFS responses were abolished by tetrodotoxin (1 micromol/L). Inhibition of NO synthase with N(omega)-nitro-L-arginine-methyl-ester (l-NAME; 100 and 300 micromol/L) prevented both EFS-evoked contractions and relaxations. L-Arginine (3 mmol/L) reversed l-NAME inhibition, primarily restoring contractions and suggesting that these require lower nitrergic transmitter levels than relaxations. 4. Pretreatment of preparations with subrelaxant concentrations of sodium nitroprusside (1 micromol/L) selectively desensitized EFS-evoked contractions without affecting relaxations, suggesting different downstream mechanisms. Nevertheless, the selective guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (3 and 10 micromol/L) inhibited both nitrergic contractions and relaxations, indicating that guanylate cyclase activation is required for both responses. 5. The results of the present study support the hypothesis that the endogenous nitrergic transmitter differentially regulates guanylate cyclase, leading to either contractions or relaxations depending on its concentrations, thus providing additional insight into the regulation of ileum contractility by nitrergic activity.

The interplay between redox signalling and proteostasis in neurodegeneration: In vivo effects of a mitochondria-targeted antioxidant in Huntington's disease mice.

Abnormal protein homeostasis (proteostasis), dysfunctional mitochondria, and aberrant redox signalling are often associated in neurodegenerative disorders, such as Huntington's (HD), Alzheimer's and Parkinson's diseases. It remains incompletely understood, however, how changes in redox signalling affect proteostasis mechanisms, including protein degradation pathways and unfolded protein responses (UPR). Here we address this open question by investigating the interplay between redox signalling and proteostasis in a mouse model of HD, and by examining the in vivo effects of the mitochondria-targeted antioxidant MitoQ. We performed behavioural tests in wild-type and R6/2 HD mice, examined markers of oxidative stress, UPR activation, and the status of key protein degradation pathways in brain and peripheral tissues. We show that R6/2 mice present widespread markers of oxidative stress, with tissue-specific changes in proteostasis that were more pronounced in the brain and muscle than in the liver. R6/2 mice presented increased levels of cytosolic and mitochondrial chaperones, particularly in muscle, indicating UPR activation. Treatment with MitoQ significantly ameliorated fine motor control of R6/2 mice, and reduced markers of oxidative damage in muscle. Additionally, MitoQ attenuated overactive autophagy induction in the R6/2 muscle, which has been associated with muscle wasting. Treatment with MitoQ did not alter autophagy markers in the brain, in agreement with its low brain bioavailability, which limits the risk of impairing neuronal protein clearance mechanisms. This study supports the hypotheses that abnormal redox signalling in muscle contributes to altered proteostasis and motor impairment in HD, and that redox interventions can improve muscle performance, highlighting the importance of peripheral therapeutics in HD.