ABSTRACT
Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons responsible for unintended or uncontrollable movements. Mutations in the leucine-rich repeat kinase 2 locus contribute to genetic forms of PD. The fruit fly Drosophila melanogaster carrying this mutation (LRRK2-Dm) is an in vivo model of PD that develops motor impairment and stands for an eligible non-mammalian paradigm to test novel therapeutic approaches. Dehydrozingerone (DHZ) is a natural phenolic compound isolated from ginger and presents anti-inflammatory, antioxidant and neuroprotective properties, making it a potential therapeutic target for PD. We administered DHZ and its C2-symmetric dimer (DHZ-DIM) at 0.5 and 1 mM for 14 and 21 days in the LRRK2-Dm, with the aim of assessing changes in rescuing motor behavior, brain dopaminergic neurons, mitochondria and synapses (T-bars). The shorter treatment with both molecules revealed efficacy at the higher dose, improving climbing behavior with a prevention of dopaminergic neuronal demise. After 21 days, a recovery of the motor disability, dopaminergic neuron loss, mitochondrial damage and T-bars failure was observed with the DHZ-DIM. Our data indicate that the DHZ-DIM exerts a more potent neuroprotective effect with respect to the monomer in LRRK2-Dm, prompting further investigation of these compounds in rodent models of PD.
Subject(s)
Disabled Persons , Motor Disorders , Neuroprotective Agents , Parkinson Disease , Styrenes , Animals , Humans , Parkinson Disease/drug therapy , Parkinson Disease/genetics , Drosophila , Neuroprotective Agents/pharmacology , Neuroprotective Agents/therapeutic use , Drosophila melanogaster/genetics , Dopaminergic Neurons , Dietary Supplements , MutationABSTRACT
It is increasingly evident that LRRK2 kinase activity is involved in oxidative stress (OS)-induced apoptosis-a type of regulated cell death and neurodegeneration, suggesting LRRK2 inhibition as a potential therapeutic target. We report that a phenolic-rich extract of avocado Persea americana var. Colinred peel (CRE, 0.01 mg/ml) restricts environmental neurotoxins paraquat (1 mM)/maneb (0.05 mM)-induced apoptosis process through blocking reactive oxygen species (ROS) signaling and concomitant inhibition of phosphorylation of LRRK2 in nerve-like cells (NLCs). Indeed, PQ + MB at 6 h exposure significantly increased ROS (57 ± 5%), oxidation of protein DJ-1cys106SOH into DJ-1Cys106SO3 ([~3.7 f(old)-(i)ncrease]), augmented p-(S935)-LRRK2 kinase (~20-f(old) (i)ncrease), induced nuclei condensation/fragmentation (28 ± 6%), increased the expression of PUMA (~6.2-fi), and activated CASPASE-3 (CASP-3, ~4-fi) proteins; but significantly decreased mitochondrial membrane potential (ΔΨm, ~48 ± 4%), all markers indicative of apoptosis compared to untreated cells. Remarkably, CRE significantly diminished both OS-signals (i.e., DCF+ cells, DJ-1Cys106SO3) as well as apoptosis markers (e.g., PUMA, CASP-3, loss of ΔΨm, p-LRRK2 kinase) in NLCs exposed to PQ + MB. Furthermore, CRE dramatically reestablishes the transient intracellular Ca2+ flow (~300%) triggered by dopamine (DA) in neuronal cells exposed to PQ + MB. We conclude that PQ + MB-induced apoptosis in NLCs through OS-mechanism, involving DJ-1, PUMA, CASP-3, LRRK2 kinase, mitochondria damage, DNA fragmentation, and alteration of DA-receptors. Our findings imply that CRE protects NLCs directly via antioxidant mechanism and indirectly by blocking LRRK2 kinase against PQ + MB stress stimuli. These data suggest that CRE might be a potential natural antioxidant.
Subject(s)
Maneb , Persea , Apoptosis , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Oxidative Stress , Paraquat/toxicity , Phosphorylation , Plant Extracts/pharmacologyABSTRACT
The most frequent missense mutations in familial Parkinson's disease (PD) occur in the highly conserved LRRK2/PARK8 gene with G2019S mutation. We previously established a fly model of PD carrying the LRRK2-G2019S mutation that exhibited the parkinsonism-like phenotypes. An herbal medicine, Gastrodia elata Blume (GE), has been reported to have neuroprotective effects in toxin-induced PD models. However, the underpinning molecular mechanisms of GE beneficiary to G2019S-induced PD remain unclear. Here, we show that these G2019S flies treated with water extracts of GE (WGE) and its bioactive compounds, gastrodin and 4-HBA, displayed locomotion improvement and dopaminergic neuron protection. WGE suppressed the accumulation and hyperactivation of G2019S proteins in dopaminergic neurons and activated the antioxidation and detoxification factor Nrf2 mostly in the astrocyte-like and ensheathing glia. Glial activation of Nrf2 antagonizes G2019S-induced Mad/Smad signaling. Moreover, we treated LRRK2-G2019S transgenic mice with WGE and found that the locomotion declines, the loss of dopaminergic neurons, and the number of hyperactive microglia were restored. WGE also suppressed the hyperactivation of G2019S proteins and regulated the Smad2/3 pathways in the mice brains. We conclude that WGE prevents locomotion defects and the neuronal loss induced by G2019S mutation via glial Nrf2/Mad signaling, unveiling a potential therapeutic avenue for PD.
Parkinson's disease is a brain disorder that leads to tremors and difficulties with balance and coordination. These symptoms are caused by the loss of neurons which release a chemical messenger that is needed to regulate movement called dopamine. Most treatments for this disease work by boosting levels of dopamine in the brain, but this can lead to severe side effects and these drugs often become less effective over time. A traditional Chinese medicine called Gastrodia elata Blume (or GE for short) has previously been reported to relieve symptoms of Parkinson's disease in both human and animal studies when administered as a decoction or formula. However, it is unclear how GE protects dopamine-producing neurons and if this mechanism involves another type of brain cell known as glia that has also been linked to Parkinson's disease. To investigate, Lin et al. studied fruit flies and mice that carry a genetic mutation that produces the symptoms and molecular characteristics of Parkinson's disease. The experiments showed that when the flies and mice were fed food containing water extracts of GE, they experienced less difficulties moving and had a higher number of intact dopamine-producing neurons. Lin et al. found that GE switched on a protein in glial cells located near dopamine-producing neurons. Activation of this protein, called Nrf2, inhibited a signaling pathway in degenerating neurons that leads to the disease state. As a result, less dopamine-producing neurons were damaged and the animals' coordination and balance were maintained. These findings suggest that GE could potentially provide an alternative or complementary therapy for Parkinson's disease, although it still needs to be studied further in humans. If the same effect is observed, the specific compounds in GE that have this protective effect could be isolated and analyzed to see if they could be used for treatment.
Subject(s)
Gastrodia/chemistry , NF-E2-Related Factor 2/metabolism , Neuroprotective Agents/pharmacology , Parkinson Disease/drug therapy , Plant Extracts/pharmacology , Signal Transduction , Animals , Benzyl Alcohols/pharmacology , Butyrates/pharmacology , Disease Models, Animal , Dopaminergic Neurons/drug effects , Dopaminergic Neurons/metabolism , Drosophila melanogaster , Glucosides/pharmacology , Locomotion/drug effects , Mice , Neuroglia/physiology , NeuroprotectionABSTRACT
Mitochondrial dysfunction has been proposed as one of the pathobiological underpinnings in Parkinson's disease. Environmental stressors, such as paraquat, induce mitochondrial dysfunction and promote reactive oxygen species production. Targeting oxidative stress pathways could prevent mitochondrial dysfunction and thereby halt the neurodegeneration in Parkinson's disease. Since curcumin is touted as an antioxidant and neuroprotective agent, the aim of this study was to investigate if curcumin is a suitable therapy to target mitochondrial dysfunction in Parkinson's disease using a paraquat-toxicity induced model in fibroblasts from LRRK2-mutation positive Parkinson's disease individuals and healthy controls. The fibroblasts were exposed to five treatment groups, (i) untreated, (ii) curcumin only, (iii) paraquat only, (iv) pre-curcumin group: with curcumin for 2hr followed by paraquat for 24hr and (v) post-curcumin group: with paraquat for 24hr followed by curcumin for 2hr. Mitochondrial function was determined by measuring three parameters of mitochondrial respiration (maximal respiration, ATP-associated respiration, and spare respiratory capacity) using the Seahorse XFe96 Extracellular Flux Analyzer. As expected, paraquat effectively disrupted mitochondrial function for all parameters. Pre-curcumin treatment improved maximal and ATP-associated respiration whereas, post-curcumin treatment had no effect. These findings indicate that curcumin may be most beneficial as a pre-treatment before toxin exposure, which has implications for its therapeutic use. These promising findings warrant future studies testing different curcumin dosages, exposure times and curcumin formulations in larger sample sizes of Parkinson's disease and control participants.
ABSTRACT
Alveolar type II (ATII) epithelial cells contain lamellar bodies (LBs) which synthesize and store lung surfactants. In animals, the inhibition or knockout of leucine-rich repeat kinase 2 (LRRK2) causes abnormal enlargement of LBs in ATII cells. This effect of LRRK2 inhibition in lung is largely accepted as being mediated directly through blocking of the kinase function; however, downstream consequences in the lung remain unknown. In this work we established an in vitro alveolar epithelial cell (AEC) model that recapitulates the in vivo phenotype of ATII cells and developed an assay to quantify changes in LB size in response to LRRK2 inhibitors. Culture of primary human AECs at the air-liquid interface on matrigel and collagen-coated transwell inserts in the presence of growth factors promoted the LB formation and apical microvilli and induced expression of LRRK2 and ATII cell markers. Treatment with a selective LRRK2 inhibitor resulted in pharmacological reduction of phospho-LRRK2 and a significant increase in LB size; effects previously reported in lungs of non-human primates treated with LRRK2 inhibitor. In summary, our human in vitro AEC model recapitulates the abnormal lung findings observed in LRRK2-perturbed animals and holds the potential for expanding current understanding of LRRK2 function in the lung.
Subject(s)
Alveolar Epithelial Cells/pathology , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/antagonists & inhibitors , Models, Biological , ATP-Binding Cassette Transporters/metabolism , Adenocarcinoma of Lung/metabolism , Alveolar Epithelial Cells/metabolism , Alveolar Epithelial Cells/ultrastructure , Cells, Cultured , Drug Evaluation, Preclinical , Gene Expression , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/genetics , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/metabolism , Lung Neoplasms/metabolism , Pulmonary Surfactant-Associated Protein C/metabolismABSTRACT
BACKGROUND: Both genetic and environmental factors contribute to Parkinson's disease (PD) risk. OBJECTIVE: We investigated the potential association of several relevant variables with PD age at onset (AAO), focusing on LRRK2 p.G2019S and GBA p.N370S mutations. METHODS: Ashkenazi Jewish (AJ) PD patients, screened for LRRK2 and GBA mutations, underwent an interview regarding exposure to the following environmental and lifestyle factors: cigarette smoking, consumption of coffee, tea and alcohol, head injury and rural living. Multivariate linear regression (adjusted for sex) was used to examine the association with AAO, and models included LRRK2 p.G2019S and GBA p.N370S mutation status (carrier/non-carriers), single environmental variable and their interactions terms, as independent variables. RESULTS: 225 Israeli AJ PD patients were enrolled: 65 LRRK2 p.G2019S mutation carriers, 60 GBA p.N370S carriers and 100 non-carries of these mutations. In the dichotomized exposure/non-exposure analyses, positive LRRK2 p.G2019S status was associated with younger AAO in all models, at nominal or near significant levels (pâ=â0.033-0.082). Smoking was associated with older AAO (pâ=â0.032), and the interaction between GBA p.N370S and history of head injury was associated with younger AAO (pâ=â0.049), both at nominal significance. There was no indication of a consistent main effect for GBA p.N370S status or significant LRRK2 p.G2019S-environmental factor interaction. In the dose-dependent analyses, increased coffee and tea consumption levels were associated with older AAO (pâ=â0.001 and pâ=â0.002, respectively). CONCLUSIONS: Our results suggest that genetic and environmental factors may affect AAO in PD patients, but validation in additional samples is required.
Subject(s)
Gene-Environment Interaction , Glucosylceramidase/genetics , Jews , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/genetics , Parkinson Disease , Adult , Age of Onset , Aged , Coffee , Drinking Behavior/physiology , Female , Heterozygote , Humans , Israel/ethnology , Jews/genetics , Jews/statistics & numerical data , Male , Middle Aged , Parkinson Disease/ethnology , Parkinson Disease/etiology , Parkinson Disease/genetics , TeaABSTRACT
Mutations in LRRK2 are currently recognized as the most common monogenetic cause of Parkinsonism. The elevation of kinase activity of LRRK2 that frequently accompanies its mutations is widely thought to contribute to its toxicity. Accordingly, many groups have developed LRRK2-specific kinase inhibitors as a potential therapeutic strategy. Given that protein phosphorylation is a reversible event, we sought to elucidate the phosphatase(s) that can reverse LRRK2-mediated phosphorylation, with the view that targeting this phosphatase(s) may similarly be beneficial. Using an unbiased RNAi phosphatase screen conducted in a Drosophila LRRK2 model, we identified PP2A as a genetic modulator of LRRK2-induced neurotoxicity. Further, we also identified ribosomal S6 kinase (S6K), a target of PP2A, as a novel regulator of LRRK2 function. Finally, we showed that modulation of PP2A or S6K activities ameliorates LRRK2-associated disease phenotype in Drosophila.
Subject(s)
Drosophila Proteins/genetics , Drosophila Proteins/physiology , Drosophila melanogaster/enzymology , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/genetics , Protein Phosphatase 2/physiology , Ribosomal Protein S6 Kinases/physiology , Animals , Animals, Genetically Modified , Cell Line , Ceramides/pharmacology , Disease Models, Animal , Drosophila Proteins/antagonists & inhibitors , Drosophila Proteins/metabolism , Drug Evaluation, Preclinical , Enzyme Activation/drug effects , Fingolimod Hydrochloride/pharmacology , Gain of Function Mutation , Gene Knockdown Techniques , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Motor Activity/drug effects , Mutation, Missense , Phosphoprotein Phosphatases/antagonists & inhibitors , Phosphoprotein Phosphatases/genetics , Phosphoprotein Phosphatases/physiology , Phosphorylation/drug effects , Protein Phosphatase 2/antagonists & inhibitors , Protein Phosphatase 2/genetics , Protein Processing, Post-Translational/drug effects , RNA Interference , RNA, Small Interfering/genetics , Recombinant Proteins/metabolism , Ribosomal Protein S6 Kinases/antagonists & inhibitors , Ribosomal Protein S6 Kinases/genetics , Ribosomal Protein S6 Kinases, 90-kDa/antagonists & inhibitors , Ribosomal Protein S6 Kinases, 90-kDa/metabolismABSTRACT
Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder worldwide affecting 2-3% of the population over 65 years. This prevalence is expected to rise as life expectancy increases and diagnostic and therapeutic protocols improve. PD encompasses a multitude of clinical, genetic, and molecular forms of the disease. Even though the mechanistic of the events leading to neurodegeneration remain largely unknown, some molecular hallmarks have been repeatedly reported in most patients and models of the disease. Neuroinflammation, protein misfolding, disrupted endoplasmic reticulum-mitochondria crosstalk, mitochondrial dysfunction and consequent bioenergetic failure, oxidative stress and autophagy deregulation, are amongst the most commonly described. Supporting these findings, numerous familial forms of PD are caused by mutations in genes that are crucial for mitochondrial and autophagy proper functioning. For instance, late and early onset PD associated to mutations in Leucine-rich repeat kinase 2 (LRRK2) and Parkin (PRKN) genes, responsible for the most frequent dominant and recessive inherited forms of PD, respectively, have emerged as promising examples of disease due to their established role in commanding bioenergetic and autophagic balance. Concomitantly, the development of animal and cell models to investigate the etiology of the disease, potential biomarkers and therapeutic approaches are being explored. One of the emerging approaches in this context is the use of patient's derived cells models, such as skin-derived fibroblasts that preserve the genetic background and some environmental cues of the patients. An increasing number of reports in these PD cell models postulate that deficient mitochondrial function and impaired autophagic flux may be determinant in PD accelerated nigral cell death in terms of limitation of cell energy supply and accumulation of obsolete and/or unfolded proteins or dysfunctional organelles. The reliance of neurons on mitochondrial oxidative metabolism and their post-mitotic nature, may explain their increased vulnerability to undergo degeneration upon mitochondrial challenges or autophagic insults. In this scenario, proper mitochondrial function and turnover through mitophagy, are gaining in strength as protective targets to prevent neurodegeneration, together with the use of patient-derived fibroblasts to further explore these events. These findings point out the presence of molecular damage beyond the central nervous system (CNS) and proffer patient-derived cell platforms to the clinical and scientific community, which enable the study of disease etiopathogenesis and therapeutic approaches focused on modifying the natural history of PD through, among others, the enhancement of mitochondrial function and autophagy.
ABSTRACT
Cerebral atrophy has been detected in patients with Parkinson's disease (PD) both with and without dementia, however differentiation based on genetic status has thus far not yielded robust findings. We assessed cortical thickness and subcortical volumes in a cohort of PD patients and healthy controls carriers of the G2019S mutation in the LRRK2 gene and the common GBA mutations, in an attempt to determine whether genetic status influences structural indexes. Cortical thickness and subcortical volumes were computed and compared between six groups of participants; idiopathic PD, GBA-PD, LRRK2-PD, non-manifesting non-carriers (NMNC), GBA-non-manifesting carriers (NMC) and LRRK2-NMC utilizing the FreeSurfer software program. All participants were cognitively intact based on a computerized cognitive assessment battery. Fifty-seven idiopathic PD patients, 9 LRRK2-PD, 12 GBA-PD, 49 NMNC, 41 LRRK2-NMC and 14 GBA-NMC participated in this study. Lower volumes among patients with PD compared to unaffected participants were detected in bilateral hippocampus, nucleus accumbens, caudate, thalamus, putamen and amygdala and the right pallidum (p = 0.016). PD patients demonstrated lower cortical thickness indexes in a majority of regions assessed compared with non-manifesting participants. No differences in cortical thickness and subcortical volumes were detected within each of the groups of participants based on genetic status. Mutations in the GBA and LRRK2 genes are not important determinants of cortical thickness and subcortical volumes in both patients with PD and non-manifesting participants. PD is associated with a general reduction in cortical thickness and sub-cortical atrophy even in cognitively intact patients.
Subject(s)
Brain/diagnostic imaging , Parkinson Disease/diagnostic imaging , Aged , Amygdala/diagnostic imaging , Amygdala/pathology , Biomarkers , Brain/pathology , Case-Control Studies , Caudate Nucleus/diagnostic imaging , Caudate Nucleus/pathology , Cerebral Cortex/diagnostic imaging , Cerebral Cortex/pathology , Cohort Studies , Family , Female , Globus Pallidus/diagnostic imaging , Globus Pallidus/pathology , Glucosylceramidase/genetics , Hippocampus/diagnostic imaging , Hippocampus/pathology , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/genetics , Male , Middle Aged , Mutation , Nucleus Accumbens/diagnostic imaging , Nucleus Accumbens/pathology , Organ Size , Parkinson Disease/genetics , Putamen/diagnostic imaging , Putamen/pathology , Thalamus/diagnostic imaging , Thalamus/pathologyABSTRACT
OBJECTIVES: A mutation in leucine-rich repeat kinase 2 is the most common cause of hereditary Parkinson's disease (PD), yet the neural mechanisms and the circuitry potentially involved are poorly understood. METHODS: We used different transcranial magnetic stimulation protocols to explore in the primary motor cortex the activity of intracortical circuits and cortical plasticity (long-term potentiation) in patients with the G2019S leucine-rich repeat kinase 2 gene mutation when compared with idiopathic PD patients and age-matched healthy subjects. Paired pulse transcranial magnetic stimulation was used to investigate short intracortical inhibition and facilitation and short afferent inhibition. Intermittent theta burst stimulation, a form of repetitive transcranial magnetic stimulation, was used to test long-term potentiation-like cortical plasticity. Leucine-rich repeat kinase 2 and idiopathic PD were tested both in ON and in OFF l-dopa therapy. RESULTS: When compared with idiopathic PD and healthy subjects, leucine-rich repeat kinase 2 PD patients showed a remarkable reduction of short intracortical inhibition in both ON and in OFF l-dopa therapy. This reduction was paralleled by an increase of intracortical facilitation in OFF l-dopa therapy. Leucine-rich repeat kinase 2 PD showed abnormal long-term potentiation-like cortical plasticity in ON l-dopa therapy. DISCUSSION: The motor cortex in leucine-rich repeat kinase 2 mutated PD patients is strongly disinhibited and hyperexcitable. These abnormalities could be a result of an impairment of inhibitory (gamma-Aminobutyric acid) transmission eventually related to altered neurotransmitter release. © 2017 International Parkinson and Movement Disorder Society.
Subject(s)
Long-Term Potentiation/physiology , Motor Cortex/physiopathology , Neural Inhibition/physiology , Parkinson Disease/physiopathology , Aged , Antiparkinson Agents/therapeutic use , Case-Control Studies , Cerebral Cortex/metabolism , Cerebral Cortex/physiopathology , Female , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/genetics , Levodopa/therapeutic use , Male , Middle Aged , Motor Cortex/metabolism , Neural Pathways/metabolism , Neural Pathways/physiopathology , Neuronal Plasticity/physiology , Parkinson Disease/drug therapy , Parkinson Disease/genetics , Parkinson Disease/metabolism , Synaptic Transmission , Transcranial Magnetic Stimulation , gamma-Aminobutyric Acid/metabolismABSTRACT
Although mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of genetic Parkinson's disease, their function is largely unknown. LRRK2 is pleiotropic in nature, shown to be involved in neurodegeneration and in more peripheral processes, including kidney functions, in rats and mice. Recent studies in zebrafish have shown conflicting evidence that removal of the LRRK2 WD40 domain may or may not affect dopaminergic neurons and/or locomotion. This study shows that â¼50% LRRK2 knockdown in zebrafish causes not only neuronal loss but also developmental perturbations such as axis curvature defects, ocular abnormalities, and edema in the eyes, lens, and otic vesicles. We further show that LRRK2 knockdown results in significant neuronal loss, including a reduction of dopaminergic neurons. Immunofluorescence demonstrates that endogenous LRRK2 is expressed in the lens, brain, heart, spinal cord, and kidney (pronephros), which mirror the LRRK2 morphant phenotypes observed. LRRK2 knockdown results further in the concomitant upregulation of ß-synuclein, PARK13, and SOD1 and causes ß-synuclein aggregation in the diencephalon, midbrain, hindbrain, and postoptic commissure. LRRK2 knockdown causes mislocalization of the Na(+) /K(+) ATPase protein in the pronephric ducts, suggesting that the edema might be linked to renal malfunction and that LRRK2 might be associated with pronephric duct epithelial cell differentiation. Combined, our study shows that LRRK2 has multifaceted roles in zebrafish and that zebrafish represent a complementary model to further our understanding of this central protein. © 2016 Wiley Periodicals, Inc.
Subject(s)
Abnormalities, Multiple/genetics , Abnormalities, Multiple/pathology , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/genetics , Neurodegenerative Diseases/genetics , Neurons/pathology , Zebrafish Proteins/genetics , beta-Synuclein/genetics , Amino Acid Sequence , Animals , Brain Chemistry/genetics , Dopaminergic Neurons , Gene Knockdown Techniques , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/biosynthesis , Locomotion , Mutation/genetics , Neurodegenerative Diseases/pathology , Parkinson Disease/genetics , Sodium-Potassium-Exchanging ATPase/genetics , Sodium-Potassium-Exchanging ATPase/metabolism , Zebrafish , Zebrafish Proteins/biosynthesisABSTRACT
OBJECTIVES: The goal of this work was to investigate iron deposition in the basal ganglia and thalamus in symptomatic and asymptomatic leucine-rich repeat kinase 2 (LRRK2) and Parkin-associated Parkinson's disease (PD), using R2* relaxometry rate. METHODS: Twenty subjects with genetic PD (four symptomatic and two asymptomatic Parkin subjects, nine symptomatic and five asymptomatic LRRK2 subjects) were compared with 20 patients with idiopathic PD (IPD) and 20 healthy subjects. Images were obtained at 3 teslas, using multi-echo T2 and T2* sequences. R2 and R2* values were calculated in the substantia nigra (SN), the striatum, the globus pallidus, and the thalamus. RESULTS: The R2* values in the SN were increased in IPD and mutation-carrying patients as compared with controls and in mutation-carrying patients as compared with IPD. Asymptomatic mutation carriers showed higher R2* values than controls and did not differ from IPD patients. No changes were seen in the other structures or in R2 values. CONCLUSION: These results are consistent with increased iron load in LRRK2- and Parkin-mutation carriers. The increased R2* in asymptomatic PD-mutation carriers suggests that iron deposition occurs early during the preclinical phase of the disease. R2* measurements may be used as markers for investigating nigrostriatal damage in preclinical mutation-carrying patients.
Subject(s)
Iron/metabolism , Magnetic Resonance Imaging/methods , Parkinson Disease/metabolism , Protein Serine-Threonine Kinases/genetics , Substantia Nigra/metabolism , Ubiquitin-Protein Ligases/genetics , Adult , Aged , Female , Globus Pallidus/metabolism , Heterozygote , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Male , Middle Aged , Mutation , Neostriatum/metabolism , Parkinson Disease/genetics , Thalamus/metabolismABSTRACT
Mutations in leucine-rich repeat kinase 2 gene (LRRK2) are implicated in autosomal dominant familial and sporadic Parkinson's disease (sPD). Given its relative frequency in PD and its putative function in several cellular pathways that are known to be impaired in the disease, we wanted to tackle LRRK2 physiological role and to address its potential as a PD therapeutic target. We investigated the impact of pharmacological inhibition of LRRK2 kinase activity in control and PD cell function. We provide evidence that physiologically LRRK2, through its kinase activity, regulates mitochondrial fission events and facilitates autophagic degradation by modulating lysosomal cellular localization. Upon LRRK2 inhibition, normal fission decreases, leading to the elongation of mitochondrial network which contributes to a poor degradation of deficient mitochondria. Moreover, LRRK2 inhibition promotes lysosomal perinuclear clustering, through Rab7 that further hinders autophagosomes degradation. These events induce a decrease in the autophagic flow, which contributed directly to a decreased proteolytic degradation of damaged mitochondria. These data resembled the results observed in sPD cells. Interestingly, the LRRK2 kinase activity is increased in sPD cells, and despite its inhibition recovers mitochondrial cellular localization, it did not improve microtubule network-dependent trafficking. Our results provide novel insights into the multiple mechanisms that dictate the association between LRRK2 and mitophagy in sPD, and contribute with new findings that could have important therapeutic implications.
Subject(s)
Autophagy/physiology , Mitochondria/metabolism , Neurons/metabolism , Parkinson Disease/metabolism , Protein Serine-Threonine Kinases/metabolism , Aged , Aged, 80 and over , Female , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Male , Middle Aged , Phosphorylation , Protein Transport , ProteolysisABSTRACT
LRRK2 mutations are a frequent cause of familial Parkinson disease (PD) and are also found in a number of sporadic PD cases. PD-linked G2019S and I2020T mutations in the kinase domain of LRRK2 result in elevated kinase activity, which is required for the toxicity of these pathogenic variants in cell and animal models of PD. We recently reported that LRRK2 interacts with and phosphorylates a number of mammalian ribosomal proteins, several of which exhibit increased phosphorylation via both G2019S and I2020T LRRK2. Blocking the phosphorylation of ribosomal protein s15 through expression of phospho-deficient T136A s15 prevents age-associated locomotor deficits and dopamine neuron loss caused by G2019S LRRK2 expression in Drosophila indicating that s15 is a pathogenic LRRK2 substrate. We previously described that G2019S LRRK2 causes an induction of bulk mRNA translation that is blocked by T136A s15 or the protein synthesis inhibitor anisomycin. Here, we report the protective effects of the eIF4E/eIF4G interaction inhibitor 4EGI-1, in preventing neurodegenerative phenotypes in G2019S LRRK2 flies, and discuss how our findings and those of other groups provide a framework to begin investigating the mechanistic impact of LRRK2 on translation.
Subject(s)
Drosophila Proteins/metabolism , Drosophila/metabolism , Hydrazones/therapeutic use , Parkinson Disease/enzymology , Protein Serine-Threonine Kinases/metabolism , Ribosomal Proteins/metabolism , Thiazoles/therapeutic use , Animals , Disease Models, Animal , Drug Evaluation, Preclinical , Hydrazones/pharmacology , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Parkinson Disease/genetics , Parkinson Disease/prevention & control , Phenotype , Protein Biosynthesis/drug effects , Thiazoles/pharmacologyABSTRACT
Oxidative stress including DNA damage, increased lipid and protein oxidation, are important features of aging and neurodegeneration suggesting that endogenous antioxidant protective pathways are inadequate or overwhelmed. Importantly, oxidative protein damage contributes to age-dependent accumulation of dysfunctional mitochondria or protein aggregates. In addition, environmental toxins such as rotenone and paraquat, which are risk factors for the pathogenesis of neurodegenerative diseases, also promote protein oxidation. The obvious approach of supplementing the primary antioxidant systems designed to suppress the initiation of oxidative stress has been tested in animal models and positive results were obtained. However, these findings have not been effectively translated to treating human patients, and clinical trials for antioxidant therapies using radical scavenging molecules such as α-tocopherol, ascorbate and coenzyme Q have met with limited success, highlighting several limitations to this approach. These could include: (1) radical scavenging antioxidants cannot reverse established damage to proteins and organelles; (2) radical scavenging antioxidants are oxidant specific, and can only be effective if the specific mechanism for neurodegeneration involves the reactive species to which they are targeted and (3) since reactive species play an important role in physiological signaling, suppression of endogenous oxidants maybe deleterious. Therefore, alternative approaches that can circumvent these limitations are needed. While not previously considered an antioxidant system we propose that the autophagy-lysosomal activities, may serve this essential function in neurodegenerative diseases by removing damaged or dysfunctional proteins and organelles.
Subject(s)
Antioxidants/physiology , Autophagy , Lysosomes/physiology , Neurodegenerative Diseases/metabolism , Aging/metabolism , Animals , Antioxidants/pharmacology , Antioxidants/therapeutic use , Antiparkinson Agents/pharmacology , Antiparkinson Agents/therapeutic use , Brain/metabolism , Clinical Trials as Topic , Disease Models, Animal , Drug Evaluation, Preclinical , Humans , Lipid Peroxidation , Nerve Tissue Proteins/metabolism , Neurodegenerative Diseases/drug therapy , Neurodegenerative Diseases/pathology , Neuroprotective Agents/pharmacology , Neuroprotective Agents/therapeutic use , Oxidation-Reduction , Oxidative Stress , Oxidoreductases/physiology , Parkinsonian Disorders/drug therapy , Parkinsonian Disorders/metabolism , Parkinsonian Disorders/pathology , Peroxynitrous Acid/metabolismABSTRACT
To develop transgenic lines for conditional expression of desired genes in rats, we generated several lines of the transgenic rats carrying the tetracycline-controlled transactivator (tTA) gene. Using a vigorous, ubiquitous promoter to drive the tTA transgene, we obtained widespread expression of tTA in various tissues. Expression of tTA was sufficient to strongly activate its reporter gene, but was below the toxicity threshold. We examined the dynamics of Doxycycline (Dox)-regulated gene expression in transgenic rats. In the two transmittable lines, tTA-mediated activation of the reporter gene was fully subject to regulation by Dox. Dox dose-dependently suppressed tTA-activated gene expression. The washout time for the effects of Dox was dose-dependent. We tested a complex regime of Dox administration to determine the optimal effectiveness and washout duration. Dox was administered at a high dose (500 microg/ml in drinking water) for two days to reach the effective concentration, and then was given at a low dose (20 microg/ml) to maintain effectiveness. This regimen of Dox administration can achieve a quick switch between ON and OFF statuses of tTA-activated gene expression. In addition, administration of Dox to pregnant rats fully suppressed postnatal tTA-activated gene expression in their offspring. Sufficient levels of Dox are present in mother's milk to produce maximal efficacy in nursing neonates. Administration of Dox to pregnant or nursing rats can provide a continual suppression of tTA-dependent gene expression during embryonic and postnatal development. The tTA transgenic rat allows for inducible and reversible gene expression in the rat; this important tool will be valuable in the development of genetic rat models of human diseases.