A high efficiency precision genome editing method with CRISPR in iPSCs.

The use of genetic engineering to generate point mutations in induced pluripotent stem cells (iPSCs) is essential for studying a specific genetic effect in an isogenic background. We demonstrate that a combination of p53 inhibition and pro-survival small molecules achieves a homologous recombination rate higher than 90% using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) in human iPSCs. Our protocol reduces the effort and time required to create isogenic lines.

Death Induced by Survival gene Elimination (DISE) correlates with neurotoxicity in Alzheimer's disease and aging.

Alzheimer's disease (AD) is characterized by progressive neurodegeneration, but the specific events that cause cell death remain poorly understood. Death Induced by Survival gene Elimination (DISE) is a cell death mechanism mediated by short (s) RNAs acting through the RNA-induced silencing complex (RISC). DISE is thus a form of RNA interference, in which G-rich 6mer seed sequences in the sRNAs (position 2-7) target hundreds of C-rich 6mer seed matches in genes essential for cell survival, resulting in the activation of cell death pathways. Here, using Argonaute precipitation and RNAseq (Ago-RP-Seq), we analyze RISC-bound sRNAs to quantify 6mer seed toxicity in several model systems. In mouse AD models and aging brain, in induced pluripotent stem cell-derived neurons from AD patients, and in cells exposed to Aß42 oligomers, RISC-bound sRNAs show a shift to more toxic 6mer seeds compared to controls. In contrast, in brains of "SuperAgers", humans over age 80 who have superior memory performance, RISC-bound sRNAs are shifted to more nontoxic 6mer seeds. Cells depleted of nontoxic sRNAs are sensitized to Aß42-induced cell death, and reintroducing nontoxic RNAs is protective. Altogether, the correlation between DISE and Aß42 toxicity suggests that increasing the levels of nontoxic miRNAs in the brain or blocking the activity of toxic RISC-bound sRNAs could ameliorate neurodegeneration.

In Vivo Prenylomic Profiling in the Brain of a Transgenic Mouse Model of Alzheimer's Disease Reveals Increased Prenylation of a Key Set of Proteins.

Dysregulation of protein prenylation has been implicated in many diseases, including Alzheimer's disease (AD). Prenylomic analysis, the combination of metabolic incorporation of an isoprenoid analogue (C15AlkOPP) into prenylated proteins with a bottom-up proteomic analysis, has allowed the identification of prenylated proteins in various cellular models. Here, transgenic AD mice were administered with C15AlkOPP through intracerebroventricular (ICV) infusion over 13 days. Using prenylomic analysis, 36 prenylated proteins were enriched in the brains of AD mice. Importantly, the prenylated forms of 15 proteins were consistently upregulated in AD mice compared to nontransgenic wild-type controls. These results highlight the power of this in vivo metabolic labeling approach to identify multiple post-translationally modified proteins that may serve as potential therapeutic targets for a disease that has proved refractory to treatment thus far. Moreover, this method should be applicable to many other types of protein modifications, significantly broadening its scope.

Pathological α-syn aggregation is mediated by glycosphingolipid chain length and the physiological state of α-syn in vivo.

GBA1 mutations that encode lysosomal ß-glucocerebrosidase (GCase) cause the lysosomal storage disorder Gaucher disease (GD) and are strong risk factors for synucleinopathies, including Parkinson's disease and Lewy body dementia. Only a subset of subjects with GBA1 mutations exhibit neurodegeneration, and the factors that influence neurological phenotypes are unknown. We find that α-synuclein (α-syn) neuropathology induced by GCase depletion depends on neuronal maturity, the physiological state of α-syn, and specific accumulation of long-chain glycosphingolipid (GSL) GCase substrates. Reduced GCase activity does not initiate α-syn aggregation in neonatal mice or immature human midbrain cultures; however, adult mice or mature midbrain cultures that express physiological α-syn oligomers are aggregation prone. Accumulation of long-chain GSLs (≥C22), but not short-chain species, induced α-syn pathology and neurological dysfunction. Selective reduction of long-chain GSLs ameliorated α-syn pathology through lysosomal cathepsins. We identify specific requirements that dictate synuclein pathology in GD models, providing possible explanations for the phenotypic variability in subjects with GCase deficiency.

Stress-Induced Cellular Clearance Is Mediated by the SNARE Protein ykt6 and Disrupted by α-Synuclein.

Age-related neurodegenerative disorders are characterized by a slow, persistent accumulation of aggregated proteins. Although cells can elicit physiological responses to enhance cellular clearance and counteract accumulation, it is unclear how pathogenic proteins evade this process in disease. We find that Parkinson's disease α-synuclein perturbs the physiological response to lysosomal stress by impeding the SNARE protein ykt6. Cytosolic ykt6 is normally autoinhibited by a unique farnesyl-mediated regulatory mechanism; however, during lysosomal stress, it activates and redistributes into membranes to preferentially promote hydrolase trafficking and enhance cellular clearance. α-Synuclein aberrantly binds and deactivates ykt6 in patient-derived neurons, thereby disabling the lysosomal stress response and facilitating protein accumulation. Activating ykt6 by small-molecule farnesyltransferase inhibitors restores lysosomal activity and reduces α-synuclein in patient-derived neurons and mice. Our findings indicate that α-synuclein creates a permissive environment for aggregate persistence by inhibiting regulated cellular clearance and provide a therapeutic strategy to restore protein homeostasis by harnessing SNARE activity.

Patient-Customized Oligonucleotide Therapy for a Rare Genetic Disease.

Genome sequencing is often pivotal in the diagnosis of rare diseases, but many of these conditions lack specific treatments. We describe how molecular diagnosis of a rare, fatal neurodegenerative condition led to the rational design, testing, and manufacture of milasen, a splice-modulating antisense oligonucleotide drug tailored to a particular patient. Proof-of-concept experiments in cell lines from the patient served as the basis for launching an "N-of-1" study of milasen within 1 year after first contact with the patient. There were no serious adverse events, and treatment was associated with objective reduction in seizures (determined by electroencephalography and parental reporting). This study offers a possible template for the rapid development of patient-customized treatments. (Funded by Mila's Miracle Foundation and others.).

Reversible Conformational Conversion of α-Synuclein into Toxic Assemblies by Glucosylceramide.

α-Synuclein (α-syn) aggregation is a key event in Parkinson's disease (PD). Mutations in glycosphingolipid (GSL)-degrading glucocerebrosidase are risk factors for PD, indicating that disrupted GSL clearance plays a key role in α-syn aggregation. However, the mechanisms of GSL-induced aggregation are not completely understood. We document the presence of physiological α-syn conformers in human midbrain dopamine neurons and tested their contribution to the aggregation process. Pathological α-syn assembly mainly occurred through the conversion of high molecular weight (HMW) physiological α-syn conformers into compact, assembly-state intermediates by glucosylceramide (GluCer), without apparent disassembly into free monomers. This process was reversible in vitro through GluCer depletion. Reducing GSLs in PD patient neurons with and without GBA1 mutations diminished pathology and restored physiological α-syn conformers that associated with synapses. Our work indicates that GSLs control the toxic conversion of physiological α-syn conformers in a reversible manner that is amenable to therapeutic intervention by GSL reducing agents.