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.

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.

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 plasmidsBasic Protocol 2: Transfection of cells with plasmids and sorting GFP‐positive cellsBasic Protocol 3: PCR screening single‐cell clones and validation of the mutant HTT expression

Technology of reprogramming of somatic cells to a pluripotent state allows generating induced pluripotent stem cells (iPSCs) and carrying out a broad range of studies. iPSCs can be obtained from patients suffering from inherited diseases to model the diseases and to study their pathological mechanisms at the molecular level after iPSC differentiation in relevant cell types. Another approach to model and study inherited diseases is using iPSCs from healthy donors and genome editing tools. The approach allows generating a panel of isogenic lines, which gives new opportunities in drug screening and toxicological testing. Moreover, iPSCs and their derivatives can be further used for substitutive cell therapy and transplantology. In this study, we generated iPSCs from human embryonic fibroblasts using episomal vectors. The lines obtained expressed pluripotency markers, had a stable karyotype – 46:XY, and did not contain episome integrations into genome. The cell lines gave rise to derivatives of three germ layers during spontaneous differentiation in vitro and in vivo .

LRRK2 is implicated in lysosomal functions, but the physiological upstream cues that engage endogenous LRRK2 activity are incompletely defined. Here we show that lysosomal swelling serves as a selective and reversible trigger for LRRK2-mediated Rab phosphorylation, without requiring membrane damage. Acute inhibition of PIKfyve, but not the general disruption of phosphoinositide signaling, induces the robust accumulation of phosphorylated Rabs across endolysosomal membranes. Rescue of swelling through pharmacological restoration of lysosomal ionic imbalances from PIKfyve inhibition suppresses LRRK2 activation without restoring lysosomal function. Mechanical lysosomal swelling from indigestible osmolyte uptake causes a dose-dependent increase in LRRK2-mediated Rab phosphorylation on both swollen and non-swollen lysosomes. Together, these findings identify LRRK2 as a sensor of lysosomal volume and mechanical stress, not specifically membrane damage or PIKfyve inhibition. As lysosomal swelling is a shared pathological feature across LRRK2-linked diseases, these results reframe LRRK2 as part of an endolysosomal surveillance system responsive to lysosomal distension.