LRRK2 Inhibition by BIIB122 in Healthy Participants and Patients with Parkinson's Disease.

BACKGROUND: Leucine-rich repeat kinase 2 (LRRK2) inhibition is a promising therapeutic approach for the treatment of Parkinson's disease (PD). OBJECTIVE: The aim of this study was to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of the potent, selective, CNS-penetrant LRRK2 inhibitor BIIB122 (DNL151) in healthy participants and patients with PD. METHODS: Two randomized, double-blind, placebo-controlled studies were completed. The phase 1 study (DNLI-C-0001) evaluated single and multiple doses of BIIB122 for up to 28 days in healthy participants. The phase 1b study (DNLI-C-0003) evaluated BIIB122 for 28 days in patients with mild to moderate PD. The primary objectives were to investigate the safety, tolerability, and plasma pharmacokinetics of BIIB122. Pharmacodynamic outcomes included peripheral and central target inhibition and lysosomal pathway engagement biomarkers. RESULTS: A total of 186/184 healthy participants (146/145 BIIB122, 40/39 placebo) and 36/36 patients (26/26 BIIB122, 10/10 placebo) were randomized/treated in the phase 1 and phase 1b studies, respectively. In both studies, BIIB122 was generally well tolerated; no serious adverse events were reported, and the majority of treatment-emergent adverse events were mild. BIIB122 cerebrospinal fluid/unbound plasma concentration ratio was ~1 (range, 0.7-1.8). Dose-dependent median reductions from baseline were observed in whole-blood phosphorylated serine 935 LRRK2 (≤98%), peripheral blood mononuclear cell phosphorylated threonine 73 pRab10 (≤93%), cerebrospinal fluid total LRRK2 (≤50%), and urine bis (monoacylglycerol) phosphate (≤74%). CONCLUSIONS: At generally safe and well-tolerated doses, BIIB122 achieved substantial peripheral LRRK2 kinase inhibition and modulation of lysosomal pathways downstream of LRRK2, with evidence of CNS distribution and target inhibition. These studies support continued investigation of LRRK2 inhibition with BIIB122 for the treatment of PD. © 2023 Denali Therapeutics Inc and The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Glucocerebrosidase Activity is not Associated with Parkinson's Disease Risk or Severity.

BACKGROUND: Mutations in the GBA gene, which encodes the lysosomal enzyme glucocerebrosidase (GCase), are risk factors for Parkinson's disease (PD). OBJECTIVE: To explore the association between GCase activity, PD phenotype, and probability for prodromal PD among carriers of mutations in the GBA and LRRK2 genes. METHODS: Participants were genotyped for the G2019S-LRRK2 and nine GBA mutations common in Ashkenazi Jews. Performance-based measures enabling the calculation of the Movement Disorder Society (MDS) prodromal probability score were collected. RESULTS: One hundred and seventy PD patients (102 GBA-PD, 38 LRRK2-PD, and 30 idiopathic PD) and 221 non-manifesting carriers (NMC) (129 GBA-NMC, 45 LRRK2-NMC, 15 GBA-LRRK2-NMC, and 32 healthy controls) participated in this study. GCase activity was lower among GBA-PD (3.15 ± 0.85 µmol/L/h), GBA-NMC (3.23 ± 0.91 µmol/L/h), and GBA-LRRK2-NMC (3.20 ± 0.93 µmol/L/h) compared to the other groups of participants, with no correlation to clinical phenotype. CONCLUSIONS: Low GCase activity does not explain the clinical phenotype or risk for prodromal PD in this cohort. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

The Delta-Specific Opioid Glycopeptide BBI-11008: CNS Penetration and Behavioral Analysis in a Preclinical Model of Levodopa-Induced Dyskinesia.

In previous work we evaluated an opioid glycopeptide with mixed µ/δ-opioid receptor agonism that was a congener of leu-enkephalin, MMP-2200. The glycopeptide analogue showed penetration of the blood-brain barrier (BBB) after systemic administration to rats, as well as profound central effects in models of Parkinson's disease (PD) and levodopa (L-DOPA)-induced dyskinesia (LID). In the present study, we tested the glycopeptide BBI-11008 with selective δ-opioid receptor agonism, an analogue of deltorphin, a peptide secreted from the skin of frogs (genus Phyllomedusa). We tested BBI-11008 for BBB-penetration after intraperitoneal (i.p.) injection and evaluated effects in LID rats. BBI-11008 (10 mg/kg) demonstrated good CNS-penetrance as shown by microdialysis and mass spectrometric analysis, with peak concentration levels of 150 pM in the striatum. While BBI-11008 at both 10 and 20 mg/kg produced no effect on levodopa-induced limb, axial and oral (LAO) abnormal involuntary movements (AIMs), it reduced the levodopa-induced locomotor AIMs by 50% after systemic injection. The N-methyl-D-aspartate receptor antagonist MK-801 reduced levodopa-induced LAO AIMs, but worsened PD symptoms in this model. Co-administration of MMP-2200 had been shown prior to block the MK-801-induced pro-Parkinsonian activity. Interestingly, BBI-11008 was not able to block the pro-Parkinsonian effect of MK-801 in the LID model, further indicating that a balance of mu- and delta-opioid agonism is required for this modulation. In summary, this study illustrates another example of meaningful BBB-penetration of a glycopeptide analogue of a peptide to achieve a central behavioral effect, providing additional evidence for the glycosylation technique as a method to harness therapeutic potential of peptides.

Varying the rate of intravenous cocaine infusion influences the temporal dynamics of both drug and dopamine concentrations in the striatum.

The faster drugs of abuse reach the brain, the greater is the risk of addiction. Even small differences in the rate of drug delivery can influence outcome. Infusing cocaine intravenously over 5 vs. 90-100 s promotes sensitization to the psychomotor and incentive motivational effects of the drug and preferentially recruits mesocorticolimbic regions. It remains unclear whether these effects are due to differences in how fast and/or how much drug reaches the brain. Here, we predicted that varying the rate of intravenous cocaine infusion between 5 and 90 s produces different rates of rise of brain drug concentrations, while producing similar peak concentrations. Freely moving male Wistar rats received acute intravenous cocaine infusions (2.0 mg/kg/infusion) over 5, 45 and 90 s. We measured cocaine concentrations in the dorsal striatum using rapid-sampling microdialysis (1 sample/min) and high-performance liquid chromatography-tandem mass spectrometry. We also measured extracellular concentrations of dopamine and other neurochemicals. Regardless of infusion rate, acute cocaine did not change concentrations of non-dopaminergic neurochemicals. Infusion rate did not significantly influence peak concentrations of cocaine or dopamine, but concentrations increased faster following 5-s infusions. We also assessed psychomotor activity as a function of cocaine infusion rate. Infusion rate did not significantly influence total locomotion, but locomotion increased earlier following 5-s infusions. Thus, small differences in the rate of cocaine delivery influence both the rate of rise of drug and dopamine concentrations, and psychomotor activity. A faster rate of rise of drug and dopamine concentrations might be an important issue in making rapidly delivered cocaine more addictive.

Delta Opioid Pharmacology in Parkinson's Disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder that compromises multiple neurochemical substrates including dopamine, norepinephrine, serotonin, acetylcholine, and glutamate systems. Loss of these transmitter systems initiates a cascade of neurological deficits beginning with motor function and ending with dementia. Current therapies primarily address the motor symptoms of the disease via dopamine replacement therapy. Exogenous dopamine replacement brings about additional challenges since after years of treatment it almost invariably gives rise to dyskinesia as a side effect. Therefore there is a clear unmet clinical need for improved PD therapeutics. Opioid receptors and their respective peptides are expressed throughout the basal ganglia and cortex where monoaminergic denervation strongly contributes to PD pathology. Delta opioid receptors are of particular interest because of their dense localization in basal ganglia and because activating this system is known to enhance locomotor activity under a variety of conditions. This chapter will outline much of the work that has demonstrated the effectiveness of delta opioid receptor activation in models of PD and its neuroprotective properties. It also discusses some of the challenges that must be addressed before moving delta opioid receptor agonists into a clinical setting.

Asphyxia-activated corticocardiac signaling accelerates onset of cardiac arrest.

The mechanism by which the healthy heart and brain die rapidly in the absence of oxygen is not well understood. We performed continuous electrocardiography and electroencephalography in rats undergoing experimental asphyxia and analyzed cortical release of core neurotransmitters, changes in brain and heart electrical activity, and brain-heart connectivity. Asphyxia stimulates a robust and sustained increase of functional and effective cortical connectivity, an immediate increase in cortical release of a large set of neurotransmitters, and a delayed activation of corticocardiac functional and effective connectivity that persists until the onset of ventricular fibrillation. Blocking the brain's autonomic outflow significantly delayed terminal ventricular fibrillation and lengthened the duration of detectable cortical activities despite the continued absence of oxygen. These results demonstrate that asphyxia activates a brainstorm, which accelerates premature death of the heart and the brain.

Microfabrication and in Vivo Performance of a Microdialysis Probe with Embedded Membrane.

Microdialysis sampling is an essential tool for in vivo neurochemical monitoring. Conventional dialysis probes are over 220 µm in diameter and have limited flexibility in design because they are made by assembly using preformed membranes. The probe size constrains spatial resolution and governs the amount of tissue damaged caused by probe insertion. To overcome these limitations, we have developed a method to microfabricate probes in Si that are 45 µm thick × 180 µm wide. The probes contain a buried, U-shaped channel that is 30 µm deep × 60 µm wide and terminates in ports for external connection. A 4 mm length of the probe is covered with a 5 µm thick nanoporous membrane. The membrane was microfabricated by deep reactive ion etching through a porous aluminum oxide layer. The microfabricated probe has cross-sectional area that is 79% less than that of the smallest conventional microdialysis probes. The probes yield 2-20% relative recovery at 100 nL/min perfusion rate for a variety of small molecules. The probe was successfully tested in vivo by sampling from the striatum of live rats. Fractions were collected at 20 min intervals (2 µL) before and after an intraperitoneal injection of 5 mg/kg amphetamine. Analysis of fractions by liquid chromatography-mass spectrometry revealed reliable detection of 14 neurochemicals, including dopamine and acetylcholine, at basal conditions. Amphetamine evoked a 43-fold rise in dopamine, a result nearly identical to a conventional dialysis probe in the same animal. The microfabricated probes have potential for sampling with higher spatial resolution and less tissue disruption than conventional probes. It may also be possible to add functionality to the probes by integrating other components, such as electrodes, optics, and additional channels.

Stimulation of δ opioid receptor and blockade of nociceptin/orphanin FQ receptor synergistically attenuate parkinsonism.

δ opioid peptide (DOP) receptors are considered a therapeutic target in Parkinson's disease, although the use of DOP agonists may be limited by side effects, including convulsions. To circumvent this issue, we evaluated whether blockade of nociceptin/orphanin FQ (N/OFQ) tone potentiated the antiparkinsonian effects of DOP agonists, thus allowing for reduction of their dosage. Systemic administration of the N/OFQ receptor (NOP) antagonist J-113397 [(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H benzimidazol-2-one] and the DOP receptor agonist SNC-80 [(+)-4-[(αR)-α-(2S,5R)-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxy-benzyl]-N-N-diethylbenzamide] revealed synergistic attenuation of motor deficits in 6-hydroxydopamine hemilesioned rats and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice. In this model, repeated administration of the combination produced reproducible antiparkinsonian effects and was not associated with rescued striatal dopamine terminals. Microdialysis studies revealed that either systemic administration or local intranigral perfusion of J-113397 and SNC-80 led to the enhancement of nigral GABA, reduction of nigral Glu, and reduction of thalamic GABA levels, consistent with the view that NOP receptor blockade and DOP receptor stimulation caused synergistic overinhibition of nigro-thalamic GABA neurons. Whole-cell recording of GABA neurons in nigral slices confirmed that NOP receptor blockade enhanced the DOP receptor-induced effect on IPSCs via presynaptic mechanisms. Finally, SNC-80 more potently stimulated stepping activity in mice lacking the NOP receptor than wild-type controls, confirming the in vivo occurrence of an NOP-DOP receptor interaction. We conclude that endogenous N/OFQ functionally opposes DOP transmission in substantia nigra reticulata and that NOP receptor antagonists might be used in combination with DOP receptor agonists to reduce their dosage while maintaining their full therapeutic efficacy.

Reducing adsorption to improve recovery and in vivo detection of neuropeptides by microdialysis with LC-MS.

Neuropeptides are an important class of neurochemicals; however, measuring their concentration in vivo by using microdialysis sampling is challenging due to their low concentration and the small samples generated. Capillary liquid chromatography with mass spectrometry (cLC-MS) can yield attomole limits of detection (LOD); however, low recovery and loss of sample to adsorptive surfaces can still hinder detection of neuropeptides. We have evaluated recovery during sampling and transfer to the cLC column for a selection of 10 neuropeptides. Adding acetonitrile to sample eliminated carryover and improved LOD by 1.4- to 60-fold. The amount of acetonitrile required was found to have an optimal value that correlated with peptide molecular weight and retention time on a reversed phase LC column. Treating AN69 dialysis membrane, which bears negative charge due to incorporated sulfonate groups, with polyethylenimine (PEI) improved recovery by 1.2- to 80-fold. The effect appeared to be due to reducing electrostatic interaction between peptides and the microdialysis probe because modification increased recovery only for peptides that carried net positive charge. The combined effects improved LOD of the entire method by 1.3- to 800-fold for the different peptides. We conclude that peptides with both charged and hydrophobic regions require combined strategies to prevent adsorption and yield the best possible detection. The method was demonstrated by determining orexin A, orexin B, and a novel isoform of rat ß-endorphin in the arcuate nucleus. Dialysate concentrations were below 10 pM for these peptides. A standard addition study on dialysates revealed that while some peptides can be accurately quantified, some are affected by the matrix.

Amphetamine stimulates movement through thalamocortical glutamate release.

The ventrolateral thalamus (VL) is a primary relay point between the basal ganglia and the primary motor cortex (M1). Using dual probe microdialysis and locomotor behavior monitoring, we investigated the contribution of VL input into M1 during amphetamine (AMPH)-stimulated monoamine release and hyperlocomotion in rats. Tetrodotoxin (10 µM) perfusion into the VL significantly lowered hyperactivity induced by AMPH (1 mg/kg i.p.). This behavioral response corresponded to reduced cortical glutamate and monoamine release. To determine which glutamate receptors the thalamocortical projections acted upon, we perfused either the α-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid (AMPA)/kainate receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX) (10 µM) or the N-methyl-D-aspartic acid (NMDA) receptor antagonist (MK-801) intracortically followed by systemic AMPH. The results show that AMPA/kainate, and to a lesser extent NMDA receptors, mediated the observed effects. As glutamate-monoamine interactions could possibly occur through local or circuit-based mechanisms, we isolated and perfused M1 tissue ex vivo to determine the extent of local glutamate-dopamine interactions. Taken together, these results demonstrate that AMPH generates hyperlocomotive states via thalamocortical signaling and that cortical AMPA receptors are an important mediator of these effects. This study utilizes dual probe microdialysis sampling and comprehensive LC-MS analysis to determine the effects of amphetamine (1 mg/kg i.p.) on thalamocortical neurotransmission. Using pharmacological tools such as local thalamic tetrodotoxin (TTX) perfusion and glutamate antagonist at the cortical level, we demonstrate that thalamocortical glutamate (acting primarily through cortical AMPA receptors) is an essential component in amphetamine-induced hyperlocomotion.

Rapid dopamine transmission within the nucleus accumbens: dramatic difference between morphine and oxycodone delivery.

While most drugs of abuse increase dopamine neurotransmission, rapid neurochemical measurements show that different drugs evoke distinct dopamine release patterns within the nucleus accumbens. Rapid changes in dopamine concentration following psychostimulant administration have been well studied; however, such changes have never been examined following opioid delivery. Here, we provide novel measures of rapid dopamine release following intravenous infusion of two opioids, morphine and oxycodone, in drug-naïve rats using fast-scan cyclic voltammetry and rapid (1 min) microdialysis coupled with high-performance liquid chromatography - tandem mass spectrometry (HPLC-MS). In addition to measuring rapid dopamine transmission, microdialysis HPLC-MS measures changes in GABA, glutamate, monoamines, monoamine metabolites and several other neurotransmitters. Although both opioids increased dopamine release in the nucleus accumbens, their patterns of drug-evoked dopamine transmission differed dramatically. Oxycodone evoked a robust and stable increase in dopamine concentration and a robust increase in the frequency and amplitude of phasic dopamine release events. Conversely, morphine evoked a brief (~ 1 min) increase in dopamine that was coincident with a surge in GABA concentration and then both transmitters returned to baseline levels. Thus, by providing rapid measures of neurotransmission, this study reveals previously unknown differences in opioid-induced neurotransmitter signaling. Investigating these differences may be essential for understanding how these two drugs of abuse could differentially usurp motivational circuitry and powerfully influence behavior.

Distinctive immunoregulatory effects of adenosine on T cells of older humans.

A role for adenosine in immunosenescence was investigated in T cells from older (≥65 yr) and younger (24-45 yr) healthy humans. Adenosine concentrations in cultures of activated T cells were significantly higher (P<0.0001) for older (145±47 nM, mean±sd) than younger (58±5.5 nM) subjects. Expression of the activation coreceptor CD28 was suppressed significantly by 0.1 to 1 µM exogenous adenosine, with greater effects of 1 µM (P<0.01) on T cells of younger (mean suppression of 67 and 65% for CD4 and CD8 T cells, respectively) than older (means of 42 and 46%) subjects. T-cell chemotaxis to CCL21 was suppressed significantly by 0.3 and 1 µM exogenous adenosine, with mean maximum decreases of 39 and 49%, respectively, for younger subjects and 28 and 31% for older subjects. Generation of IL-2 and IFN-γ by T cells of younger and older subjects was suppressed substantially only at adenosine levels of 3 µM or higher. Lower baseline expression of CD28 and chemotaxis to CCL21 and S1P for T cells from older subjects attributable to endogenous adenosine were reversed completely by two different A(2A) adenosine receptor antagonists without affecting T cells of younger subjects. Adenosine is an endogenous T-cell immunosuppressor in older humans, and A(2A) antagonists reverse adenosine-induced T-cell deficiencies of aging.

In vivo neurochemical monitoring using benzoyl chloride derivatization and liquid chromatography-mass spectrometry.

In vivo neurochemical monitoring using microdialysis sampling is important in neuroscience because it allows correlation of neurotransmission with behavior, disease state, and drug concentrations in the intact brain. A significant limitation of current practice is that different assays are utilized for measuring each class of neurotransmitter. We present a high performance liquid chromatography (HPLC)-tandem mass spectrometry method that utilizes benzoyl chloride for determination of the most common low molecular weight neurotransmitters and metabolites. In this method, 17 analytes were separated in 8 min. The limit of detection was 0.03-0.2 nM for monoamine neurotransmitters, 0.05-11 nM for monoamine metabolites, 2-250 nM for amino acids, 0.5 nM for acetylcholine, 2 nM for histamine, and 25 nM for adenosine at sample volume of 5 µL. Relative standard deviation for repeated analysis at concentrations expected in vivo averaged 7% (n = 3). Commercially available (13)C benzoyl chloride was used to generate isotope-labeled internal standards for improved quantification. To demonstrate utility of the method for study of small brain regions, the GABA(A) receptor antagonist bicuculline (50 µM) was infused into a rat ventral tegmental area while recording neurotransmitter concentration locally and in nucleus accumbens, revealing complex GABAergic control over mesolimbic processes. To demonstrate high temporal resolution monitoring, samples were collected every 60 s while neostigmine, an acetylcholine esterase inhibitor, was infused into the medial prefrontal cortex. This experiment revealed selective positive control of acetylcholine over cortical glutamate.

Mass spectrometry "sensor" for in vivo acetylcholine monitoring.

Developing sensors for in vivo chemical monitoring is a daunting challenge. An alternative approach is to couple sampling methods with online analytical techniques; however, such approaches are generally hampered by lower temporal resolution and slow analysis. In this work, microdialysis sampling was coupled with segmented flow electrospray ionization mass spectrometry (ESI-MS) to perform in vivo chemical monitoring. The use of segmented flow to prevent Taylor dispersion of collected zones and rapid analysis with direct ESI-MS allowed 5 s temporal resolution to be achieved. The MS "sensor" was applied to monitor acetylcholine in the brain of live rats. The detection limit of 5 nM was sufficient to monitor basal acetylcholine as well as dynamic changes elicited by microinjection of neostigmine, an inhibitor of acetylcholinesterase, that evoked rapid increases in acetycholine and tetrodotoxin, a blocker of Na(+) channels, that lowered the acetylcholine concentration. The versatility of the sensor was demonstrated by simultaneously monitoring metabolites and infused drugs.

Microdialysis and mass spectrometric monitoring of dopamine and enkephalins in the globus pallidus reveal reciprocal interactions that regulate movement.

Pallidal dopamine, GABA and the endogenous opioid peptides enkephalins have independently been shown to be important controllers of sensorimotor processes. Using in vivo microdialysis coupled to liquid chromatography-mass spectrometry and a behavioral assay, we explored the interaction between these three neurotransmitters in the rat globus pallidus. Amphetamine (3 mg/kg i.p.) evoked an increase in dopamine, GABA and methionine/leucine enkephalin. Local perfusion of the dopamine D(1) receptor antagonist SCH 23390 (100 µM) fully prevented amphetamine stimulated enkephalin and GABA release in the globus pallidus and greatly suppressed hyperlocomotion. In contrast, the dopamine D(2) receptor antagonist raclopride (100 µM) had only minimal effects suggesting a greater role for pallidal D(1) over D(2) receptors in the regulation of movement. Under basal conditions, opioid receptor blockade by naloxone perfusion (10 µM) in the globus pallidus stimulated GABA and inhibited dopamine release. Amphetamine-stimulated dopamine release and locomotor activation were attenuated by naloxone perfusion with no effect on GABA. These findings demonstrate a functional relationship between pallidal dopamine, GABA and enkephalin systems in the control of locomotor behavior under basal and stimulated conditions. Moreover, these findings demonstrate the usefulness of liquid chromatography-mass spectrometry as an analytical tool when coupled to in vivo microdialysis.

Collection, storage, and electrophoretic analysis of nanoliter microdialysis samples collected from awake animals in vivo.

Microdialysis sampling is an important tool for chemical monitoring in living systems. Temporal resolution is an important figure of merit that is determined by sampling frequency, assay sensitivity, and dispersion of chemical zones during transport from sampling device to fraction collector or analytical system. Temporal resolution has recently been improved by segmenting flow into plugs, so that nanoliter fractions are collected at intervals of 0.1-2 s, thus eliminating temporal distortion associated with dispersion in continuous flow. Such systems, however, have yet to be used with behaving subjects. Furthermore, long-term storage of nanoliter samples created by segmented flow has not been reported. In this work, we have addressed these challenges. A microdialysis probe was integrated to a plug generator that could be stably mounted onto behaving animals. Long-term storage of dialysate plugs was achieved by collecting plugs into high-purity perfluoroalkoxy tubes, placing the tube into hexane and then freezing at -80°C. Slow warming with even temperatures prevented plug coalescence during sample thawing. As a demonstration of the system, plugs were collected from the striatum of behaving rats using a 0.5-mm-long microdialysis probe. Resulting plugs were analyzed 1-4 days later by chip-based electrophoresis. To improve throughput of plug analysis over previous work, the speed of electrophoretic separation was increased by using forced air cooling and 1-butyl-2,3-dimethylimidazolium tetrafluoroborate as a separation buffer additive, allowing resolution of six neuroactive amino acids in 30 s. Concentration changes induced by K(+) microinjections were monitored with 10 s temporal resolution. The improvements reported in this work make it possible to apply segmented flow microdialysis to the study of behaving animals and enable experiments where the analytical system cannot be placed close to the animal.

Further evidence for an involvement of nociceptin/orphanin FQ in the pathophysiology of Parkinson's disease: a behavioral and neurochemical study in reserpinized mice.

The contribution of nociceptin/orphanin FQ (N/OFQ) to reserpine-induced Parkinsonism was evaluated in mice. A battery of motor tests revealed that reserpine caused dose-dependent and long-lasting motor impairment. Endogenous N/OFQ sustained this response because N/OFQ peptide (NOP) receptor knockout (NOP(-/-) ) mice were less susceptible to the hypokinetic action of reserpine than wild-type (NOP(+/+) ) animals. Microdialysis revealed that reserpine elevated glutamate and reduced GABA levels in substantia nigra reticulata, and that resistance to reserpine in NOP(-/-) mice was accompanied by a milder increase in glutamate and lack of inhibition of GABA levels. To substantiate this genetic evidence, the NOP receptor antagonist 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H benzimidazol-2-one (J-113397) simultaneously reduced akinesia and nigral glutamate levels in reserpinized NOP(+/+) mice, being ineffective in NOP(-/-) mice. Moreover, repeated J-113397 administration in reserpinized mice resulted in faster recovery of baseline motor performance which was, however, accompanied by a loss of acute antiakinetic response. The short-term beneficial effect of J-113397 was paralleled by normalization of nigral glutamate levels, whereas loss of acute response was paralleled by loss of the ability of J-113397 to inhibit glutamate levels. We conclude that endogenous N/OFQ contributes to reserpine-induced Parkinsonism, and that sustained NOP receptor blockade produces short-term motor improvement accompanied by normalization of nigral glutamate release.

Quantitative Measurements of LRRK2 in Human Cerebrospinal Fluid Demonstrates Increased Levels in G2019S Patients.

Leucine-rich repeat kinase 2 (LRRK2) mutations are among the most significant genetic risk factors for developing late onset Parkinson's disease (PD). To understand whether a therapeutic can modulate LRRK2 levels as a potential disease modifying strategy, it is important to have methods in place to measure the protein with high sensitivity and specificity. To date, LRRK2 measurements in cerebrospinal fluid (CSF) have used extracellular vesicle enrichment via differential ultracentrifugation and western blot detection. Our goal was to develop a methodology which could be deployed in a clinical trial, therefore throughput, robustness and sensitivity were critical. To this end, we developed a Stable Isotope Standard Capture by Anti-peptide Antibody (SISCAPA) assay which is capable of detecting LRRK2 from 1 ml of human CSF. The assay uses a commercially available LRRK2 monoclonal antibody (N241A/34) and does not require extracellular vesicle enrichment steps. The assay includes stable isotope peptide addition which allows for absolute quantitation of LRRK2 protein. We determined that the assay performed adequately for CSF measurements and that blood contamination from traumatic lumbar puncture does not pose a serious analytical challenge. We then applied this technique to 106 CSF samples from the MJFF LRRK2 Cohort Consortium which includes healthy controls, sporadic PD patients and LRRK2 mutation carriers with and without PD. Of the 105 samples that had detectable LRRK2 signal, we found that the PD group with the G2019S LRRK2 mutation had significantly higher CSF LRRK2 levels compared to all other groups. We also found that CSF LRRK2 increased with the age of the participant. Taken together, this work represents a step forward in our ability to measure LRRK2 in a challenging matrix like CSF which has implications for current and future LRRK2 therapeutic clinical trials.

The Current State-of-the Art of LRRK2-Based Biomarker Assay Development in Parkinson's Disease.

Evidence is mounting that LRRK2 function, particularly its kinase activity, is elevated in multiple forms of Parkinson's disease, both idiopathic as well as familial forms linked to mutations in the LRRK2 gene. However, sensitive quantitative markers of LRRK2 activation in clinical samples remain at the early stages of development. There are several measures of LRRK2 activity that could potentially be used in longitudinal studies of disease progression, as inclusion/exclusion criteria for clinical trials, to predict response to therapy, or as markers of target engagement. Among these are levels of LRRK2, phosphorylation of LRRK2 itself, either by other kinases or via auto-phosphorylation, its in vitro kinase activity, or phosphorylation of downstream substrates. This is advantageous on many levels, in that multiple indices of elevated kinase activity clearly strengthen the rationale for targeting this kinase with novel therapeutic candidates, and provide alternate markers of activation in certain tissues or biofluids for which specific measures are not detectable. However, this can also complicate interpretation of findings from different studies using disparate measures. In this review we discuss the current state of LRRK2-focused biomarkers, the advantages and disadvantages of the current pallet of outcome measures, the gaps that need to be addressed, and the priorities that the field has defined.