Dose-dependent reduction of somatic expansions but not Htt aggregates by di-valent siRNA-mediated silencing of MSH3 in HdhQ111 mice.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by CAG trinucleotide repeat expansions in exon 1 of the HTT gene. In addition to germline CAG expansions, somatic repeat expansions in neurons also contribute to HD pathogenesis. The DNA mismatch repair gene, MSH3, identified as a genetic modifier of HD onset and progression, promotes somatic CAG expansions, and thus presents a potential therapeutic target. However, what extent of MSH3 protein reduction is needed to attenuate somatic CAG expansions and elicit therapeutic benefits in HD disease models is less clear. In our study, we employed potent di-siRNAs to silence mouse Msh3 mRNA expression in a dose-dependent manner in HdhQ111/+ mice and correlated somatic Htt CAG instability with MSH3 protein levels from simultaneously isolated DNA and protein after siRNA treatment. Our results reveal a linear correlation with a proportionality constant of ~ 1 between the prevention of somatic Htt CAG expansions and MSH3 protein expression in vivo, supporting MSH3 as a rate-limiting step in somatic expansions. Intriguingly, despite a 75% reduction in MSH3 protein levels, striatal nuclear HTT aggregates remained unchanged. We also note that evidence for nuclear Msh3 mRNA that is inaccessible to RNA interference was found, and that MSH6 protein in the striatum was upregulated following MSH3 knockdown in HdhQ111/+ mice. These results provide important clues to address critical questions for the development of therapeutic molecules targeting MSH3 as a potential therapeutic target for HD.

Nigrostriatal tau pathology in parkinsonism and Parkinson's disease.

While Parkinson's disease remains clinically defined by cardinal motor symptoms resulting from nigrostriatal degeneration, it is now appreciated that the disease commonly consists of multiple pathologies, but it is unclear where these co-pathologies occur early in disease and whether they are responsible for the nigrostriatal degeneration. For the past number of years, we have been studying a well-characterized cohort of subjects with motor impairment that we have termed mild motor deficits. Motor deficits were determined on a modified and validated Unified Parkinson's Disease Rating Scale III but were insufficient in degree to diagnose Parkinson's disease. However, in our past studies, cases in this cohort had a selection bias, as both a clinical syndrome in between no motor deficits and Parkinson's disease, plus nigral Lewy pathology as defined post-mortem, were required for inclusion. Therefore, in the current study, we only based inclusion on the presence of a clinical phenotype with mild motor impairment insufficient to diagnose Parkinson's disease. Then, we divided this group further based upon whether or not subjects had a synucleinopathy in the nigrostriatal system. Here we demonstrate that loss of nigral dopaminergic neurons, loss of putamenal dopaminergic innervation and loss of the tyrosine hydroxylase-phenotype in the substantia nigra and putamen occur equally in mild motor deficit groups with and without nigral alpha-synuclein aggregates. Indeed, the common feature of these two groups is that both have similar degrees of AT8 positive phosphorylated tau, a pathology not seen in the nigrostriatal system of age-matched controls. These findings were confirmed with early (tau Ser208 phosphorylation) and late (tau Ser396/Ser404 phosphorylation) tau markers. This suggests that the initiation of nigrostriatal dopaminergic neurodegeneration occurs independently of alpha-synuclein aggregation and can be tau mediated.

ASO-mediated knockdown or kinase inhibition of G2019S-Lrrk2 modulates lysosomal tubule-associated antigen presentation in macrophages.

Genetic variation around the LRRK2 gene affects risk for both familial and sporadic Parkinson's disease (PD). LRRK2 levels have become an appealing target for potential PD therapeutics with LRRK2 antisense oligonucleotides (ASOs) now moving toward clinical trials. However, LRRK2 has been suggested to play a fundamental role in peripheral immunity, and it is currently unknown if targeting increased LRRK2 levels in peripheral immune cells will be beneficial or deleterious. Here it was observed that G2019S macrophages exhibited increased stimulation-dependent lysosomal tubule formation (LTF) and MHC-II trafficking from the perinuclear lysosome to the plasma membrane in an mTOR-dependent manner with concomitant increases in pro-inflammatory cytokine release. Both ASO-mediated knockdown of mutant Lrrk2 and LRRK2 kinase inhibition ameliorated this phenotype and decreased these immune responses in control cells. Given the critical role of antigen presentation, lysosomal function, and cytokine release in macrophages, it is likely LRRK2-targeting therapies with systemic activity may have therapeutic value with regard to mutant LRRK2, but deleterious effects on the peripheral immune system, such as altered pathogen control in these cells, should be considered when reducing levels of non-mutant LRRK2.

Transient exposure to rotenone causes degeneration and progressive parkinsonian motor deficits, neuroinflammation, and synucleinopathy.

Individuals with Parkinson's disease (PD) typically receive a diagnosis once they have developed motor symptoms, at which point there is already significant loss of substantia nigra dopamine neurons, α-synuclein accumulation in surviving neurons, and neuroinflammation. Consequently, the point of clinical presentation may be too late to initiate disease-modifying therapy. In contrast to this clinical reality, animal models often involve acute neurodegeneration and potential therapies are tested concurrently or shortly after the pathogenic insult has begun rather than later when diagnostic clinical symptoms emerge. Therefore, we sought to develop a model that reflects the clinical situation more accurately. Middle-aged rats (7-9 months-old) received a single daily intraperitoneal injection of rotenone for 5 consecutive days and were observed over the next 8-9 months. Rotenone-treated rats showed transient motor slowing and postural instability during exposure but recovered within 9 days of rotenone cessation. Rats remained without behavioral deficits for 3-4 months, then developed progressive motor abnormalities over the ensuing months. As motor abnormalities began to emerge 3 months after rotenone exposure, there was significant loss of nigral dopaminergic neurons and significant microglial activation. There was delayed accumulation of α-synuclein in neurons of the substantia nigra and frontal cortex, which was maximal at 9 months post-rotenone. In summary, a brief temporally-remote exposure to rotenone causes delayed and progressive behavioral and neuropathological changes similar to Parkinson's disease. This model mimics the human clinical situation, in which pathogenesis is well-established by the time diagnostic motor deficits appear. As such, this model may provide a more relevant experimental system in which to test disease-modifying therapeutics.

Perspective on the current state of the LRRK2 field.

Almost 2 decades after linking LRRK2 to Parkinson's disease, a vibrant research field has developed around the study of this gene and its protein product. Recent studies have begun to elucidate molecular structures of LRRK2 and its complexes, and our understanding of LRRK2 has continued to grow, affirming decisions made years ago to therapeutically target this enzyme for PD. Markers of LRRK2 activity, with potential to monitor disease progression or treatment efficacy, are also under development. Interestingly, there is a growing understanding of the role of LRRK2 outside of the central nervous system in peripheral tissues such as gut and immune cells that may also contribute to LRRK2 mediated pathology. In this perspective, our goal is to take stock of LRRK2 research by discussing the current state of knowledge and critical open questions in the field.

Totally tubular: ASO-mediated knock-down of G2019S -Lrrk2 modulates lysosomal tubule-associated antigen presentation in macrophages.

Genetic variation around the LRRK2 gene affects risk of both familial and sporadic Parkinson's disease (PD). LRRK2 levels have become an appealing target for potential PD-therapeutics with LRRK2 antisense oligonucleotides (ASOs) now in clinical trials. However, LRRK2 has been suggested to play a fundamental role in peripheral immunity, and it is currently unknown if targeting increased LRRK2 levels in peripheral immune cells will be beneficial or deleterious. Furthermore, the precise role of LRRK2 in immune cells is currently unknown, although it has been suggested that LRRK2-mediated lysosomal function may be crucial to immune responses. Here, it was observed that G2019S macrophages exhibited increased stimulation-dependent lysosomal tubule formation (LTF) and MHC-II trafficking from the perinuclear lysosome to the plasma membrane in an mTOR dependent manner with concomitant increases in pro-inflammatory cytokine release. Both ASO-mediated knock down of mutant Lrrk 2 and LRRK2 kinase inhibition ameliorated this phenotype and decreased these immune responses in control cells. Given the critical role of antigen presentation, lysosomal function, and cytokine release in macrophages, it is likely LRRK2-targetting therapies may have therapeutic value with regards to mutant LRRK2 but deleterious effects on the peripheral immune system, such as altered pathogen control and infection resolution.

Mixed pathology as a rule, not exception: Time to reconsider disease nosology.

Parkinson's disease is a progressive neurodegenerative disorder that is associated with motor and nonmotor symptoms. Accumulation of misfolded α-synuclein is considered a key pathological feature during disease initiation and progression. While clearly deemed a synucleinopathy, the development of amyloid-ß plaques, tau-containing neurofibrillary tangles, and even TDP-43 protein inclusions occur within the nigrostriatal system and in other brain regions. In addition, inflammatory responses, manifested by glial reactivity, T-cell infiltration, and increased expression of inflammatory cytokines, plus other toxic mediators derived from activated glial cells, are currently recognized as prominent drivers of Parkinson's disease pathology. However, copathologies have increasingly been recognized as the rule (>90%) and not the exception, with Parkinson's disease cases on average exhibiting three different copathologies. While microinfarcts, atherosclerosis, arteriolosclerosis, and cerebral amyloid angiopathy may have an impact on disease progression, α-synuclein, amyloid-ß, and TDP-43 pathology do not seem to contribute to progression.

Beta2-adrenoreceptor agonist clenbuterol produces transient decreases in alpha-synuclein mRNA but no long-term reduction in protein.

ß2-adrenoreceptor (ß2AR) agonists have been associated with a decreased risk of developing Parkinson's disease (PD) and are hypothesized to decrease expression of both alpha-synuclein mRNA (Snca) and protein (α-syn). Effects of ß2AR agonist clenbuterol on the levels of Snca mRNA and α-syn protein were evaluated in vivo (rats and mice) and in rat primary cortical neurons by two independent laboratories. A modest decrease in Snca mRNA in the substantia nigra was observed after a single acute dose of clenbuterol in rats, however, this decrease was not maintained after multiple doses. In contrast, α-syn protein levels remained unchanged in both single and multiple dosing paradigms. Furthermore, clenbuterol did not decrease Snca in cultured rat primary cortical neurons, or decrease Snca or α-syn in mice. Additionally, compared to the single-dose paradigm, repeat dosing resulted in substantially lower levels of clenbuterol in plasma and brain tissue in rodents. Based on our observations of a transient decrease in Snca and no effect on α-syn protein in this preclinical study, these data support the conclusion that clenbuterol is not likely a viable disease-modifying strategy for PD.

Evaluation of ABT-888 in the amelioration of α-synuclein fibril-induced neurodegeneration.

The accumulation of α-synuclein inclusions in vulnerable neuronal populations pathologically defines Lewy body diseases including Parkinson's disease. Recent pre-clinical studies suggest poly(ADP-ribose) polymerase-1 activation and the subsequent generation of poly(ADP-ribose) polymer represent key steps in the formation of toxic α-synuclein aggregates and neurodegeneration. Several studies suggest that the inhibition of poly(ADP-ribose) polymerase-1 activity via the poly(ADP-ribose) polymerase-1/2 small molecule inhibitor ABT-888 (Veliparib), a drug in clinical trials for different cancers, may prevent or ameliorate α-synuclein fibril-induced aggregation, inclusion formation and dopaminergic neurodegeneration. Herein, we evaluated the effects of poly(ADP-ribose) polymer on α-synuclein fibrillization in vitro, the effects of ABT-888 on the formation of fibril-seeded α-synuclein inclusions in primary mouse cortical neurons and the effects of an in-diet ABT-888 dosage regimen with the intracranial injection of α-synuclein fibrils into the mouse dorsal striatum. We found that poly(ADP-ribose) polymer minimally but significantly increased the rate of spontaneously formed α-synuclein fibrils in vitro. Machine-learning algorithms that quantitatively assessed α-synuclein inclusion counts in neurons, both in primary cultures and in the brains of fibril-injected mice, did not reveal differences between ABT-888- and vehicle-treated groups. The in-diet administered ABT-888 molecule demonstrated outstanding brain penetration in mice; however, dopaminergic cell loss in the substantia nigra caused by α-synuclein fibril injections in the striatum was similar between ABT-888- and vehicle-treated groups. α-Synuclein fibril-induced loss of dopaminergic fibres in the dorsal striatum was also similar between ABT-888- and vehicle-treated groups. We conclude that additional pre-clinical evaluation of ABT-888 may be warranted to justify further exploration of ABT-888 for disease modification in Lewy body diseases.

Lysophosphatidylcholine acyltransferase 1 promotes pathology and toxicity in two distinct cell-based alpha-synuclein models.

Alpha-synuclein (αSyn) pathology is a hallmark of Parkinson's disease. Here we show that lysophosphatidylcholine acyltransferase 1 (LPCAT1) is a regulator of αSyn pathology and cytotoxicity. LPCAT1 is upregulated by αSyn E35K E46K E61K (3K) in human M17 neuroblastoma cells and primary rat cortical neurons, and in postmortem brain tissue from PD patients with confirmed αSyn aggregate pathology. Suppression of LPCAT1 reduces αSyn accumulations and toxicity in our neuroblastoma αSyn 3K overexpression model. Further overexpression of LPCAT1 promotes pS129 αSyn positive aggregation in primary neurons in the αSyn pre-formed fibril (PFF) model. A phospholipid product of LPCAT1 enzymatic activity, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, similarly promotes neuronal PFF seeded aggregation. Using a pH sensitive PFF model we provide evidence that αSyn fibrils have altered endo-lysosomal processing under LPCAT1 enhancement, suggesting less aggregate degradation. Our data demonstrates that LPCAT1 and associated phospholipids can regulate αSyn pathology.

Discovery of small-molecule positive allosteric modulators of Parkin E3 ligase.

Pharmacological activation of the E3 ligase Parkin represents a rational therapeutic intervention for the treatment of Parkinson's disease. Here we identify several compounds that enhance the activity of wildtype Parkin in the presence of phospho-ubiquitin and act as positive allosteric modulators (PAMs). While these compounds activate Parkin in a series of biochemical assays, they do not act by thermally destabilizing Parkin and fail to enhance the Parkin translocation rate to mitochondria or to enact mitophagy in cell-based assays. We conclude that in the context of the cellular milieu the therapeutic window to pharmacologically activate Parkin is very narrow.

Evaluation of Strategies for Measuring Lysosomal Glucocerebrosidase Activity.

Mutations in GBA1, which encode for the protein glucocerebrosidase (GCase), are the most common genetic risk factor for Parkinson's disease and dementia with Lewy bodies. In addition, growing evidence now suggests that the loss of GCase activity is also involved in onset of all forms of Parkinson's disease, dementia with Lewy bodies, and other dementias, such as progranulin-linked frontal temporal dementia. As a result, there is significant interest in developing GCase-targeted therapies that have the potential to stop or slow progression of these diseases. Despite this interest in GCase as a therapeutic target, there is significant inconsistency in the methodology for measuring GCase enzymatic activity in disease-modeling systems and patient populations, which could hinder progress in developing GCase therapies. In this review, we discuss the different strategies that have been developed to assess GCase activity and highlight the specific strengths and weaknesses of these approaches as well as the gaps that remain. We also discuss the current and potential role of these different methodologies in preclinical and clinical development of GCase-targeted therapies. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Wild-type GBA1 increases the α-synuclein tetramer-monomer ratio, reduces lipid-rich aggregates, and attenuates motor and cognitive deficits in mice.

Loss-of-function mutations in acid beta-glucosidase 1 (GBA1) are among the strongest genetic risk factors for Lewy body disorders such as Parkinson's disease (PD) and Lewy body dementia (DLB). Altered lipid metabolism in PD patient-derived neurons, carrying either GBA1 or PD αS mutations, can shift the physiological α-synuclein (αS) tetramer-monomer (T:M) equilibrium toward aggregation-prone monomers. A resultant increase in pSer129+ αS monomers provides a likely building block for αS aggregates. 3K αS mice, representing a neuropathological amplification of the E46K PD-causing mutation, have decreased αS T:M ratios and vesicle-rich αS+ aggregates in neurons, accompanied by a striking PD-like motor syndrome. We asked whether enhancing glucocerebrosidase (GCase) expression could benefit αS dyshomeostasis by delivering an adeno-associated virus (AAV)-human wild-type (wt) GBA1 vector into the brains of 3K neonates. Intracerebroventricular AAV-wtGBA1 at postnatal day 1 resulted in prominent forebrain neuronal GCase expression, sustained through 6 mo. GBA1 attenuated behavioral deficits both in working memory and fine motor performance tasks. Furthermore, wtGBA1 increased αS solubility and the T:M ratio in both 3K-GBA mice and control littermates and reduced pS129+ and lipid-rich aggregates in 3K-GBA. We observed GCase distribution in more finely dispersed lysosomes, in which there was increased GCase activity, lysosomal cathepsin D and B maturation, decreased perilipin-stabilized lipid droplets, and a normalized TFEB translocation to the nucleus, all indicative of improved lysosomal function and lipid turnover. Therefore, a prolonged increase of the αS T:M ratio by elevating GCase activity reduced the lipid- and vesicle-rich aggregates and ameliorated PD-like phenotypes in mice, further supporting lipid modulating therapies in PD.

Alpha-synuclein research: defining strategic moves in the battle against Parkinson's disease.

With the advent of the genetic era in Parkinson's disease (PD) research in 1997, α-synuclein was identified as an important player in a complex neurodegenerative disease that affects >10 million people worldwide. PD has been estimated to have an economic impact of $51.9 billion in the US alone. Since the initial association with PD, hundreds of researchers have contributed to elucidating the functions of α-synuclein in normal and pathological states, and these remain critical areas for continued research. With this position paper the authors strive to achieve two goals: first, to succinctly summarize the critical features that define α-synuclein's varied roles, as they are known today; and second, to identify the most pressing knowledge gaps and delineate a multipronged strategy for future research with the goal of enabling therapies to stop or slow disease progression in PD.

SCD Inhibition Protects from α-Synuclein-Induced Neurotoxicity But Is Toxic to Early Neuron Cultures.

Here, we report the independent discovery and validation of stearoyl-CoA desaturase (SCD) as a modulator of α-synuclein (αSyn)-induced pathology and toxicity in cell-based Parkinson's disease (PD) models. We identified SCD as top altered gene from transcriptional profiling in primary neurons exogenously expressing αSyn with the amplified familial PD mutation 3K. Thus, we sought to further explore SCD as a therapeutic target in neurodegeneration. We report that SCD inhibitors are toxic to early human and rat neuron cultures while displaying minimal toxicity to late cultures. The fatty acid product of SCD, oleic acid (OLA), fully rescues this toxicity in early cultures, suggesting on-target toxicity. Furthermore, SCD inhibition rescues αSyn 3K-induced toxicity in late primary neurons. We also confirm that SCD inhibitors reduce formation of αSyn accumulations, while OLA increases these accumulations in an αSyn 3K neuroblastoma model. However, we identify a caveat with this model where αSyn 3K levels can be suppressed by high SCD inhibitor concentrations, obscuring true effect size. Further, we show that both SCD1 or SCD5 knock-down reduce αSyn 3K accumulations and toxicity, making both a putative drug target. Overall, we confirm key findings of published data on SCD inhibition and its benefits in αSyn accumulation and stress models. The differential neurotoxicity induced by SCD inhibition based on neuron culture age must be accounted for when researching SCD in neuron models and has potential clinical implications. Lastly, our gene profiling studies also revealed novel putative genes connected to αSyn neurotoxicity that are worth further study.

Decreased glucocerebrosidase activity and substrate accumulation of glycosphingolipids in a novel GBA1 D409V knock-in mouse model.

Multiple mutations have been described in the human GBA1 gene, which encodes the lysosomal enzyme beta-glucocerebrosidase (GCase) that degrades glucosylceramide and is pivotal in glycosphingolipid substrate metabolism. Depletion of GCase, typically by homozygous mutations in GBA1, is linked to the lysosomal storage disorder Gaucher's disease (GD) and distinct or heterozygous mutations in GBA1 are associated with increased Parkinson's disease (PD) risk. While numerous genes have been linked to heritable PD, GBA1 mutations in aggregate are the single greatest risk factor for development of idiopathic PD. The importance of GCase in PD necessitates preclinical models in which to study GCase-related mechanisms and novel therapeutic approaches, as well as to elucidate the molecular mechanisms leading to enhanced PD risk in GBA1 mutation carriers. The aim of this study was to develop and characterize a novel GBA1 mouse model and to facilitate wide accessibility of the model with phenotypic data. Herein we describe the results of molecular, biochemical, histological, and behavioral phenotyping analyses in a GBA1 D409V knock-in (KI) mouse. This mouse model exhibited significantly decreased GCase activity in liver and brain, with substantial increases in glycosphingolipid substrates in the liver. While no changes in the number of dopamine neurons in the substantia nigra were noted, subtle changes in striatal neurotransmitters were observed in GBA1 D409V KI mice. Alpha-synuclein pathology and inflammation were not observed in the nigrostriatal system of this model. In summary, the GBA1 D409V KI mouse model provides an ideal model for studies aimed at pharmacodynamic assessments of potential therapies aiming to restore GCase.

Optimization of small-scale sample preparation for high-throughput OpenArray analysis.

OpenArray is one of the most high-throughput qPCR platforms available but its efficiency can be limited by sample preparation methods that are slow and costly. To optimize the sample workflow for high-throughput qPCR processing by OpenArray, small-scale sample preparation methods were compared for compatibility with this system to build confidence in a method that maintains quality and accuracy while using less starting material and saving time and money. This study is the first to show that the Cells-to-CT kit can be used to prepare samples within the dynamic range of OpenArray directly from cultured cells in a single well of a 96-well plate when used together with a cDNA preamplification PCR step. Use of Cells-to-CT produced results of similar quality and accuracy to that of a preparation method using purified RNA in less than half the sample preparation time. While Cells-to-CT samples also exhibited slightly increased variance, which affects the ability of OpenArray to distinguish small differences in gene expression, overall gene expression mean results correlated well between small-scale methods. This work demonstrates that Cells-to-CT with preamplification can be used to reliably prepare samples for OpenArray analysis while saving time, money, and starting material.

The Parkinson's disease-associated gene ITPKB protects against α-synuclein aggregation by regulating ER-to-mitochondria calcium release.

Inositol-1,4,5-triphosphate (IP3) kinase B (ITPKB) is a ubiquitously expressed lipid kinase that inactivates IP3, a secondary messenger that stimulates calcium release from the endoplasmic reticulum (ER). Genome-wide association studies have identified common variants in the ITPKB gene locus associated with reduced risk of sporadic Parkinson's disease (PD). Here, we investigate whether ITPKB activity or expression level impacts PD phenotypes in cellular and animal models. In primary neurons, knockdown or pharmacological inhibition of ITPKB increased levels of phosphorylated, insoluble α-synuclein pathology following treatment with α-synuclein preformed fibrils (PFFs). Conversely, ITPKB overexpression reduced PFF-induced α-synuclein aggregation. We also demonstrate that ITPKB inhibition or knockdown increases intracellular calcium levels in neurons, leading to an accumulation of calcium in mitochondria that increases respiration and inhibits the initiation of autophagy, suggesting that ITPKB regulates α-synuclein pathology by inhibiting ER-to-mitochondria calcium transport. Furthermore, the effects of ITPKB on mitochondrial calcium and respiration were prevented by pretreatment with pharmacological inhibitors of the mitochondrial calcium uniporter complex, which was also sufficient to reduce α-synuclein pathology in PFF-treated neurons. Taken together, these results identify ITPKB as a negative regulator of α-synuclein aggregation and highlight modulation of ER-to-mitochondria calcium flux as a therapeutic strategy for the treatment of sporadic PD.

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.

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.