Clinical-Pharmacogenetic Predictive Models for Time to Occurrence of Levodopa Related Motor Complications in Parkinson’s Disease

The NLRP3 inflammasome signaling pathway is a major contributor to the neuroinflammatory process in the central nervous system. Oxidative stress and mitochondrial dysfunction are key pathophysiological processes of many chronic neurodegenerative diseases, including Parkinson’s disease (PD). However, the inter-relationship between mitochondrial defects and neuroinflammation is not well understood. In the present study, we show that impaired mitochondrial function can augment the NLRP3 inflammasome-driven proinflammatory cascade in microglia. Primary mouse microglia treated with the common inflammogen LPS increased NLRP3 and pro-IL-1β expression. Interestingly, exposure of LPS-primed microglial cells to the mitochondrial complex-I inhibitory pesticides rotenone and tebufenpyrad specifically potentiated the NLRP3 induction, ASC speck formation and pro-IL-1β processing to IL-1β in a dose-dependent manner, indicating that mitochondrial impairment heightened the NLRP3 inflammasome-mediated proinflammatory response in microglia. The neurotoxic pesticide-induced NLRP3 inflammasome activation was accompanied by bioenergetic defects and lysosomal dysfunction in microglia. Furthermore, the pesticides enhanced mitochondrial ROS generation in primary microglia, while amelioration of mitochondria-derived ROS by the mitochondria-targeted antioxidant mito-apocynin completely abolished IL-1β release, indicating mitochondrial ROS drives potentiation of the NLRP3 inflammasome in microglia. Exposure to conditioned media obtained from mitochondrial inhibitor-treated, LPS-primed microglial cells, but not unprimed cells, induced dopaminergic neurodegeneration in cultured primary mesencephalic and human dopaminergic neuronal cells (LUHMES). Notably, our in vivo results with chronic rotenone rodent models of PD further support the activation of proinflammatory NLRP3 inflammasome signaling due to mitochondrial dysfunction. Collectively, our results demonstrate that mitochondrial impairment in microglia can amplify NLRP3 inflammasome signaling, which augments the dopaminergic neurodegenerative process.

Introduction More than 50 years after its introduction by George C. Cotzias (Cotzias et al., 1967), levodopa still is the most effective treatment for the motor symptoms of Parkinson’s disease. In studies including patients in the pre-levodopa era, early treatment with levodopa proved to increase life expectancy (Diamond et al., 1987). However, long-term management of patients on chronic levodopa is hampered by the occurrence of motor fluctuations and dyskinesias which, as the disease progresses, usually become major causes of disability and reduced quality of life (Chapuis et al., 2005). To date, the pathophysiological mechanisms underlying the occurrence of levodopa-induced complications are still poorly understood. In animal studies, the occurrence of motor fluctuations and dyskinesias has been associated with the extent of nigral neuronal loss and with the duration and dose of levodopa therapy, as independent factors (Boyce et al., 1990; Guigoni et al., 2005; Jenner, 2008). Motor complications have been described to occur very early after the initiation of levodopa therapy in monkeys and humans with MPTP-induced parkinsonism, who display rapid and severe depletion of dopaminergic neurons (Williams, 1984; Ballard et al., 1985). However, current experimental models of levodopa-induced motor complications cannot replicate the pathophysiological changes that occur during the progressive neurodegenerative process of idiopathic Parkinson’s disease, mainly due to the rapid and extensive nigral denervation. Thus, the issue of the relationship between levodopa and motor complications should be addressed by clinical studies in patients with Parkinson’s disease. A number of large drug trials reported that the initial treatment with a dopamine agonist was associated with a lower incidence of motor complications than initial treatment with levodopa (Parkinson Study Group, 2000; Rascol et al., 2000; Fahn et al., 2004; reviewed in Stowe et al., 2008). Likewise, higher rates of dyskinesias were reported in association with higher cumulative doses of levodopa (Hauser et al., 2006; Olanow et al., 2013) and longer duration of levodopa treatment (Miyawaki et al., 1997; Grandas et al., 1999; Schrag and Quinn, 2000). Overall, these observations led clinicians to consider levodopa therapy as an additional source of disability within the natural history of Parkinson’s disease (Poewe and Mahlknecht, 2009) and supported the clinical practice of delaying the initiation of levodopa therapy as long as possible to reduce the risk of motor complications (Fahn, 1999, 2006; Kieburtz, 2008). The ELLDOPA study was specifically designed to provide conclusive evidence as to whether levodopa could be safely initiated early or should be delayed in newly diagnosed patients with Parkinson’s disease (Fahn, 1999). The ELLDOPA study was specifically designed to provide conclusive evidence as to whether levodopa could be safely initiated early or should be delayed in newly diagnosed patients with Parkinson’s disease (Fahn, 1999). Unfortunately, although the ELLDOPA study did not find any clinical or imaging evidence indicating that early use of levodopa could negatively impact the course of the disease (Fahn et al., 2004), it failed to change the patterns of treatment of Parkinson’s disease (as neurologists remained concerned about levodopa’s likelihood to induce motor complications) (Fahn, 2006). Therefore, the question as to whether the risk of levodopa-induced motor complications is primarily associated with levodopa therapy or disease progression itself remained unanswered. Theoretically, the differences between the effects of treatment-related variables (i.e. levodopa therapy duration versus daily dose) and the effects of disease progression (i.e. disease duration versus severity) in promoting motor complications without confounders (such as concomitant dopamine agonist therapy) could be conclusively demonstrated only by a trial including patients with Parkinson’s disease left untreated for several years after onset of disease and then followed-up after the initiation of levodopa. Obviously, such a study design is not ethically acceptable. Early motor complications have been described in series including patients from the pre-levodopa era, who had longer disease duration at the initiation of levodopa therapy (Ahlskog and Muenter, 2011). However, the exact relationship between disease-related variables and levodopa dose could not be established, because of the tendency towards more aggressive levodopa dosing (due to the initial lack of DOPA decarboxylase inhibitors), and the lack of systematic assessment of motor fluctuation rates (Ahlskog and Muenter, 2011) and of motor symptom severity using validated rating tools [e.g. the Unified Parkinson’s Disease Rating Scale (UPDRS); Fahn et al., 2004; Poewe and Mahlknecht, 2009]. In sub-Saharan African countries, the access to medication is limited and the initiation of levodopa therapy often occurs several years after the onset of motor symptoms (Cilia et al., 2011; Dotchin et al., 2011), closely resembling what is described in series containing patients with Parkinson’s disease from the pre-levodopa era (Ahlskog and Muenter, 2011). In this scenario, we conducted a 4-year naturalistic study investigating a large cohort of patients with Parkinson’s disease from a sub-Saharan African country (Ghana), whose primary objective was to disentangle the relative role played by treatment-related variables and disease progression per se in the pathophysiology of motor complications. Materials and methods Participants All subjects consecutively attending three out-patient clinics in different regions of Ghana between December 2008 and November 2012 were examined and screened for any movement disorder by local neurologists (A.A., F.S.S.) or by a medical officer (M.C.). Parkinsonism was suspected by the local clinician on the basis of the presence of at least three of the four cardinal features (i.e. resting tremor, rigidity, bradykinesia, and postural or gait abnormality). Patients were then assessed in consecutive order by a neurologist specialized in movement disorders (R.C.) and by another movement disorder specialist (M.A., M.F., G.P.), who made the diagnosis according to current criteria (Hughes et al., 1992; Litvan et al., 2003). In case of any doubt or disagreement between the investigators, the patient was admitted to hospital for in-depth clinical evaluation and additional investigation (e.g. neuroimaging). Onset of the disease was defined at the first appearance of any motor symptom, as reported by the patient, a family member or a clinician. In case of any doubt or uncertainty, we adopted a recall technique tailored to major events in the patient’s life. Considering the relatively high average number of offspring and grandchildren, we used information on offspring to investigate the relationship between the year of onset and either major preschool milestones of development of grandchildren (e.g. birth, first unassisted steps) or other meaningful events in the family (e.g. wedding of offspring). Early onset was defined as age at onset ≤50 years. Family history for Parkinson’s disease was limited to first-degree relatives. Clinical work-up included the UPDRS from part I to part IV (Fahn and Elton, 1987) and Hoehn and Yahr (HY) staging (Hoehn and Yahr, 1967). Major milestones of disease progression were assessed using the UPDRS items of part I (psychosis, item 2 score ≥ 2; depression or apathy, sum of items 3 + 4 ≥ 4), part II (dysphagia, item 7 score ≥ 2; falls, item 13 score ≥ 2) and part III (postural instability, item 30 score ≥ 2) (as described in Merola et al., 2011). Dementia was diagnosed according to the Diagnostic and Statistical Manual, Fourth Edition, Text Revision (DSM-IV-TR) criteria (American Psychiatric Association, 2000). Motor phenotype at disease presentation was assessed in the ‘OFF’ state (Jankovic et al., 1990). Acute levodopa challenge was performed using dispersible levodopa/benserazide 100 mg + 50 mg, giving either a 150-mg or 200-mg levodopa dose, according to body weight (≤ or >70 kg, respectively); all patients were assessed after 12-h medication withdrawal (OFF state) and 90 min after levodopa intake (ON state). In drug-naïve patients, levodopa therapy was initiated immediately, starting from 50 mg once daily and slowly titrating up to 100 mg three times daily, 20–30 min before meals. If further treatment was required, an additional 50 or 100 mg of levodopa could be added to each of the doses, as appropriate, to achieve adequate control of motor symptoms. At each visit, the presence of motor fluctuations and dyskinesias was assessed by recall and prolonged direct observation, and time of their first occurrence was recorded. Patients were observed for a minimum of 4 h after the administration of dispersible levodopa/benserazide to monitor motor response. In case of uncertainty about either the response to levodopa therapy or the presence of motor fluctuations or dyskinesias, patients were admitted to hospital. Motor fluctuations were defined as predictable wearing-off, unpredictable ON–OFF fluctuations and sudden OFF periods according to UPDRS part IV (Fahn and Elton, 1987). Dyskinesias were defined as abnormal involuntary movements, including chorea and dystonia, that could be peak dose or diphasic; OFF-related dystonia was not included. In-between the follow-up visits by the principal investigator, local doctors visited all patients every 2 months to review therapy tolerability, onset of motor complications, and to provide levodopa supplies free of charge (Cilia et al., 2011). Follow-up visits were performed by the principal investigator every 6 months during the study period. A large cohort of consecutive patients with Parkinson’s disease (n = 2282) assessed for the first time at the Parkinson Institute in Milan during the same 4-year period was considered as control group for demographic and general clinical features. During this period, a total of nine neurologists examined on average three new patients every work week (of whom 42–48% were diagnosed as probable idiopathic Parkinson’s disease) for a total of 46 weeks in a year. Nested matched subgroups were then used to investigate the frequency and severity of major motor and non-motor symptoms, including the relationship between levodopa therapy and the onset of motor complications. Ethics The study was performed in agreement with the principles of the Declaration of Helsinki and the protocol was approved by the local Ethics Committee. We obtained written informed consent from every patient; an additional consent was requested for video recording. Informed consent was translated into local Ghanaian dialect whenever required and/or it was provided by a first-degree relative in cases with clinical dementia. Statistical analysis Data were analysed using the software program SPSS (Windows Release 17.0; SPSS Inc.). We conducted a cross-sectional data analysis and a prospective cohort study. A two-sided P-value of 40.5) between disease severity and levodopa daily dose. d,eBest predictive models for motor complications were those including Levodopa dose, duration of levodopa and duration of disease at their onset. Significant values (p 4 years. In agreement with cross-sectional findings, wearing-off and dyskinesias appeared very early, after a median levodopa duration of 6 months and at a median disease duration of 7 years (IQR, 4.3–9). As paradigmatic case, we describe a 69-year-old patient with Parkinson’s disease with a 12-year history of untreated disease and severe motor disability, who developed wearing-off phenomena 24 h after the introduction of levodopa (each levodopa dose lasted <3 h), whereas peak-dose dyskinesias took 4–5 weeks to appear; levodopa dose was 150 mg four times/day (6.5 mg/kg/day) (Supplementary Video Segment 2). Discussion In the present 4-year naturalistic study, we had the unique opportunity to observe the effects of chronic levodopa therapy on a large sample of patients with Parkinson’s disease untreated for years and produced compelling evidence that the duration of levodopa therapy itself is not a risk factor for the occurrence of ‘levodopa-induced’ motor complications. The results were consistently provided by a cross-sectional analysis of data collected at the baseline assessment and further supported by the subsequent prospective follow-up of a subgroup of newly diagnosed, untreated patients with Parkinson’s disease. In the first part of the study, we showed that the onset of motor complications occurred at comparable disease duration in the two populations, despite the large difference in the duration of exposure to levodopa. In line with data from community-based studies (Schrag and Quinn, 2000; Evans et al., 2011), motor fluctuations and dyskinesias occurred after a mean of 6 to 7 years from onset regardless of the timing of initiation of adequate levodopa therapy. Since the 1980s, there has been much debate about whether levodopa could be safely started early or late in the course of the disease (Fahn, 1999, 2006). A number of studies suggested that dyskinesias could be best predicted by high daily levodopa dose (Fahn et al., 2004) and longer duration of levodopa treatment (Miyawaki et al., 1997; Schrag and Quinn, 2000) and that delaying the initiation of levodopa was the best therapeutic strategy to prevent motor fluctuations and dyskinesias (Fahn and Calne, 1978; Melamed, 1986; Fahn, 1999; Montastruc et al., 1999; Rascol et al., 2000; Fahn et al., 2004). This induced neurologists to withhold the introduction of adequate levodopa therapy as long as possible (especially in younger patients) and patients to avoid it, generating a sort of widespread ‘levodopa phobia’ (Kurlan, 2005). However, whether the delay of onset of motor complications in patients initially treated with a dopamine agonist was primarily due to delaying levodopa or rather to the lower levodopa dose needed to compensate motor symptoms remained unclear. The putative protective role of delayed levodopa was challenged by post hoc analyses of the CALM-PD trial (Parkinson Study Group, 2000), which revealed that the incidence of dyskinesias after initiating levodopa was independent of the initial treatment, after adjusting for disease duration and levodopa daily dose (Constantinescu et al., 2007). Cumulative levodopa dose has been suggested to be an independent predictor of dyskinesias in patients with Parkinson’s disease (Hauser et al., 2006). However, whether this is primarily related to the duration of treatment or to the daily dose remained unclear. In an experimental study in 6-hydroxydopamine-lesioned rats, animals treated with higher levodopa doses for a short period developed dyskinesias earlier than rats treated chronically with lower doses and exposed to higher cumulative doses (Tsironis et al., 2008). Taken as a whole, these findings are in line with our data and further support the hypothesis that the timing of initiation of levodopa therapy does not modulate the risk of motor complications. Accordingly, the main variables we need to focus on are individual daily levodopa dose and disease progression itself. After a mean of 6 years from onset of motor symptoms, the rates of motor fluctuations and dyskinesias in Ghanaian patients on chronic levodopa therapy were 56% and 14%, respectively. Considerable variability in motor complication rates has been reported in literature so far, due to important differences in the population assessed, in levodopa dosage, in time from onset to the initiation of levodopa, and in disease duration and severity at assessment (Schrag and Quinn, 2000; Hauser et al., 2006; Stowe et al., 2008). The motor complication rates in series containing patients from the pre-levodopa era are similar to those found in Ghana after a comparable interval from onset to the initiation of levodopa (58% versus 56% of the present study; Ahsklog and Muenter, 2001). On the other hand, series containing pre-levodopa era patients reported a considerably higher frequency of early dyskinesias compared to the modern era series and to our ‘modern pre-levodopa era’ study, as ∼50% of patients developed dyskinesias after a median time of 6 months since the initiation of chronic levodopa therapy (Ahsklog and Muenter, 2001). Cotzias et al. (1969) were the first who reported the early appearance of involuntary movements after the initiation of levodopa therapy in 50% of patients with Parkinsonism, describing them as ‘dose dependent in each case’. However, in contrast to the present study, patients from the pre-levodopa era had considerably longer median disease duration at introduction of levodopa (6–10 years versus 4 years of the present study) and there was the bias of more aggressive levodopa dosing (reviewed in Ahsklog and Muenter, 2001; Fahn, 2006). Therefore, it was not possible to disentangle the long duration of the disease from the very high levodopa daily dose and establish which was the most relevant factor for the early development of dyskinesias. Similarly, early motor complications in patients with longstanding and untreated Parkinson’s disease have been described even in the modern era in association with high individual levodopa daily doses (Onofrj et al., 1998). In agreement with the modern-era literature, our multivariate analysis confirmed that the individual levodopa dosing regimen is a major risk factor for motor fluctuations and dyskinesias (Sharma et al., 2008; Olanow et al., 2013). Despite the collinearity between levodopa daily dose and disease severity, the early appearance of motor complications was not associated with levodopa overdosing in the Ghanaian cohort, as levodopa was slowly titrated up and kept at a relatively low dose. In particular, mean levodopa daily dose was slightly lower than the recommended initial target dose considered to be the threshold for dyskinesias in the post hoc analysis of the STRIDE-PD study (365 mg daily of the present study versus 400 mg daily in Olanow et al., 2013). This is even more evident when we consider that Ghanaian patients had longer disease duration at the initiation of levodopa (4 versus 2 years, respectively) and that none of them had concomitant dopamine agonist therapy compared to the majority of patients from the drug trial (∼60% in Olanow et al., 2013), thus unbalancing individual levodopa-equivalent daily dose even further. Overall, we believe that the primary cause for the early appearance of motor complications in the Ghanaian Parkinson’s disease population was disease progression rather than the relatively high levodopa daily dose. This hypothesis is further supported by a recent imaging study showing that identical doses of levodopa induced increasingly larger changes in striatal dopamine levels as the duration of motor symptoms increased, due to a compensatory mechanism secondary to the reduction in buffering capacity and presynaptic reuptake of dopamine that occurs as Parkinson’s disease progresses (de la Fuente-Fernández et al., 2004). In line with our clinical data, the authors concluded that the primary cause of peak-dose dyskinesias was disease progression and not levodopa per se (de la Fuente-Fernández et al., 2004). Hence, it seems that we should emphasize the role of substantia nigra pars compacta neuronal loss and the subsequent synaptic dopamine depletion rather than levodopa therapy per se to shed light on the pathophysiology of ‘levodopa-induced’ motor complications. According to presynaptic models of levodopa-induced dyskinesias, the extent of dopamine denervation and striatal dopamine reduction increases the sensitivity of post synaptic receptors and the downstream signalling pathway (Carta and Bezard, 2011), and regulates the level and duration of exposure to levodopa that is required to induce dyskinesias (Jenner, 2008). From a clinical perspective, it is generally assumed that the severity of motor symptoms reflects the extent of nigrostriatal neuronal loss. In contrast with experimental studies, however, we did not observe a strong association between the severity of parkinsonism and the latency to the onset of dyskinesias, at both multivariate analysis of cross sectional data and during the prospective follow-up of patients. The post hoc analyses of the STRIDE-PD trial are consistent with our observations, as the severity of motor symptoms had no predictive role for dyskinesias (Olanow et al., 2013). Interestingly, Cotzias et al. (1969) similarly reported in their pivotal study on levodopa that ‘the most severe involuntary movements were induced among the patients whose disease had the longest duration, not necessarily among those with the most severe disease’. This discrepancy might be explained considering that patients with different degrees of neuronal loss may present with comparable severity of motor symptoms, as compensatory mechanisms may modulate the length of the pre-motor stage of Parkinson’s disease considerably (de la Fuente-Fernández et al., 2011). In particular, younger age at onset is associated with better presynaptic and postsynaptic compensatory mechanisms aimed at keeping normal striatal dopamine. Thus, early-onset patients may tolerate greater severity of substantia nigra pars compacta neuronal loss before dopamine levels decrease below the estimated threshold for the first motor symptoms to appear (de la Fuente-Fernández et al., 2011). Therefore, it could be argued that the higher risk of dyskinesias associated with younger age at Parkinson’s disease onset, consistently described so far (Kempster et al., 2007; Olanow et al., 2013) and confirmed in the present study, might be due to a greater extent of neuronal loss in patients with similar severity of motor symptoms. Overall, these data support the idea that it is the duration of Parkinson’s disease rather than the severity of motor symptoms that is closely associated with the extent of neuronal loss. In turn, it is the extent of substantia nigra pars compacta neuronal loss that causes the decrease in striatal dopamine levels that predisposes to the development of motor complications once a patient with Parkinson’s disease is exposed to levodopa. Nevertheless, preclinical (Guigoni et al., 2005) and clinical (Linazasoro et al., 2009) studies show that similar extension of neuronal loss may lead to involuntary movements in some individuals and not in others after exposure to similar levodopa doses, suggesting that individual predisposition is likely to play a role in addition to disease-related variables, including genetic factors (reviewed in Manson et al., 2012). Further evidence is provided by the Ghanaian patient who developed peak-dose dyskinesias after the first-ever levodopa intake despite her mild disease severity, which is in contrast to the relatively delayed appearance of dykinesias in patients with more severe motor symptoms. This paradigmatic case supports the hypothesis that the induction of levodopa-induced dyskinesias (defined as ‘priming’ process) does not require chronic dopaminergic treatment and brain sensitization (Cotzias, 1971; Huot et al., 2013), but may occur even after the first-ever dose of levodopa in a dopamine-depleted brain (Nadjar et al., 2009). Nigrostriatal degeneration has generally been considered to be mandatory in the pathogenesis of dyskinesias in view of the absence of dyskinesias in animals or humans with intact dopamine system despite long-term exposure to levodopa (Boyce et al., 1990; Jenner, 2008), including patients with DOPA-responsive dystonia due to GCH1 mutations (Nutt et al., 2001; Trender-Gerhard et al., 2009). However, levodopa-induced dyskinesias may be generated under extreme conditions even when basal ganglia are normal, as shown in animals and humans with preserved substantia nigra pars compacta neuronal density that received very high doses of levodopa (Hwang et al., 2001; Togasaki et al., 2005; Jenner, 2008). On the other hand, dyskinesias may develop in the absence of substantia nigra pars compacta neuronal loss even after exposure to low levodopa doses in the dopamine-depleted brain of patients with tyrosine hydroxylase deficiency, most likely via postsynaptic dopamine receptor super-sensitivity (Carta and Bezard, 2011; Bézard et al., 2013; Pons et al., 2013). Taken as a whole, these data suggest that striatal dopamine levels and levodopa dose are inversely related in the equation predicting dyskinesias, in a continuum that does not include the duration of levodopa therapy. Age at onset and individual genetically-inherited predisposing factors have a modulatory effect (Manson et al., 2012). This study has some limitations that should be acknowledged. First, we used a control Parkinson’s disease population with a different genetic and environmental background (Cilia et al., 2011, 2012). Therefore, we cannot definitely exclude the possibility that Parkinson’s disease in black Africans may have a different phenotype and response to levodopa and further studies are needed to confirm the present findings without the bias related to the assessment of different populations. Nevertheless, we found that major demographic and clinical characteristics 5 years after onset were overall comparable, suggesting that potential differences related to the geographical setting did not play a substantial role in the current findings. In particular, our extensive analysis did not reveal any significant difference in terms of mean age at onset, frequency of major non-motor features, and severity of motor symptoms, including response to acute levodopa challenge, non-levodopa-responsive motor symptoms, activities of daily living and progression to advanced disease stages. As the awareness of the disease and the skills of local doctors are progressively increasing in several sub-Saharan African countries, the disease is diagnosed at earlier stages and levodopa therapy started accordingly. Therefore, it is conceivable that in the near future it will be possible to perform a direct comparison between patients with an early-start compared to those with a delayed-start of levodopa. Second, the different access to medications between the two populations forced us to add a control subgroup of patients with Parkinson’s disease who had never been exposed to dopamine agonists or COMT inhibitors and thus complicated the design of study analyses. Finally, this study was not designed as a community-based trial and inclusion bias might have occurred. In the attempt to make the study cohort as representative as possible of the general Parkinson’s disease population in the sub-Saharan African region, we conducted a long-term naturalistic study observing outpatients consecutively referred not only to two neurology clinics in large cities of densely populated regions, but also to a small clinic in a rural area of the country. Nonetheless, the higher frequency of patients presenting with a tremor-dominant phenotype reasonably reflects the low awareness of Parkinson’s disease that still induces most doctors and patients to place stronger emphasis on resting tremor as the ‘cardinal symptom’ leading to suspect Parkinson’s disease (as gait and balance disturbances are generally attributed to normal ageing) so that non-tremor-dominant subtypes are likely to be under-represented. This observation underlines further the need to promote educational initiatives in developing countries (The Movement Disorders Society, 2013). The different phenotype at presentation might have had a confounding effect on clinical variables, including the risk of motor complications. It has been recently suggested that the tremor-dominant presenting phenotype may be a negative predictor of dyskinesias (Kipfer et al., 2011; Zhang et al., 2013). In a clinicopathological study, the onset of dyskinesias was delayed by ∼2 years in patients presenting with a tremor-dominant motor phenotype compared to those with a non-tremor phenotype (although this difference was not statistically significant, Selikhova et al., 2009). Accordingly, we might assume that, if the Ghanaian cohort had had a similar proportion of patients with non-tremor-dominant phenotype to the Italian control population, the main finding of the present study would have been strengthened even further, as the rate of motor complications would have been expected to be higher and their appearance even earlier than what we found. Overall, we believe that our naturalistic case-control study including a matched control population from a Western country was the only possible way to provide conclusive answers about the relationship between levodopa therapy and disease progression in the pathophysiology of motor complications, overcoming the limitations of previous studies. Conclusion The present study provides evidence that motor fluctuations and dyskinesias are not associated with the duration of exposure to levodopa therapy, but rather to disease progression itself. Therefore, there is no reason to delay the initiation of adequate levodopa therapy in patients with Parkinson’s disease. In contrast with past drug trials, recent experimental studies suggest that the best therapeutic option to delay the molecular changes in gene expression, synaptic morphology and abnormal corticostriatal connectivity associated with dyskinesias may be early initiation of levodopa treatment (Marin et al., 2009). We emphasize the importance of considering the pharmacokinetic properties of levodopa and recommend (i) to adjust the individual levodopa dose regimen per kilogram of body weight to reduce the risk of dyskinesias (Zappia et al., 2002; Sharma et al., 2008; Olanow et al., 2013); and (ii) to consider the potential benefits of dietary protein redistribution during the daytime to minimize levodopa requirement and improve the control of motor fluctuations (Mena and Cotzias, 1975; Cereda et al., 2010). Further studies are needed to better understand the natural course of Parkinson’s disease without the confounding effect of medications, and the pathophysiology of motor and non-motor symptoms. Whether the answers will come from Africa, time will tell. Supplementary Material Supplementary Data

Levodopa is the most effective drug for the treatment of Parkinson's disease. However, the long-term use of this dopamine precursor is complicated by highly disabling fluctuations and dyskinesias. Although preclinical and clinical findings suggest pulsatile stimulation of striatal postsynaptic receptors as a key mechanism underlying levodopa-induced dyskinesias, their pathogenesis is still unclear. In recent years, evidence from animal models of Parkinson's disease has provided important information to understand the effect of specific receptor and post-receptor molecular mechanisms underlying the development of dyskinetic movements. Recent preclinical and clinical data from promising lines of research focus on the differential role of presynaptic versus postsynaptic mechanisms, dopamine receptor subtypes, ionotropic and metabotropic glutamate receptors, and non-dopaminergic neurotransmitter systems in the pathophysiology of levodopa-induced dyskinesias. Copyright © 2010 Elsevier Ltd. All rights reserved.