Alterations in the LRRK2-Rab pathway in urinary extracellular vesicles as Parkinson's disease and pharmacodynamic biomarkers.

Expression or phosphorylation levels of leucine-rich repeat kinase 2 (LRRK2) and its Rab substrates have strong potential as disease or pharmacodynamic biomarkers. The main objective of this study is therefore to assess the LRRK2-Rab pathway for use as biomarkers in human, non-human primate (NHP) and rat urine. With urine collected from human subjects and animals, we applied an ultracentrifugation based fractionation protocol to isolate small urinary extracellular vesicles (uEVs). We used western blot with antibodies directed against total and phosphorylated LRRK2, Rab8, and Rab10 to measure these LRRK2 and Rab epitopes in uEVs. We confirm the presence of LRRK2 and Rab8/10 in human and NHP uEVs, including total LRRK2 as well as phospho-LRRK2, phospho-Rab8 and phospho-Rab10. We also confirm LRRK2 and Rab expression in rodent uEVs. We quantified LRRK2 and Rab epitopes in human cohorts and found in a first cohort that pS1292-LRRK2 levels were elevated in individuals carrying the LRRK2 G2019S mutation, without significant differences between healthy and PD groups, whether for LRRK2 G2019S carriers or not. In a second cohort, we found that PD was associated to increased Rab8 levels and decreased pS910-LRRK2 and pS935-LRRK2. In animals, acute treatment with LRRK2 kinase inhibitors led to decreased pT73-Rab10. The identification of changes in Rab8 and LRRK2 phosphorylation at S910 and S935 heterologous phosphosites in uEVs of PD patients and pT73-Rab10 in inhibitor-dosed animals further reinforces the potential of the LRRK2-Rab pathway as a source of PD and pharmacodynamic biomarkers in uEVs.

Centrosomal cohesion deficits as cellular biomarker in lymphoblastoid cell lines from LRRK2 Parkinson's disease patients.

Leucine-rich repeat kinase 2 (LRRK2) is a promising therapeutic target for the treatment of Parkinson's disease (PD), and orally bioavailable, brain penetrant and highly potent LRRK2 kinase inhibitors are in early stages of clinical testing. Detection of LRRK2 phosphorylation, as well as phosphorylation of Rab10, a LRRK2 kinase substrate, have been proposed as target engagement biomarkers for LRRK2 inhibitor clinical trials. However, these readouts do not seem able to stratify patients based on enhanced LRRK2 kinase activity. Here, we describe a robust cell biological assay based on centrosomal cohesion alterations which were observed in peripheral blood mononuclear cell-derived lymphoblastoid cell lines (LCLs) from patients with G2019S LRRK2 mutations as compared with healthy controls, and could also be detected in a subset of sporadic PD patient samples. We suggest that LCLs may be a valuable resource for LRRK2 research, and that determination of centrosomal cohesion deficits may assist in the stratification of a subset of sporadic PD patients.

Freezing/festination during motor tasks in early-stage Parkinson's disease: A prospective study.

BACKGROUND: Parkinsonian patients have a tendency to speed up during repetitive motor tasks (festination) and to experience sudden motor blocks (freezing). In this article, we prospectively studied the appearance and progression of these phenomena in 30 early-stage PD patients. METHODS: A total of 30 controls and early-stage PD patients were assessed in the "off-drug" condition at baseline and 2 years later. Freezing of gait was evaluated using a standardized gait trajectory with the usual triggers. Patients also performed diadochokinetic tasks with 3 different effectors (repetitive, antiphase movements for the hands and feet, and repetitive syllable production for the orofacial effector) at frequencies ranging from 1 to 7 Hz (in random order). The primary endpoint was the occurrence of freezing and festination. RESULTS: At baseline, freezing was observed in 6.5% of the trials in PD patients (43% of the patients) and 2.3% of the trials in controls, and festination was observed in 5.7% of the trials in patients (53% of the patients) and 0.8% of the trials in controls. These proportions were slightly higher in patients 2 years later. None of the patients presented freezing of gait at baseline, but 2 displayed this condition 2 years later. These phenomena occurred more frequently for the limb effectors than for the orofacial effector. Freezing and festination were associated with the akinetic-rigid subtype, although tremor-dominant patients displayed greater rhythm variability outside episodes. CONCLUSION: Freezing and festination of the upper and lower limbs are observed soon after the diagnosis of PD and may be early biomarkers for disease progression. © 2016 International Parkinson and Movement Disorder Society.

Auditory cueing of gait initiation in Parkinson's disease patients with freezing of gait.

OBJECTIVE: Impaired gait initiation (GI) in patients with advanced Parkinson's disease (PD) is a typical functional sign of akinesia. Failure to initiate the first step is frequently presented by patients with freezing of gait (FOG) and is often considered a sub-type of freezing. The literature on the effects of cueing of GI preparation and execution remains controversial. Our objective was to establish whether auditory cueing improves the preparation and/or execution of GI in PD patients with a history of FOG. METHODS: We recorded first-step preparation and execution in 30 PD patients with confirmed FOG under two randomised conditions: self-triggered (ST) gait and gait cued by a sound beep in off- and on-dopa conditions. Anticipatory postural adjustments (APAs) were evaluated by monitoring the trajectory of the centre of pressure. RESULTS: We compared the patients with 30 patients without history of FOG and 30 healthy controls (HCs). l-Dopa only slightly improved the characteristics of APAs in freezers but was effective to improve gait hypokinesia. Auditory cueing was effective in improving step preparation in freezers, who showed adequate APAs more frequently. As seen with HCs and patients without FOG, patients released their APAs more quickly when auditory cueing was applied. However, cueing did not have a significant effect on step length. Clinically, auditory cueing also improved start hesitation in freezers. CONCLUSIONS: Auditory cueing improved step preparation but not step execution in PD patients. SIGNIFICANCE: A failure to link step preparation and execution during GI may explain the poor first-step execution seen in PD freezers.

Involvement of the immune system, endocytosis and EIF2 signaling in both genetically determined and sporadic forms of Parkinson's disease.

The leucine-rich repeat kinase 2 (LRRK2) G2019S mutation is a common genetic cause of Parkinson's disease (PD). Although patients with sporadic PD and individuals with LRRK2-linked PD display the classical PD phenotype, it is not known whether or not the same biological pathways are deregulated in each context. By using transcriptome profiling, we investigated the deregulation of various biological pathways in a total of 47 peripheral blood mononuclear cell (PBMC) samples from patients with sporadic PD, patients heterozygous for the LRRK2 G2019S mutation compared to healthy controls. We found that the deregulation patterns were indeed similar in PBMCs obtained from patients with sporadic PD and from LRRK2 G2019S carriers, with dysfunctions in mitochondrial pathways, cell survival signaling, cancerization, endocytosis signaling and iron metabolism. Analysis of our PBMC data and other publicly available transcriptome datasets (for whole blood samples) showed that deregulation of the immune system, endocytosis and eukaryotic initiation factor 2 (EIF2) signaling are the main features of transcriptome profiles in PD (since they are also present in the transcriptome of dopaminergic neurons from patients). Transcriptome analysis of PBMCs is thus valuable for (i) characterizing the pathophysiological pathways shared by genetic and sporadic forms of PD and (ii) identifying potential biomarkers and therapeutic targets. This minimally invasive approach opens up tremendous perspectives for better diagnosis and therapy of neurodegenerative diseases because it can be applied from the earliest stages of the disease onwards.

Are gait initiation parameters early markers of Huntington's disease in pre-manifest mutation carriers?

Huntington's disease (HD) pre-manifest mutation carriers (PMCs) present early-onset gait disturbances. Gait initiation encompasses the preparation and execution of the first step. By using paradigms with and without external cues, a gait initiation analysis can highlight the interaction between motor and cognitive aspects of movement preparation and execution. Hence, gait initiation disorders may constitute particularly interesting early markers of HD. The objective of the present study was to quantify gait initiation in PMCs. In a case-control study, 17 PMCs (median age: 36.5) were compared with a group of 25 healthy controls (HCs, median age: 36) for gait initiation and a group of 57 HCs (median age: 38) for gait. Presymptomatic mutation carriers displayed a shorter first step duration and lower-amplitude postural adjustments. For the first step duration and speed, these impairments were more pronounced under self-triggered (ST) conditions. The PMCs displayed a lower gait speed, cadence and stride length and higher stride-to-stride variability. The latter parameter seemed capable of differentiating between PMCs and HCs with adequate sensitivity (0.81) and specificity (0.87). We confirmed the early-onset impairment of gait in general and first step execution in particular in PMCs (particularly under ST conditions). The temporal parameters of step execution (e.g. duration) and spatial parameters of postural adjustment (e.g. a backward shift in the centre of pressure) may be worth investigating as early markers of HD. However, two such parameters (stride-to-stride variability and first step duration under ST conditions) already appear to be sufficiently reliable diagnostic tools for differentiating between PMCs and HCs.

Kinematic angular parameters in PD: reliability of joint angle curves and comparison with healthy subjects.

BACKGROUND: Most previous biomechanical studies of Parkinson's disease (PD) have been restricted to the description of spatiotemporal parameters and certain peak values for angular parameters. The reliability of joint angle curves and comparisons with control data are of major interest in PD, since variability in gait cycle timing is a feature of this pathology. METHODS: We used a video motion analysis system to record kinematic, spatiotemporal and angular parameters in 32 'off-drug' PD patients. The reliability of the patients' lower limb joint angle curves in the sagittal plane were analysed using the intra-class correlation coefficient (ICC), together with fast Fourier transform (FFT) analysis and hierarchical classification for discarding deviant curves. Lastly, we compared average curves (using a mixed model and the bootstrap method) for the less-affected and more-affected sides of PD patients and then compared the patient data with the results from 30 age-matched controls. RESULTS: The ICC-based procedure was easily applicable. Only 9.4% and 12.5% of the patients' hip and knee curves (respectively) were deemed to be unreliable. However, the PD patients' very high cycle-to-cycle variability in the sagittal plane ankle curves prevented us from applying to this joint. For the knee joint, the curves for the most disabled patients (who walked at below 0.5 m/s) were not reliable. We did not find any differences between the less and more disabled sides. The differences between patient and control curves concerned the double-support time during the stance phase and the time point for maximum knee flexion during the swing phase. Patients and controls differed in terms of the hip extension phase, with lower values in PD. CONCLUSION: We have developed the use of validated statistic tools for unambiguously comparing PD patients and controls in terms of joint angle curve differences.

Effect of age on anticipatory postural adjustments in unilateral arm movement.

Aging is frequently accompanied by a deterioration in postural control. Accordingly, the elderly adopt postural strategies in order to maintain balance. The purpose of this study was to compare anticipatory postural adjustments in (healthy) 10 young and 10 elderly subjects using electromyography (EMG) and biomechanical parameters. While standing on a force platform, subjects performed voluntary, arm-raising movements under five conditions: self-paced at three different velocities, self-paced with load and an externally triggered, both at maximal velocity. The force platform provided information on vertical torque (T(z)) and center of pressure anteroposterior displacements (COP). EMG activity was recorded from the biceps femoris, quadriceps, tibialis anterior and soleus muscles. Voluntary movements were associated with an early COP backward shift and an anticipatory T(z). At low velocity, elderly subjects did not show any impairment in stability. At maximal velocity, T(z) was delayed in all conditions in the elderly group, whereas COP latency was reduced only in the self-paced condition without load. Despite this decrease in anticipation, the movement was performed at the same velocity as in younger subjects. The elderly adopted various muscle strategies in order to perform the same movement with less stability. In the self-paced condition, elderly subjects used a hip strategy, whereas young subjects used an ankle strategy. In the triggered condition, the strategy corresponded to increased activation of certain thigh muscles, rather than a sequence modification. Hence, local muscle strategies were used to counteract the overall delay in postural preparation revealed by biomechanical parameters.

Vertical torque allows recording of anticipatory postural adjustments associated with slow, arm-raising movements.

BACKGROUND: When performed in the upright position, voluntary arm-raising movements perturb balance. The maintenance of equilibrium requires postural adjustments, some of which can be anticipatory. It is usually suggested that the role of anticipatory postural adjustments is to stabilise the whole body centre of mass. During movements performed at low velocity (i.e. with a lower inertial perturbation), anticipatory postural adjustments have not systematically been detected by classical recording methods (mainly electromyography). The aim of this study was to use vertical torque to characterise anticipatory postural adjustments in slow movement and to determine the significance of this biomechanical parameter. METHODS: Twenty healthy subjects performed self-paced, right arm-raising movements at low and high velocities. Movements were recorded by an optoelectronic system enabling the synchronization of video, force plate and electromyographic data. The force platform provided information on vertical torque and centre of foot pressure anteroposterior displacement. Electromyography activity was recorded from the right anterior deltoid and the bilateral biceps femoris, tibialis anterior and soleus muscles. FINDINGS: Rapid, voluntary, unilateral movements were associated with an early centre of pressure backward shift, anticipatory vertical torque and electromyographic activities. In slow movements, only the anticipatory changes in vertical torque were consistently observed, with the same latency as in rapid movement. INTERPRETATION: The existence of vertical torque in slow movement (when stabilisation of the centre of mass is not necessary) shows that this parameter does not serve to minimise the centre of mass displacement but rather contributes to the generation of arm movement.

[Anticipatory postural adjustments associated with arm flexion: interest of vertical torque]. / Ajustements posturaux anticipés lors de la flexion du membre supérieur: intérêt du moment de torsion.

OBJECTIVE: To find a biomechanical parameter able to characterize postural adjustments in different movement conditions. METHODS: The arm-raising movement performed during the upright human position imposes a vertical torque (Tz) that can be measured by a force plate-form. This torque was studied in ten healthy young subjects with opto electronic system Vicon 370. The subjects stood on a force platform, performed shoulder flexion of their right arm, to grasp a handle in front of them, in five conditions : self-paced at 3 different velocities (slow, medium, maximal), triggered by an auditory signal, loaded (1 kg attached to the wrist), all at maximal velocity. In a sixth condition, the arm was passively displaced by an experimenter. RESULTS: Tz displayed a negative phase (counter-clock wise body rotation) in all conditions. A positive phase occurred prior to the negative one, and preceded movement onset only if the movement was voluntary. In the triggered condition, the positive phase of Tz was delayed (- 60 ms) compared to the self-paced condition at maximal velocity (- 155 ms). However Tz onset latency was modified neither by load nor velocity. Tz amplitude increased with increasing velocity, load and in a reaction time condition. CONCLUSIONS: The vertical torque Tz, especially its positive phase gives useful informations about the latency, duration and intensity of the postural preparation related to a voluntary movement, according to the movement parameters. Tz is therefore able to characterise postural adjustments in all conditions, even with low movement velocity.