Single molecule array measures of LRRK2 kinase activity in serum link Parkinson's disease severity to peripheral inflammation.

BACKGROUND: LRRK2-targeting therapeutics that inhibit LRRK2 kinase activity have advanced to clinical trials in idiopathic Parkinson's disease (iPD). LRRK2 phosphorylates Rab10 on endolysosomes in phagocytic cells to promote some types of immunological responses. The identification of factors that regulate LRRK2-mediated Rab10 phosphorylation in iPD, and whether phosphorylated-Rab10 levels change in different disease states, or with disease progression, may provide insights into the role of Rab10 phosphorylation in iPD and help guide therapeutic strategies targeting this pathway. METHODS: Capitalizing on past work demonstrating LRRK2 and phosphorylated-Rab10 interact on vesicles that can shed into biofluids, we developed and validated a high-throughput single-molecule array assay to measure extracellular pT73-Rab10. Ratios of pT73-Rab10 to total Rab10 measured in biobanked serum samples were compared between informative groups of transgenic mice, rats, and a deeply phenotyped cohort of iPD cases and controls. Multivariable and weighted correlation network analyses were used to identify genetic, transcriptomic, clinical, and demographic variables that predict the extracellular pT73-Rab10 to total Rab10 ratio. RESULTS: pT73-Rab10 is absent in serum from Lrrk2 knockout mice but elevated by LRRK2 and VPS35 mutations, as well as SNCA expression. Bone-marrow transplantation experiments in mice show that serum pT73-Rab10 levels derive primarily from circulating immune cells. The extracellular ratio of pT73-Rab10 to total Rab10 is dynamic, increasing with inflammation and rapidly decreasing with LRRK2 kinase inhibition. The ratio of pT73-Rab10 to total Rab10 is elevated in iPD patients with greater motor dysfunction, irrespective of disease duration, age, sex, or the usage of PD-related or anti-inflammatory medications. pT73-Rab10 to total Rab10 ratios are associated with neutrophil degranulation, antigenic responses, and suppressed platelet activation. CONCLUSIONS: The extracellular serum ratio of pT73-Rab10 to total Rab10 is a novel pharmacodynamic biomarker for LRRK2-linked innate immune activation associated with disease severity in iPD. We propose that those iPD patients with higher serum pT73-Rab10 levels may benefit from LRRK2-targeting therapeutics that mitigate associated deleterious immunological responses.

Single molecule array measures of LRRK2 kinase activity in serum link Parkinson's disease severity to peripheral inflammation.

Background: LRRK2-targeting therapeutics that inhibit LRRK2 kinase activity have advanced to clinical trials in idiopathic Parkinson's disease (iPD). LRRK2 phosphorylates Rab10 on endolysosomes in phagocytic cells to promote some types of immunological responses. The identification of factors that regulate LRRK2-mediated Rab10 phosphorylation in iPD, and whether phosphorylated-Rab10 levels change in different disease states, or with disease progression, may provide insights into the role of Rab10 phosphorylation in iPD and help guide therapeutic strategies targeting this pathway. Methods: Capitalizing on past work demonstrating LRRK2 and phosphorylated-Rab10 interact on vesicles that can shed into biofluids, we developed and validated a high-throughput single-molecule array assay to measure extracellular pT73-Rab10. Ratios of pT73-Rab10 to total Rab10 measured in biobanked serum samples were compared between informative groups of transgenic mice, rats, and a deeply phenotyped cohort of iPD cases and controls. Multivariable and weighted correlation network analyses were used to identify genetic, transcriptomic, clinical, and demographic variables that predict the extracellular pT73-Rab10 to total Rab10 ratio. Results: pT73-Rab10 is absent in serum from Lrrk2 knockout mice but elevated by LRRK2 and VPS35 mutations, as well as SNCA expression. Bone-marrow transplantation experiments in mice show that serum pT73-Rab10 levels derive primarily from circulating immune cells. The extracellular ratio of pT73-Rab10 to total Rab10 is dynamic, increasing with inflammation and rapidly decreasing with LRRK2 kinase inhibition. The ratio of pT73-Rab10 to total Rab10 is elevated in iPD patients with greater motor dysfunction, irrespective of disease duration, age, sex, or the usage of PD-related or anti-inflammatory medications. pT73-Rab10 to total Rab10 ratios are associated with neutrophil activation, antigenic responses, and the suppression of platelet activation. Conclusions: The extracellular ratio of pT73-Rab10 to total Rab10 in serum is a novel pharmacodynamic biomarker for LRRK2-linked innate immune activation associated with disease severity in iPD. We propose that those iPD patients with higher serum pT73-Rab10 levels may benefit from LRRK2-targeting therapeutics to mitigate associated deleterious immunological responses.

Biochemical Characteristics of iPSC-Derived Dopaminergic Neurons from N370S GBA Variant Carriers with and without Parkinson's Disease.

GBA variants increase the risk of Parkinson's disease (PD) by 10 times. The GBA gene encodes the lysosomal enzyme glucocerebrosidase (GCase). The p.N370S substitution causes a violation of the enzyme conformation, which affects its stability in the cell. We studied the biochemical characteristics of dopaminergic (DA) neurons generated from induced pluripotent stem cells (iPSCs) from a PD patient with the GBA p.N370S mutation (GBA-PD), an asymptomatic GBA p.N370S carrier (GBA-carrier), and two healthy donors (control). Using liquid chromatography with tandem mass spectrometry (LC-MS/MS), we measured the activity of six lysosomal enzymes (GCase, galactocerebrosidase (GALC), alpha-glucosidase (GAA), alpha-galactosidase (GLA), sphingomyelinase (ASM), and alpha-iduronidase (IDUA)) in iPSC-derived DA neurons from the GBA-PD and GBA-carrier. DA neurons from the GBA mutation carrier demonstrated decreased GCase activity compared to the control. The decrease was not associated with any changes in GBA expression levels in DA neurons. GCase activity was more markedly decreased in the DA neurons of GBA-PD patient compared to the GBA-carrier. The amount of GCase protein was decreased only in GBA-PD neurons. Additionally, alterations in the activity of the other lysosomal enzymes (GLA and IDUA) were found in GBA-PD neurons compared to GBA-carrier and control neurons. Further study of the molecular differences between the GBA-PD and the GBA-carrier is essential to investigate whether genetic factors or external conditions are the causes of the penetrance of the p.N370S GBA variant.

Generation of induced pluripotent stem cell line, ICGi033-A, by reprogramming peripheral blood mononuclear cells from a patient with Huntington's disease.

Huntington's disease (HD) is a hereditary autosomal dominant neurodegenerative disease caused by the polyglutamine stretch expansion in the huntingtin (HTT) protein. In HD, dysregulation of multiple cellular processes occurs, resulting in the death of medium spiny neurons of striatum. A line of induced pluripotent stem cells (iPSCs) ICGi033-A was obtained from peripheral blood mononuclear cells of a patient carrying 77 CAG repeats in the HTT gene. The iPSCs express pluripotency markers, have a normal karyotype, and differentiate into three germ layers: endoderm, ectoderm, mesoderm.

The Cutting Edge of Disease Modeling: Synergy of Induced Pluripotent Stem Cell Technology and Genetically Encoded Biosensors.

The development of cell models of human diseases based on induced pluripotent stem cells (iPSCs) and a cell therapy approach based on differentiated iPSC derivatives has provided a powerful stimulus in modern biomedical research development. Moreover, it led to the creation of personalized regenerative medicine. Due to this, in the last decade, the pathological mechanisms of many monogenic diseases at the cell level have been revealed, and clinical trials of various cell products derived from iPSCs have begun. However, it is necessary to reach a qualitatively new level of research with cell models of diseases based on iPSCs for more efficient searching and testing of drugs. Biosensor technology has a great application prospect together with iPSCs. Biosensors enable researchers to monitor ions, molecules, enzyme activities, and channel conformation in live cells and use them in live imaging and drug screening. These probes facilitate the measurement of steady-state concentrations or activity levels and the observation and quantification of in vivo flux and kinetics. Real-time monitoring of drug action in a specific cellular compartment, organ, or tissue type; the ability to screen at the single-cell resolution; and the elimination of the false-positive results caused by low drug bioavailability that is not detected by in vitro testing methods are a few of the benefits of using biosensors in drug screening. Here, we discuss the possibilities of using biosensor technology in combination with cell models based on human iPSCs and gene editing systems. Furthermore, we focus on the current achievements and problems of using these methods.

A Human Induced Pluripotent Stem Cell-Derived Isogenic Model of Huntington's Disease Based on Neuronal Cells Has Several Relevant Phenotypic Abnormalities.

Huntington's disease (HD) is a severe neurodegenerative disorder caused by a CAG triplet expansion in the first exon of the HTT gene. Here we report the introduction of an HD mutation into the genome of healthy human embryonic fibroblasts through CRISPR/Cas9-mediated homologous recombination. We verified the specificity of the created HTT-editing system and confirmed the absence of undesirable genomic modifications at off-target sites. We showed that both mutant and control isogenic induced pluripotent stem cells (iPSCs) derived by reprogramming of the fibroblast clones can be differentiated into striatal medium spiny neurons. We next demonstrated phenotypic abnormalities in the mutant iPSC-derived neural cells, including impaired neural rosette formation and increased sensitivity to growth factor withdrawal. Moreover, using electron microscopic analysis, we detected a series of ultrastructural defects in the mutant neurons, which did not contain huntingtin aggregates, suggesting that these defects appear early in HD development. Thus, our study describes creation of a new isogenic iPSC-based cell system that models HD and recapitulates HD-specific disturbances in the mutant cells, including some ultrastructural features implemented for the first time.

Generation of GABAergic striatal neurons by a novel iPSC differentiation protocol enabling scalability and cryopreservation of progenitor cells.

Cell models are promising tools for studying hereditary human neurodegenerative diseases. Neuronal derivatives of pluripotent stem cells provide the opportunity to investigate different stages of the neurodegeneration process. Therefore, easy and large-scale production of relevant cell types is a crucial barrier to overcome. In this work, we present an alternative protocol for iPSC differentiation into GABAergic medium spiny neurons (MSNs). The first stage involved dual-SMAD signalling inhibition through treatment with SB431542 and LDN193189, which results in the generation of neuroectodermal cells. Moreover, we used bFGF as a neuronal survival factor and dorsomorphin to inhibit BMP signalling. The combined treatment of dorsomorphin and SB431542 significantly enhanced neuronal induction, which was confirmed by the increased expression of the telencephalic-specific markers SOX1 and OTX2 as well as the forebrain marker PAX6. The next stage involved the derivation of actively proliferating MSN progenitor cells. An important feature of our protocol at this stage is the ability to perform prolonged cultivation of precursor cells at a high density without losing phenotypic properties. Moreover, the protocol enables multiple expansion steps (> 180 days cultivation) and cryopreservation of MSN progenitors. Therefore, this method allows quick production of a large number of neurons that are relevant for basic research, large-scale drug screening, and toxicological studies.

Introducing an Expanded Trinucleotide Repeat Tract into the Human Genome for Huntington's Disease Modeling In Vitro.

In neurodegeneration studies, researchers are faced with problems such as limited material availability and late disease manifestation. Cell models provide the opportunity to investigate molecular mechanisms of pathogenesis. Moreover, genome editing technologies enable generation of isogenic cell models of hereditary diseases. Our protocol outlines an approach for introducing an expanded CAG repeat tract into the first exon of the HTT gene, the Huntington's disease causing mutation. The protocol allows modeling the disease at various severity levels by introducing different numbers of CAG repeats. Furthermore, the protocol can be applicable for modeling other diseases caused by trinucleotide repeat expansion. It is important to note there are many difficulties with cloning repeated sequences and amplification of GC-rich regions. Here, we also propose troubleshooting options, which overcome these problems. The protocol is based on CRISPR/Cas9-mediated homologous recombination with a uniquely designed donor plasmid harboring an expanded CAG tract flanked with long homology arms. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Design and assembling donor and CRISPR/Cas9-expressing plasmids Basic Protocol 2: Transfection of cells with plasmids and sorting GFP-positive cells Basic Protocol 3: PCR screening single-cell clones and validation of the mutant HTT expression.

Introducing an expanded CAG tract into the huntingtin gene causes a wide spectrum of ultrastructural defects in cultured human cells.

Modeling of neurodegenerative diseases in vitro holds great promise for biomedical research. Human cell lines harboring a mutations in disease-causing genes are thought to recapitulate early stages of the development an inherited disease. Modern genome-editing tools allow researchers to create isogenic cell clones with an identical genetic background providing an adequate "healthy" control for biomedical and pharmacological experiments. Here, we generated isogenic mutant cell clones with 150 CAG repeats in the first exon of the huntingtin (HTT) gene using the CRISPR/Cas9 system and performed ultrastructural and morphometric analyses of the internal organization of the mutant cells. Electron microscopy showed that deletion of three CAG triplets or an HTT gene knockout had no significant influence on the cell structure. The insertion of 150 CAG repeats led to substantial changes in quantitative and morphological parameters of mitochondria and increased the association of mitochondria with the smooth and rough endoplasmic reticulum while causing accumulation of small autolysosomes in the cytoplasm. Our data indicate for the first time that expansion of the CAG repeat tract in HTT introduced via the CRISPR/Cas9 technology into a human cell line initiates numerous ultrastructural defects that are typical for Huntington's disease.

Modern Genome Editing Technologies in Huntington's Disease Research.

The development of new revolutionary technologies for directed gene editing has made it possible to thoroughly model and study NgAgo human diseases at the cellular and molecular levels. Gene editing tools like ZFN, TALEN, CRISPR-based systems, NgAgo and SGN can introduce different modifications. In gene sequences and regulate gene expression in different types of cells including induced pluripotent stem cells (iPSCs). These tools can be successfully used for Huntington's disease (HD) modeling, for example, to generate isogenic cell lines bearing different numbers of CAG repeats or to correct the mutation causing the disease. This review presents common genome editing technologies and summarizes the progress made in using them in HD and other hereditary diseases. Furthermore, we will discuss prospects and limitations of genome editing in understanding HD pathology.