The R1441C mutation of LRRK2 disrupts GTP hydrolysis

Mutations in Leucine Rich Repeat Kinase 2 (LRRK2) are the leading genetic cause of Parkinson's disease (PD). LRRK2 is predicted to contain kinase and GTPase enzymatic domains, with recent evidence suggesting that the kinase activity of LRRK2 is central to the pathogenic process associated with this protein. The GTPase domain of LRRK2 plays an important role in the regulation of kinase activity. To investigate how the GTPase domain might be related to disease, we examined the GTP binding and hydrolysis properties of wild type and a mutant form of LRRK2. We show that LRRK2 immunoprecipitated from cells has a detectable GTPase activity that is disrupted by a familial mutation associated with PD located within the GTPase domain, R1441C.     

Disturbance of Iron Metabolism as a Contributing Factor to SN Hyperechogenicity in Parkinson’s Disease: Implications for Idiopathic and Monogenetic Forms

A number of investigations have provided evidence for a central role of iron in the pathogenesis of Parkinson’s disease (PD). Recently it could be demonstrated that iron related changes of the substantia nigra may be one important factor contributing to the hyperechogenicity typicall visualized by transcranial sonography in idiopathic PD. Moreover, also patients with monogenetically caused PD show this hyperechogenicity, although to a lesser extent. According to numerous findings and experiments it seems plausible that iron also contributes to the pathophysiological cascades in the monogenetic forms of PD. Therefore, it is not only essential to acknowledge the pivotal role of iron for PD, but also to enhance the effort in finding therapeutic strategies to prevent the impact of iron on neurodegenerative processes. Moreover, early detection of subjects at risk is essential for the application of therapeutic strategies at a stage at which neuroprotection is still possible. Special issue dedicated to Dr. Moussa Youdim     

The Effects of Piroxicam in the Attenuation of MPP+/MPTP Toxicity In Vitro and In Vivo

Several lines of evidence support the neuroprotective action of cyclooxygenase-2 (COX-2) inhibitors in various models of Parkinson’s disease (PD). In the current study, we investigated the neuroprotective properties of several COX inhibitors against 1-methyl-4-phenylpyridinium (MPP+) in neuroblastoma Neuro 2A (N-2A) cells in vitro and the protection against degeneration of substantia nigra pars compacta (SNc) dopaminergic (DA) neurons after the administration of 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) in C57/BL6 male mice. The data obtained demonstrate a lack of protective effects observed by COX 1-2 inhibitors ibuprofen and acetylsalicylic acid against MPP+ toxicity in N-2A, where piroxicam was protective in a dose dependent manner (MPP+ control: 15 ± 2% MPP+ piroxicam: 5 mM 89 ± 4%). The data also indicate a drop in mitochondrial oxygen (O₂) consumption and ATP during MPP+ toxicity with no restoration of mitochondrial function concurrent to a heightened concentration of somatic ATP during piroxicam rescue. These findings indicate that the neuroprotective effects of COX inhibitors against MPP+ are not consistent, but that piroxicam may work through an unique mechanism to propel anaerobic energy metabolism. On the other hand, using mice, piroxicam (20 mg/kg) was effective against MPTP-induced dopaminergic degeneration in the (SNc) and loss of locomotive function in mice. Administering a 3 day pre-treatment of piroxicam (20 mg/kg) was effective in antagonizing the losses in SNc tyrosine hydroxylase protein expression, SNc DA concentration and associated anomaly in ambulatory locomotor activity. It was concluded from these findings that piroxicam is unique among COX inhibitors in providing very significant neuroprotection against MPP+ in vitro and in vivo.     

LRRK2 mutations and neurotoxicant susceptibility

Interactions between genetic and environmental factors are thought to contribute to the pathogenesis of the majority of Parkinson’s disease (PD) cases. However, our understanding of these interactions is at an early stage. Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of hereditary PD. Penetrance of LRRK2 mutations is incomplete and variable, suggesting that other environmental or genetic factors may contribute to the development of the disorder. Recently, using animal models, several attempts have been made to understand if LRRK2 may mediate sensitivity to environmental neurotoxicants. Here, we critically review the most current data on how LRRK2 mutations influence neurotoxicity in PD models.     

Mitochondria and Neurodegeneration

Many lines of evidence suggest that mitochondria have a central role in ageing-related neurodegenerative diseases. However, despite the evidence of morphological, biochemical and molecular abnormalities in mitochondria in various tissues of patients with neurodegenerative disorders, the question “is mitochondrial dysfunction a necessary step in neurodegeneration?” is still unanswered. In this review, we highlight some of the major neurodegenerative disorders (Alzheimer’s disease, Parkinson’s disease, Amyotrophic lateral sclerosis and Huntington’s disease) and discuss the role of the mitochondria in the pathogenetic cascade leading to neurodegeneration.     

Correlations between indices of P300 EEG potential,cognitive tests,and variational pulsometry in Parkinsonian patients

The parameters of an event-related EEG potential (ERP), P300 wave, are now extensively used as objective neurophysiological indices of the state of cognitive functions. At the same time, information on the effects of the autonomic nervous system on the parameters of P300 is limited. In Parkinson’s disease clinics, in addition to the leading motor disorders, more or less clear psychoemotional, cognitive, and autonomic (in particular cardiovascular) impairments are usually observed. This allows one to study the dependence between the cardiovascular dysfunction and intensity of cognitive disorders in Parkinsonian patients. In our study on this contingent, we analyzed correlations between the parameters of P300 potential, indices of the state of the cognitive sphere (determined using a questionnaire, Mini Mental State Examination, MMSE, and a Luriya’s test), and indices of variational pulsometry. Thirty-five Parkinsonian patients (49 to 74 years, severity of disease 1.5 to 3.0 by the international classification) were examined. We found a negative influence of excessive sympathetic tonus in cardiovascular control on the state of cognitive functions. The latency of P300 potential was longer in patients with greater intensities of sympathetic influences on the cardiovascular system. The coefficients of correlation of the latency of P300 with the amplitude of mode of R-R intervals (AMo), index of tension in the regulatory systems by Baevskii (IT), and index of autonomic balance by Baevskii (IAB) were 0.52 (P < 0.01), 0.36 (P < 0.05), and 0.37 (P < 0.05), respectively. The above autonomic indices demonstrated significant negative correlations with the volume of short-term memory measured by Luriya’s test. The P300 latency, in turn, showed negative correlations with the memory volume estimated by the MMSE scale and Luriya’s test. With increase in the age of patients, the degree of the above-mentioned correlations between the P300 latency, memory volume (by Luriya’s test), and parameters of variational pulsometry increased. Our data emphasize the expedience of “routine” studies of the balance of sympathetic and parasympathetic control in pathological states accompanied by clear or subclinical cognitive disorders. Early recognition of cardiovascular dysfunction and its corresponding therapeutic correction should improve the state of brain functions and quality of life in patients suffering from neurodegenerative diseases, in particular from Parkinson’s disease. Neirofiziologiya/Neurophysiology, Vol. 40, No. 1, pp. 43–52, January–February, 2008.     

Age-dependent and cell-population-restricted LRRK2 expression in normal mouse spleen

Leucine-rich repeat kinase 2 (LRRK2) is the causal molecule of familial Parkinson’s disease (PD), but its true physiological function remains unknown. In the normal mouse, LRRK2 is expressed in kidney, spleen, and lung at much higher levels than in brain, suggesting that LRRK2 may play an important role in these organs. Analysis of age-related changes in LRRK2 expression demonstrated that expression in kidney, lung, and various brain regions was constant throughout adult life. On the other hand, expression of both LRRK2 mRNA and protein decreased markedly in spleen in an age-dependent manner. Analysis of purified spleen cells indicated that B lymphocytes were the major population expressing LRRK2, and that T lymphocytes showed no expression. Consistently, the B lymphocyte surface marker CD19 exhibited an age-dependent decrease of mRNA expression in spleen. These results suggest a possibly novel function of LRRK2 in the immune system, especially in B lymphocytes.     

The Parkinson’s disease kinase LRRK2 autophosphorylates its GTPase domain at multiple sites

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of inherited Parkinson’s disease (PD). The protein is large and complex, but pathogenic mutations cluster in a region containing GTPase and kinase domains. LRRK2 can autophosphorylate in vitro within a dimer pair, although the significance of this reaction is unclear. Here, we mapped the sites of autophosphorylation within LRRK2 and found several potential phosphorylation sites within the GTPase domain. Using mass spectrometry, we found that Thr1343 is phosphorylated and, using kinase dead versions of LRRK2, show that this is an autophosphorylation site. However, we also find evidence for additional sites in the GTPase domain and in other regions of the protein suggesting that there may be multiple autophosphorylation sites within LRRK2. These data suggest that the kinase and GTPase activities of LRRK2 may exhibit complex autoregulatory interdependence.     

Dopaminergic Agonists: Possible Neurorescue Drugs Endowed with Independent and Synergistic Multisites of Action

Dopaminergic agonists have been usually used as adjunctive therapy for the cure of Parkinson’s disease (PD). It is generally believed that treatment with these drugs is symptomatic rather then curative and does not stop or delay the progression of neuronal degeneration. However, several DA agonists of the DA D2–receptor family (including D2, D3 and D4-subtypes) have recently been shown to possess neuroprotective properties in different in vitro and in vivo experimental PD models. Here we summarize some recent data from our and other groups underlining the wide pharmacological spectrum of DA agonists currently used for treating PD patients. In particular, the mechanism of action of different DA agonists does not appear to be restricted to the stimulation of selective DA receptor subtypes being these drugs endowed with intrinsic, independent, and peculiar antioxidant effects. This activity may represent an additional pharmacological property contributing to their clinical efficacy in PD. Special issue dedicated to Dr. Moussa Youdim.     

Cell death pathways in Parkinson’s disease: proximal triggers, distal effectors, and final steps

Parkinson’s disease (PD) is a common neurodegenerative disorder. Neuronal cell death in PD is still poorly understood, despite a wealth of potential pathogenic mechanisms and pathways. Defects in several cellular systems have been implicated as early triggers that start cells down the road toward neuronal death. These include abnormal protein accumulation, particularly of alpha-synuclein; altered protein degradation via multiple pathways; mitochondrial dysfunction; oxidative stress; neuroinflammation; and dysregulated kinase signaling. As dysfunction in these systems mounts, pathways that are more explicitly involved in cell death become recruited. These include JNK signaling, p53 activation, cell cycle re-activation, and signaling through bcl-2 family proteins. Eventually, neurons become overwhelmed and degenerate; however, even the mechanism of final cell death in PD is still unsettled. In this review, we will discuss cell death triggers and effectors that are relevant to PD, highlighting important unresolved issues and implications for the development of neuroprotective therapies.     

Protective Effects of Asiatic Acid on Rotenone- or H<Subscript>2</Subscript>O<Subscript>2</Subscript>-Induced Injury in SH-SY5Y Cells

Parkinson’s disease (PD) is a progressive neurodegenerative disorder with a prevalence of 1–2% in people over the age of 50. Mitochondrial dysfunction occurred in PD patients showing a 15–30% loss of activity in complex I. Asiatic acid (AA), a triterpenoid, is an antioxidant and used for depression treatment, but the effect of AA against PD-like damage has never been reported. In the present study, we investigated the protective effects of AA against H₂O₂ or rotenone-induced cellular injury and mitochondrial dysfunction in SH-SY5Y cells. Mitochondrial membrane potential (MMP) and the expression of voltage-dependent anion channel (VDAC) were detected with or without AA pretreatment following cellular injury to address the possible mechanisms of AA neuroprotection. The results showed that pre-treatment of AA (0.01–100 nM) protected cells against the toxicity induced by rotenone or H₂O₂. In addition, MMP dissipation occurred following the exposure of rotenone, which could be prevented by AA treatment. More interestingly, pre-administration of AA inhibited the elevation of VDAC mRNA and protein levels induced by rotenone(100 nM) or H₂O₂ (300 μM).These data indicate that AA could protect neuronal cells against mitochondrial dysfunctional injury and suggest that AA might be developed as an agent for PD prevention or therapy. Special issue article in honor of Dr. Akitane Mori.     

Genes and the Environment in Neurodegeneration

Neurodegenerative diseases are a heterogeneous group of pathologies which includes complex multifactorial diseases, monogenic disorders and disorders for which inherited, sporadic and transmissible forms are known. Factors associated with predisposition and vulnerability to neurodegenerative disorders may be described usefully within the context of gene–environment interplay. There are many identified genetic determinants for neurodegeneration, and it is possible to duplicate many elements of recognized human neurodegenerative disorders in animal models of the disease. However, there are similarly several identifiable environmental influences on outcomes of the genetic defects; and the course of a progressive neurodegenerative disorder can be greatly modified by environmental elements. In this review we highlight some of the major neurodegenerative disorders (Alzheimer’s disease, Parkinson’s disease, Amyotrophic lateral sclerosis, Huntington’s disease, and prion diseases.) and discuss possible links of gene–environment interplay including, where implicated, mitochondrial genes.     

Reactive Macrophages Increase Oxidative Stress and Alpha-Synuclein Nitration During Death of Dopaminergic Neuronal Cells in Co-Culture: Relevance to Parkinson’s Disease

Parkinson’s disease (PD) is characterized by progressive degeneration of dopaminergic neurons and a substantial decrease in the neurotransmitter dopamine in the nigro-striatal region of the brain. Increased markers of oxidative stress, activated microglias and elevated levels of pro-inflammatory cytokines have been identified in the brains of patients with PD. Although the precise mechanism of loss of neurons in PD remains unclear, these findings suggest that microglial activation may contribute directly to loss of dopaminergic neurons in PD patients. In the present study, we tested the hypothesis that activated microglia induces nitric oxide-dependent oxidative stress which subsequently causes death of dopaminergic neuronal cells in culture. We employed lipopolysaccharide (LPS) stimulated mouse macrophage cells (RAW 264.7) as a reactive microglial model and SH-SY5Y cells as a model for human dopaminergic neurons. LPS stimulation of macrophages led to increased production of nitric oxide in a time and dose dependent manner as well as subsequent generation of other reactive nitrogen species such as peroxynitrite anions. In co-culture conditions, reactive macrophages stimulated SH-SY5Y cell death characterized by increased peroxynitrite concentrations and nitration of alpha-synuclein within SH-SY5Y cells. Importantly 1400W, an inhibitor of the inducible nitric oxide synthase provided protection from cell death via decreasing the levels of nitrated alpha-synuclein. These results suggest that reactive microglias could induce oxidative stress in dopaminergic neurons and such oxidative stress may finally lead to nitration of alpha-synuclein and death of dopaminergic neurons in PD.     

Interleukin-6 Serum Levels in Patients with Parkinson’s Disease

Several lines of evidence suggest that neuroimmune mechanisms may be involved in the neurodegenerative process of Parkinson’s disease (PD). Interleukin-6 (IL-6) is increased in the nigrostriatal region and in the cerebrospinal fluid of patients with PD. IL-6 serum level was evaluated in PD patients. The effects of levodopa treatment and disease severity on IL-6 were also studied. The IL-6 levels were similar between PD patients (treated and not treated) and controls. However, there was a negative correlation of IL-6 levels and the activities of daily living scale (P < 0.05), indicating that patients with more severe disease have higher levels of this cytokine. No correlation involving levodopa treatment and IL-6 serum level was found. The results suggest that only marginal effects of IL-6 occur on the peripheral immune system, and that the role of IL-6 and others neuroimmune factors needs to be well elucidated on PD.     

Voltage-gated Calcium Channels Provide an Alternate Route for Iron Uptake in Neuronal Cell Cultures

Recent studies suggest that iron enters cardiomyocytes via the L-type voltage-gated calcium channel (VGCC). The neuronal VGCC may also provide iron entry. As with calcium, extraneous iron is associated with the pathology and progression of neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease. VGCCs, ubiquitously expressed, may be an important route of excessive entry for both iron and calcium, contributing to cell toxicity or death. We evaluated the uptake of ⁴⁵Ca²⁺ and ⁵⁵Fe²⁺ into NGF-treated rat PC12, and murine N-2α cells. Iron not only competed with calcium for entry into these cells, but iron uptake (similar to calcium uptake) was inhibited by nimodipine, a specific L-type VGCC blocker, and enhanced by FPL 64176, an L-VGCC activator, in a dose-dependent manner. Taken together, these data suggest that voltage-gated calcium channels are an alternate route for iron entry into neuronal cells under conditions that promote cellular iron overload toxicity. Special issue dedicated to Dr. Moussa Youdim.     

Mitochondrial Dysfunction in Neurodegenerative Diseases

Mitochondria play a pivotal role in mammalian cell metabolism, hosting a number of important biochemical pathways including oxidative phosphorylation. As might be expected from this fundamental contribution to cell function, abnormalities of mitochondrial metabolism are a common cause of human disease. Primary mutations of mitochondrial DNA result in a diverse group of disorders often collectively referred to as the mitochondrial encephalomyopathies. Perhaps more importantly in numerical terms are those neurodegenerative diseases caused by mutations of nuclear genes encoding mitochondrial proteins. Finally there are mitochondrial abnormalities induced by secondary events e.g. oxidative stress that may contribute to senescence, and environmental toxins that may cause disease either alone or in combination with a genetic predisposition. Special issue article in honor of Dr. Anna Maria Giuffrida-Stella.     

Applications of electron paramagnetic resonance to studies of neurological disease

Electron paramagnetic resonance spectroscopy (EPR) has the potential to give much detail on the structure of the paramagnetic transition ion coordination sites, principally of Cu²⁺, in a number of proteins associated with central nervous system diseases. Since these sites have been implicated in misfolding/mis-oligomerisation events associated with neurotoxic molecular species and/or the catalysis of damaging redox reactions in neurodegeneration, an understanding of their structure is important to the development of therapeutic agents. Nevertheless EPR, by its nature an in vitro technique, has its limitations in the study of such complex biochemical systems involving self-associating proteins that are sensitive to their chemical environment. These limitations are at the instrumental and theoretical level, which must be understood and the EPR data interpreted in the light of other biophysical and biochemical studies if useful conclusions are to be drawn. Australian Society for Biophysics Special Issue: Metals and Membranes in Neuroscience, held in Melbourne on 11 July 2007.     

Modeling synucleinopathies in genetically modified animals: Successes and failures

The synuclein family and particularly α-synuclein takes a central part in aetiology and pathogenesis of Parkinson’s disease—one of the most common human neurodegenerative diseases. The pathological changes in certain other neurodegenerative diseases are also linked to changes in the metabolism and function of α-synuclein, hence comprising a new group of diseases—synucleinopathies. The molecular and cellular mechanisms that are involved in the development of neurodegeneration in synucleinopathies are still largely unknown. As a result, the therapeutic approaches to the treatment of synucleinopathies are inadequately tampered. The development of models of neurodegenerative process in laboratory animals plays a crucial role in the study of these molecular mechanisms. Recently a special emphasis was placed on transgenic animal models with modified expression of genes, whose mutations are associated with inherited forms of human neurodegenerative diseases. The current review is devoted to the analysis of different models of synucleinopathies as a result of genetic modifications of α-synuclein expression.     

Identification of chemicals to inhibit the kinase activity of leucine-rich repeat kinase 2 (LRRK2), a Parkinson's disease-associated protein

Parkinson’s disease (PD) is a late-onset neurodegenerative disease which occurs at more than 1% in populations aging 65-years and over. Recently, leucine-rich repeat kinase 2 (LRRK2) has been identified as a causative gene for autosomal dominantly inherited familial PD cases. LRRK2 G2019S which is a prevalent mutant found in familial PD patients with LRRK2 mutations, exhibited kinase activity stronger than that of the wild type, suggesting the LRRK2 kinase inhibitor as a potential PD therapeutics. To develop such therapeutics, we initially screened a small chemical library and selected compound 1, whose IC₅₀ is about 13.2 μM. To develop better inhibitors, we tested five of the compound 1 derivatives and found a slightly better inhibitor, compound 4, whose IC₅₀ is 4.1 μM. The cell-based assay showed that these two chemicals inhibited oxidative stress-induced neurotoxicity caused by over-expression of a PD-specific LRRK2 mutant, G2019S. In addition, the structural analysis of compound 4 suggested hydrogen bond interactions between compound 4 and Ala 1950 residue in the backbone of the ATP binding pocket of LRRK2 kinas domain. Therefore, compound 4 may be a promising lead compound to further develop a PD therapeutics based on LRRK2 kinase inhibition.