A Drug Combination Rescues Frataxin-Dependent Neural and Cardiac Pathophysiology in FA Models.

Friedreich's ataxia (FA) is an inherited multisystemic neuro- and cardio-degenerative disorder. Seventy-four clinical trials are listed for FA (including past and present), but none are considered FDA/EMA-approved therapy. To date, FA therapeutic strategies have focused along two main lines using a single-drug approach: a) increasing frataxin and b) enhancing downstream pathways, including antioxidant levels and mitochondrial function. Our novel strategy employed a combinatorial approach to screen approved compounds to determine if a combination of molecules provided an additive or synergistic benefit to FA cells and/or animal models. Eight single drug molecules were administered to FA fibroblast patient cells: nicotinamide riboside, hemin, betamethasone, resveratrol, epicatechin, histone deacetylase inhibitor 109, methylene blue, and dimethyl fumarate. We measured their individual ability to induce FXN transcription and mitochondrial biogenesis in patient cells. Single-drug testing highlighted that dimethyl fumarate and resveratrol increased these two parameters. In addition, the simultaneous administration of these two drugs was the most effective in terms of FXN mRNA and mitobiogenesis increase. Interestingly, this combination also improved mitochondrial functions and reduced reactive oxygen species in neurons and cardiomyocytes. Behavioral tests in an FA mouse model treated with dimethyl fumarate and resveratrol demonstrated improved rotarod performance. Our data suggest that dimethyl fumarate is effective as a single agent, and the addition of resveratrol provides further benefit in some assays without showing toxicity. Therefore, they could be a valuable combination to counteract FA pathophysiology. Further studies will help fully understand the potential of a combined therapeutic strategy in FA pathophysiology.

Hsp90 Inhibition: A Promising Therapeutic Approach for ARSACS.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a neurodegenerative disease caused by mutations in the SACS gene, encoding the 520 kDa modular protein sacsin, which comprises multiple functional sequence domains that suggest a role either as a scaffold in protein folding or in proteostasis. Cells from patients with ARSACS display a distinct phenotype including altered organisation of the intermediate filament cytoskeleton and a hyperfused mitochondrial network where mitochondrial respiration is compromised. Here, we used vimentin bundling as a biomarker of sacsin function to test the therapeutic potential of Hsp90 inhibition with the C-terminal-domain-targeted compound KU-32, which has demonstrated mitochondrial activity. This study shows that ARSACS patient cells have significantly increased vimentin bundling compared to control, and this was also present in ARSACS carriers despite them being asymptomatic. We found that KU-32 treatment significantly reduced vimentin bundling in carrier and patient cells. We also found that cells from patients with ARSACS were unable to maintain mitochondrial membrane potential upon challenge with mitotoxins, and that the electron transport chain function was restored upon KU-32 treatment. Our preliminary findings presented here suggest that targeting the heat-shock response by Hsp90 inhibition alleviates vimentin bundling and may represent a promising area for the development of therapeutics for ARSACS.

The translocator protein (TSPO) is prodromal to mitophagy loss in neurotoxicity.

Dysfunctional mitochondria characterise Parkinson's Disease (PD). Uncovering etiological molecules, which harm the homeostasis of mitochondria in response to pathological cues, is therefore pivotal to inform early diagnosis and therapy in the condition, especially in its idiopathic forms. This study proposes the 18 kDa Translocator Protein (TSPO) to be one of those. Both in vitro and in vivo data show that neurotoxins, which phenotypically mimic PD, increase TSPO to enhance cellular redox-stress, susceptibility to dopamine-induced cell death, and repression of ubiquitin-dependent mitophagy. TSPO amplifies the extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) signalling, forming positive feedback, which represses the transcription factor EB (TFEB) and the controlled production of lysosomes. Finally, genetic variances in the transcriptome confirm that TSPO is required to alter the autophagy-lysosomal pathway during neurotoxicity.

NH-sulfoximine: A novel pharmacological inhibitor of the mitochondrial F1 Fo -ATPase, which suppresses viability of cancerous cells.

BACKGROUND AND PURPOSE: The mitochondrial F1 Fo -ATPsynthase is pivotal for cellular homeostasis. When respiration is perturbed, its mode of action everts becoming an F1 Fo -ATPase and therefore consuming rather producing ATP. Such a reversion is an obvious target for pharmacological intervention to counteract pathologies. Despite this, tools to selectively inhibit the phases of ATP hydrolysis without affecting the production of ATP remain scarce. Here, we report on a newly synthesised chemical, the NH-sulfoximine (NHS), which achieves such a selectivity. EXPERIMENTAL APPROACH: The chemical structure of the F1 Fo -ATPase inhibitor BTB-06584 was used as a template to synthesise NHS. We assessed its pharmacology in human neuroblastoma SH-SY5Y cells in which we profiled ATP levels, redox signalling, autophagy pathways and cellular viability. NHS was given alone or in combination with either the glucose analogue 2-deoxyglucose (2-DG) or the chemotherapeutic agent etoposide. KEY RESULTS: NHS selectively blocks the consumption of ATP by mitochondria leading a subtle cytotoxicity associated via the concomitant engagement of autophagy which impairs cell viability. NHS achieves such a function independently of the F1 Fo -ATPase inhibitory factor 1 (IF1). CONCLUSION AND IMPLICATIONS: The novel sulfoximine analogue of BTB-06584, NHS, acts as a selective pharmacological inhibitor of the mitochondrial F1 Fo -ATPase. NHS, by blocking the hydrolysis of ATP perturbs the bioenergetic homoeostasis of cancer cells, leading to a non-apoptotic type of cell death.

Mitochondria form contact sites with the nucleus to couple prosurvival retrograde response.

Mitochondria drive cellular adaptation to stress by retro-communicating with the nucleus. This process is known as mitochondrial retrograde response (MRR) and is induced by mitochondrial dysfunction. MRR results in the nuclear stabilization of prosurvival transcription factors such as the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Here, we demonstrate that MRR is facilitated by contact sites between mitochondria and the nucleus. The translocator protein (TSPO) by preventing the mitophagy-mediated segregation o mitochonria is required for this interaction. The complex formed by TSPO with the protein kinase A (PKA), via the A-kinase anchoring protein acyl-CoA binding domain containing 3 (ACBD3), established the tethering. The latter allows for cholesterol redistribution of cholesterol in the nucleus to sustain the prosurvival response by blocking NF-κB deacetylation. This work proposes a previously unidentified paradigm in MRR: the formation of contact sites between mitochondria and nucleus to aid communication.

Bi-allelic JAM2 Variants Lead to Early-Onset Recessive Primary Familial Brain Calcification.

Primary familial brain calcification (PFBC) is a rare neurodegenerative disorder characterized by a combination of neurological, psychiatric, and cognitive decline associated with calcium deposition on brain imaging. To date, mutations in five genes have been linked to PFBC. However, more than 50% of individuals affected by PFBC have no molecular diagnosis. We report four unrelated families presenting with initial learning difficulties and seizures and later psychiatric symptoms, cerebellar ataxia, extrapyramidal signs, and extensive calcifications on brain imaging. Through a combination of homozygosity mapping and exome sequencing, we mapped this phenotype to chromosome 21q21.3 and identified bi-allelic variants in JAM2. JAM2 encodes for the junctional-adhesion-molecule-2, a key tight-junction protein in blood-brain-barrier permeability. We show that JAM2 variants lead to reduction of JAM2 mRNA expression and absence of JAM2 protein in patient's fibroblasts, consistent with a loss-of-function mechanism. We show that the human phenotype is replicated in the jam2 complete knockout mouse (jam2 KO). Furthermore, neuropathology of jam2 KO mouse showed prominent vacuolation in the cerebral cortex, thalamus, and cerebellum and particularly widespread vacuolation in the midbrain with reactive astrogliosis and neuronal density reduction. The regions of the human brain affected on neuroimaging are similar to the affected brain areas in the myorg PFBC null mouse. Along with JAM3 and OCLN, JAM2 is the third tight-junction gene in which bi-allelic variants are associated with brain calcification, suggesting that defective cell-to-cell adhesion and dysfunction of the movement of solutes through the paracellular spaces in the neurovascular unit is a key mechanism in CNS calcification.

Xeroderma pigmentosum: overview of pharmacology and novel therapeutic strategies for neurological symptoms.

Xeroderma pigmentosum (XP) encompasses a group of rare diseases characterized in most cases by malfunction of nucleotide excision repair (NER), which results in an increased sensitivity to UV radiation in affected individuals. Approximately 25-30% of XP patients present with neurological symptoms, such as sensorineural deafness, mental deterioration and ataxia. Although it is known that dysfunctional DNA repair is the primary pathogenesis in XP, growing evidence suggests that mitochondrial pathophysiology may also occur. This appears to be secondary to dysfunctional NER but may contribute to the neurodegenerative process in these patients. The available pharmacological treatments in XP mostly target the dermal manifestations of the disease. In the present review, we outline how current understanding of the pathophysiology of XP could be used to develop novel therapies to counteract the neurological symptoms. Moreover, the coexistence of cancer and neurodegeneration present in XP led us to focus on possible new avenues targeting mitochondrial pathophysiology. LINKED ARTICLES: This article is part of a themed section on Mitochondrial Pharmacology: Featured Mechanisms and Approaches for Therapy Translation. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.22/issuetoc.

Complexity of the Genetics and Clinical Presentation of Spinocerebellar Ataxia 17.

Spinocerebellar ataxia type 17 (SCA17) is a rare autosomal dominant neurodegenerative disease caused by a CAG repeat expansion in the TATA-box binding protein gene (TBP). The disease has a varied age at onset and clinical presentation. It is distinct from other SCAs for its association with dementia, psychiatric symptoms, and some patients presenting with chorea. For this reason, it is also called Huntington's disease-like 4 (HDL-4). Here we examine the distribution of SCA17 allele repeat sizes in a United Kingdom-based cohort with ataxia and find that fully penetrant pathogenic alleles are very rare (5 in 1,316 chromosomes; 0.38%). Phenotype-genotype correlation was performed on 30 individuals and the repeat structure of their TBP genes was examined. We found a negative linear correlation between total CAG repeat length and age at disease onset and, unlike SCA1, there was no correlation between the longest contiguous CAG tract and age at disease onset. We were unable to identify any particular phenotypic trait that segregated with particular CAG/CAA repeat tract structures or repeat lengths. One individual within the cohort was homozygous for variable penetrance range SCA17 alleles. This patient had a similar age at onset to heterozygotes with the same repeat sizes, but also presented with a rapidly progressive dementia. A pair of monozygotic twins within the cohort presented 3 years apart with the sibling with the earlier onset having a more severe phenotype with dementia and chorea in addition to the ataxia observed in their twin. This appears to be a case of variable expressivity, possibly influenced by other environmental or epigenetic factors. Finally, there was an asymptomatic father with a severely affected child with an age at onset in their twenties. Despite this, they share the same expanded allele repeat sizes and sequences, which would suggest that there is marked difference in the penetrance of this 51-repeat allele. We therefore propose that the variable penetrance range extend from 48 repeats to incorporate this allele. This study shows that there is variability in the presentation and penetrance of the SCA17 phenotype and highlights the complexity of this disorder.

Calcium Deregulation: Novel Insights to Understand Friedreich's Ataxia Pathophysiology.

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

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

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

mTOR independent alteration in ULK1 Ser758 phosphorylation following chronic LRRK2 kinase inhibition.

Unc-51 Like Kinase 1 (ULK1) is a critical regulator of the biogenesis of autophagosomes, the central component of the catabolic macroautophagy pathway. Regulation of ULK1 activity is dependent upon several phosphorylation events acting to repress or activate the enzymatic function of this protein. Phosphorylation of Ser758 ULK1 has been linked to repression of autophagosome biogenesis and was thought to be exclusively dependent upon mTOR complex 1 kinase activity. In the present study, a novel regulation of Ser758 ULK1 phosphorylation is reported following prolonged inhibition of the Parkinson's disease linked protein leucine rich repeat kinase 2 (LRRK2). Here, modulation of Ser758 ULK1 phosphorylation following LRRK2 inhibition is decoupled from the repression of autophagosome biogenesis and independent of mTOR complex 1 activity.

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

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

A reduction in Drp1-mediated fission compromises mitochondrial health in autosomal recessive spastic ataxia of Charlevoix Saguenay.

The neurodegenerative disease autosomal recessive spastic ataxia of Charlevoix Saguenay (ARSACS) is caused by loss of function of sacsin, a modular protein that is required for normal mitochondrial network organization. To further understand cellular consequences of loss of sacsin, we performed microarray analyses in sacsin knockdown cells and ARSACS patient fibroblasts. This identified altered transcript levels for oxidative phosphorylation and oxidative stress genes. These changes in mitochondrial gene networks were validated by quantitative reverse transcription PCR. Functional impairment of oxidative phosphorylation was then demonstrated by comparison of mitochondria bioenergetics through extracellular flux analyses. Moreover, staining with the mitochondrial-specific fluorescent probe MitoSox suggested increased levels of superoxide in patient cells with reduced levels of sacsin.Key to maintaining mitochondrial health is mitochondrial fission, which facilitates the dynamic exchange of mitochondrial components and separates damaged parts of the mitochondrial network for selective elimination by mitophagy. Fission is dependent on dynamin-related protein 1 (Drp1), which is recruited to prospective sites of division where it mediates scission. In sacsin knockdown cells and ARSACS fibroblasts, we observed a decreased incidence of mitochondrial associated Drp1 foci. This phenotype persists even when fission is induced by drug treatment. Mitochondrial-associated Drp1 foci are also smaller in sacsin knockdown cells and ARSACS fibroblasts. These data suggest a model for ARSACS where neurons with reduced levels of sacsin are compromised in their ability to recruit or retain Drp1 at the mitochondrial membrane leading to a decline in mitochondrial health, potentially through impaired mitochondrial quality control.

Targeting lipid peroxidation and mitochondrial imbalance in Friedreich's ataxia.

Friedreich's ataxia (FRDA) is an autosomal recessive disorder, caused by reduced levels of the protein frataxin. This protein is located in the mitochondria, where it functions in the biogenesis of iron-sulphur clusters (ISCs), which are important for the function of the mitochondrial respiratory chain complexes. Moreover, disruption in iron biogenesis may lead to oxidative stress. Oxidative stress can be the cause and/or the consequence of mitochondrial energy imbalance, leading to cell death. Fibroblasts from two FRDA mouse models, YG8R and KIKO, were used to analyse two different categories of protective compounds: deuterised poly-unsaturated fatty acids (dPUFAs) and Nrf2-inducers. The former have been shown to protect the cell from damage induced by lipid peroxidation and the latter trigger the well-known Nrf2 antioxidant pathway. Our results show that the sensitivity to oxidative stress of YG8R and KIKO mouse fibroblasts, resulting in cell death and lipid peroxidation, can be prevented by d4-PUFA and Nrf2-inducers (SFN and TBE-31). The mitochondrial membrane potential (ΔΨm) of YG8R and KIKO fibroblasts revealed a difference in their mitochondrial pathophysiology, which may be due to the different genetic basis of the two models. This suggests that variable levels of reduced frataxin may act differently on mitochondrial pathophysiology and that these two cell models could be useful in recapitulating the observed differences in the FRDA phenotype. This may reflect a different modulatory effect towards cell death that will need to be investigated further.

Mitochondrial Ca(2+) in neurodegenerative disorders.

Functional mitochondria are vital to accomplish their key role in the cell, by maintaining the energy metabolism, buffering of the Ca(2+) signal and directing the cell death mechanism. Mitochondrial Ca(2+) can stimulate ATP production or trigger the opening of mitochondrial permeability transition pore and activating the cell death cascade. Mitochondrial Ca(2+) uptake play a crucial role in neurons by buffering excessive Ca(2+) from the cytosol at the time of the transmission of the signal. Changes in the maintenance of mitochondrial Ca(2+) may trigger neuronal cell death. Abnormality in mitochondrial Ca(2+) handling has been detected in a range of neurodegenerative diseases, and emerging evidence from disease models suggests that mitochondrial Ca(2+) may play a role in disease pathogenesis. In this review, we assess how mitochondrial Ca(2+) imbalance may be a trigger in common neurodegenerative disease.

TSPO interacts with VDAC1 and triggers a ROS-mediated inhibition of mitochondrial quality control.

The 18-kDa TSPO (translocator protein) localizes on the outer mitochondrial membrane (OMM) and participates in cholesterol transport. Here, we report that TSPO inhibits mitochondrial autophagy downstream of the PINK1-PARK2 pathway, preventing essential ubiquitination of proteins. TSPO abolishes mitochondrial relocation of SQSTM1/p62 (sequestosome 1), and consequently that of the autophagic marker LC3 (microtubule-associated protein 1 light chain 3), thus leading to an accumulation of dysfunctional mitochondria, altering the appearance of the network. Independent of cholesterol regulation, the modulation of mitophagy by TSPO is instead dependent on VDAC1 (voltage-dependent anion channel 1), to which TSPO binds, reducing mitochondrial coupling and promoting an overproduction of reactive oxygen species (ROS) that counteracts PARK2-mediated ubiquitination of proteins. These data identify TSPO as a novel element in the regulation of mitochondrial quality control by autophagy, and demonstrate the importance for cell homeostasis of its expression ratio with VDAC1.

Point mutations in the transmembrane region of the clic1 ion channel selectively modify its biophysical properties.

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

Inhibition of LRRK2 kinase activity stimulates macroautophagy.

Leucine Rich Repeat Kinase 2 (LRRK2) is one of the most important genetic contributors to Parkinson's disease. LRRK2 has been implicated in a number of cellular processes, including macroautophagy. To test whether LRRK2 has a role in regulating autophagy, a specific inhibitor of the kinase activity of LRRK2 was applied to human neuroglioma cells and downstream readouts of autophagy examined. The resulting data demonstrate that inhibition of LRRK2 kinase activity stimulates macroautophagy in the absence of any alteration in the translational targets of mTORC1, suggesting that LRRK2 regulates autophagic vesicle formation independent of canonical mTORC1 signaling. This study represents the first pharmacological dissection of the role LRRK2 plays in the autophagy/lysosomal pathway, emphasizing the importance of this pathway as a marker for LRRK2 physiological function. Moreover it highlights the need to dissect autophagy and lysosomal activities in the context of LRRK2 related pathologies with the final aim of understanding their aetiology and identifying specific targets for disease modifying therapies in patients.

Activation of PARP by oxidative stress induced by ß-amyloid: implications for Alzheimer's disease.

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

Beta-amyloid activates PARP causing astrocytic metabolic failure and neuronal death.

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