Loss of TREM2 rescues hyperactivation of microglia, but not lysosomal deficits and neurotoxicity in models of progranulin deficiency.

Haploinsufficiency of the progranulin (PGRN)-encoding gene (GRN) causes frontotemporal lobar degeneration (GRN-FTLD) and results in microglial hyperactivation, TREM2 activation, lysosomal dysfunction, and TDP-43 deposition. To understand the contribution of microglial hyperactivation to pathology, we used genetic and pharmacological approaches to suppress TREM2-dependent transition of microglia from a homeostatic to a disease-associated state. Trem2 deficiency in Grn KO mice reduced microglia hyperactivation. To explore antibody-mediated pharmacological modulation of TREM2-dependent microglial states, we identified antagonistic TREM2 antibodies. Treatment of macrophages from GRN-FTLD patients with these antibodies led to reduced TREM2 signaling due to its enhanced shedding. Furthermore, TREM2 antibody-treated PGRN-deficient microglia derived from human-induced pluripotent stem cells showed reduced microglial hyperactivation, TREM2 signaling, and phagocytic activity, but lysosomal dysfunction was not rescued. Similarly, lysosomal dysfunction, lipid dysregulation, and glucose hypometabolism of Grn KO mice were not rescued by TREM2 ablation. Synaptic loss and neurofilament light-chain (NfL) levels, a biomarker for neurodegeneration, were further elevated in the Grn/Trem2 KO cerebrospinal fluid (CSF). These findings suggest that TREM2-dependent microglia hyperactivation in models of GRN deficiency does not promote neurotoxicity, but rather neuroprotection.

Membrane lipid remodeling modulates γ-secretase processivity.

Imbalances in the amounts of amyloid-ß peptides (Aß) generated by the membrane proteases ß- and γ-secretase are considered as a trigger of Alzheimer's disease (AD). Cell-free studies of γ-secretase have shown that increasing membrane thickness modulates Aß generation but it has remained unclear if these effects are translatable to cells. Here we show that the very long-chain fatty acid erucic acid (EA) triggers acyl chain remodeling in AD cell models, resulting in substantial lipidome alterations which included increased esterification of EA in membrane lipids. Membrane remodeling enhanced γ-secretase processivity, resulting in the increased production of the potentially beneficial Aß37 and/or Aß38 species in multiple cell lines. Unexpectedly, we found that the membrane remodeling stimulated total Aß secretion by cells expressing WT γ-secretase but lowered it for cells expressing an aggressive familial AD mutant γ-secretase. We conclude that EA-mediated modulation of membrane composition is accompanied by complex lipid homeostatic changes that can impact amyloidogenic processing in different ways and elicit distinct γ-secretase responses, providing critical implications for lipid-based AD treatment strategies.

Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9.

The bacterial CRISPR/Cas9 system allows sequence-specific gene editing in many organisms and holds promise as a tool to generate models of human diseases, for example, in human pluripotent stem cells. CRISPR/Cas9 introduces targeted double-stranded breaks (DSBs) with high efficiency, which are typically repaired by non-homologous end-joining (NHEJ) resulting in nonspecific insertions, deletions or other mutations (indels). DSBs may also be repaired by homology-directed repair (HDR) using a DNA repair template, such as an introduced single-stranded oligo DNA nucleotide (ssODN), allowing knock-in of specific mutations. Although CRISPR/Cas9 is used extensively to engineer gene knockouts through NHEJ, editing by HDR remains inefficient and can be corrupted by additional indels, preventing its widespread use for modelling genetic disorders through introducing disease-associated mutations. Furthermore, targeted mutational knock-in at single alleles to model diseases caused by heterozygous mutations has not been reported. Here we describe a CRISPR/Cas9-based genome-editing framework that allows selective introduction of mono- and bi-allelic sequence changes with high efficiency and accuracy. We show that HDR accuracy is increased dramatically by incorporating silent CRISPR/Cas-blocking mutations along with pathogenic mutations, and establish a method termed 'CORRECT' for scarless genome editing. By characterizing and exploiting a stereotyped inverse relationship between a mutation's incorporation rate and its distance to the DSB, we achieve predictable control of zygosity. Homozygous introduction requires a guide RNA targeting close to the intended mutation, whereas heterozygous introduction can be accomplished by distance-dependent suboptimal mutation incorporation or by use of mixed repair templates. Using this approach, we generated human induced pluripotent stem cells with heterozygous and homozygous dominant early onset Alzheimer's disease-causing mutations in amyloid precursor protein (APP(Swe)) and presenilin 1 (PSEN1(M146V)) and derived cortical neurons, which displayed genotype-dependent disease-associated phenotypes. Our findings enable efficient introduction of specific sequence changes with CRISPR/Cas9, facilitating study of human disease.

Immunophilin FKBP52 induces Tau-P301L filamentous assembly in vitro and modulates its activity in a model of tauopathy.

The Tau protein is the major component of intracellular filaments observed in a number of neurodegenerative diseases known as tauopathies. The pathological mutant of Tau containing a proline-to-leucine mutation at position 301 (P301L) leads to severe human tauopathy. Here, we assess the impact of FK506-binding protein with a molecular mass of ∼52 kDa (FKBP52), an immunophilin protein that interacts with physiological Tau, on Tau-P301L activity. We identify a direct interaction of FKBP52 with Tau-P301L and its phosphorylated forms and demonstrate FKBP52's ability to induce the formation of Tau-P301L oligomers. EM analysis shows that Tau-P301L oligomers, induced by FKBP52, can assemble into filaments. In the transgenic zebrafish expressing the human Tau-P301L mutant, FKBP52 knockdown is sufficient to redrive defective axonal outgrowth and branching related to Tau-P301L expression in spinal primary motoneurons. This result correlates with a significant reduction of pT181 pathological phosphorylated Tau and with recovery of the stereotypic escape response behavior. Collectively, FKBP52 appears to be an endogenous candidate that directly interacts with the pathogenic Tau-P301L and modulates its function in vitro and in vivo.

Tackling neurodegeneration in vitro with omics: a path towards new targets and drugs.

Drug discovery is a generally inefficient and capital-intensive process. For neurodegenerative diseases (NDDs), the development of novel therapeutics is particularly urgent considering the long list of late-stage drug candidate failures. Although our knowledge on the pathogenic mechanisms driving neurodegeneration is growing, additional efforts are required to achieve a better and ultimately complete understanding of the pathophysiological underpinnings of NDDs. Beyond the etiology of NDDs being heterogeneous and multifactorial, this process is further complicated by the fact that current experimental models only partially recapitulate the major phenotypes observed in humans. In such a scenario, multi-omic approaches have the potential to accelerate the identification of new or repurposed drugs against a multitude of the underlying mechanisms driving NDDs. One major advantage for the implementation of multi-omic approaches in the drug discovery process is that these overarching tools are able to disentangle disease states and model perturbations through the comprehensive characterization of distinct molecular layers (i.e., genome, transcriptome, proteome) up to a single-cell resolution. Because of recent advances increasing their affordability and scalability, the use of omics technologies to drive drug discovery is nascent, but rapidly expanding in the neuroscience field. Combined with increasingly advanced in vitro models, which particularly benefited from the introduction of human iPSCs, multi-omics are shaping a new paradigm in drug discovery for NDDs, from disease characterization to therapeutics prediction and experimental screening. In this review, we discuss examples, main advantages and open challenges in the use of multi-omic approaches for the in vitro discovery of targets and therapies against NDDs.

Cell-type-specific loops linked to RNA polymerase II elongation in human neural differentiation.

DNA is folded into higher-order structures that shape and are shaped by genome function. The role of long-range loops in the establishment of new gene expression patterns during cell fate transitions remains poorly understood. Here, we investigate the link between cell-specific loops and RNA polymerase II (RNA Pol II) during neural lineage commitment. We find thousands of loops decommissioned or gained de novo upon differentiation of human induced pluripotent stem cells (hiPSCs) to neural progenitor cells (NPCs) and post-mitotic neurons. During hiPSC-to-NPC and NPC-to-neuron transitions, genes changing from RNA Pol II initiation to elongation are >4-fold more likely to anchor cell-specific loops than repressed genes. Elongated genes exhibit significant mRNA upregulation when connected in cell-specific promoter-enhancer loops but not invariant promoter-enhancer loops or promoter-promoter loops or when unlooped. Genes transitioning from repression to RNA Pol II initiation exhibit a slight mRNA increase independent of loop status. Our data link cell-specific loops and robust RNA Pol II-mediated elongation during neural cell fate transitions.

A multi-looping chromatin signature predicts dysregulated gene expression in neurons with familial Alzheimer's disease mutations.

Mammalian genomes fold into tens of thousands of long-range loops, but their functional role and physiologic relevance remain poorly understood. Here, using human post-mitotic neurons with rare familial Alzheimer's disease (FAD) mutations, we identify hundreds of reproducibly dysregulated genes and thousands of miswired loops prior to amyloid accumulation and tau phosphorylation. Single loops do not predict expression changes; however, the severity and direction of change in mRNA levels and single-cell burst frequency strongly correlate with the number of FAD-gained or -lost promoter-enhancer loops. Classic architectural proteins CTCF and cohesin do not change occupancy in FAD-mutant neurons. Instead, we unexpectedly find TAATTA motifs amenable to binding by DLX homeodomain transcription factors and changing noncoding RNAPolII signal at FAD-dynamic promoter-enhancer loops. DLX1/5/6 mRNA levels are strongly upregulated in FAD-mutant neurons coincident with a shift in excitatory-to-inhibitory gene expression and miswiring of multi-loops connecting enhancers to neural subtype genes. DLX1 overexpression is sufficient for loop miswiring in wildtype neurons, including lost and gained loops at enhancers with tandem TAATTA arrays and singular TAATTA motifs, respectively. Our data uncover a genome structure-function relationship between multi-loop miswiring and dysregulated excitatory and inhibitory transcriptional programs during lineage commitment of human neurons homozygously-engineered with rare FAD mutations.

Proteomics of mouse brain endothelium uncovers dysregulation of vesicular transport pathways during aging.

Age-related decline in brain endothelial cell (BEC) function contributes critically to neurological disease. Comprehensive atlases of the BEC transcriptome have become available, but results from proteomic profiling are lacking. To gain insights into endothelial pathways affected by aging, we developed a magnetic-activated cell sorting-based mouse BEC enrichment protocol compatible with proteomics and resolved the profiles of protein abundance changes during aging. Unsupervised cluster analysis revealed a segregation of age-related protein dynamics with biological functions, including a downregulation of vesicle-mediated transport. We found a dysregulation of key regulators of endocytosis and receptor recycling (most prominently Arf6), macropinocytosis and lysosomal degradation. In gene deletion and overexpression experiments, Arf6 affected endocytosis pathways in endothelial cells. Our approach uncovered changes not picked up by transcriptomic studies, such as accumulation of vesicle cargo and receptor ligands, including Apoe. Proteomic analysis of BECs from Apoe-deficient mice revealed a signature of accelerated aging. Our findings provide a resource for analysing BEC function during aging.

Peripheral expression of brain-penetrant progranulin rescues pathologies in mouse models of frontotemporal lobar degeneration.

Progranulin (PGRN) haploinsufficiency is a major risk factor for frontotemporal lobar degeneration with TAR DNA-binding protein 43 (TDP-43) pathology (FTLD-GRN). Multiple therapeutic strategies are in clinical development to restore PGRN in the CNS, including gene therapy. However, a limitation of current gene therapy approaches aimed to alleviate FTLD-associated pathologies may be their inefficient brain exposure and biodistribution. We therefore developed an adeno-associated virus (AAV) targeting the liver (L) to achieve sustained peripheral expression of a transferrin receptor (TfR) binding, brain-penetrant (b) PGRN variant [AAV(L):bPGRN] in two mouse models of FTLD-GRN, namely, Grn knockout and GrnxTmem106b double knockout mice. This therapeutic strategy avoids potential safety and biodistribution issues of CNS-administered AAVs and maintains sustained concentrations of PGRN in the brain after a single dose. AAV(L):bPGRN treatment reduced several FTLD-GRN-associated pathologies including severe motor function deficits, aberrant TDP-43 phosphorylation, dysfunctional protein degradation, lipid metabolism, gliosis, and neurodegeneration in the brain. The potential translatability of our findings was tested in an in vitro model using cocultured human induced pluripotent stem cell (hiPSC)-derived microglia lacking PGRN and TMEM106B and wild-type hiPSC-derived neurons. As in mice, aberrant TDP-43, lysosomal dysfunction, and neuronal loss were ameliorated after treatment with exogenous TfR-binding protein transport vehicle fused to PGRN (PTV:PGRN). Together, our studies suggest that peripherally administered brain-penetrant PGRN replacement strategies ameliorate FTLD-GRN relevant phenotypes including TDP-43 pathology, neurodegeneration, and behavioral deficits. Our data provide preclinical proof of concept for the use of this AAV platform for treatment of FTLD-GRN and potentially other CNS disorders.

In vivo imaging of disease-related mitochondrial dynamics in a vertebrate model system.

Mitochondria provide ATP, maintain calcium homeostasis, and regulate apoptosis. Neurons, due to their size and complex geometry, are particularly dependent on the proper functioning and distribution of mitochondria. Thus disruptions of these organelles and their transport play a central role in a broad range of neurodegenerative diseases. While in vitro studies have greatly expanded our knowledge of mitochondrial dynamics, our understanding in vivo remains limited. To address this shortcoming, we developed tools to study mitochondrial dynamics in vivo in optically accessible zebrafish. We demonstrate here that our newly generated tools, including transgenic "MitoFish," can be used to study the in vivo "life cycle" of mitochondria and allows identifying pharmacological and genetic modulators of mitochondrial dynamics. Furthermore we observed profound mitochondrial transport deficits in real time in a zebrafish tauopathy model. By rescuing this phenotype using MARK2 (microtubule-affinity regulating kinase 2), we provide direct in vivo evidence that this kinase regulates axonal transport in a Tau-dependent manner. Thus, our approach allows detailed studies of the dynamics of mitochondria in their natural environment under normal and disease conditions.

Reproducible and scalable differentiation of highly pure cortical neurons from human induced pluripotent stem cells.

Human-induced-pluripotent-stem-cell (hiPSC)-derived neurons are valuable for investigating brain physiology and disease. Here, we present a protocol to differentiate hiPSCs into cortical neurons with high yield and purity. We describe neural induction via dual-SMAD inhibition, followed by spot-based differentiation to provide high quantities of neural precursors. We detail their enrichment, expansion, and purification to avoid unwanted cell fates and provide optimal conditions for neural rosette proliferation. These differentiated neurons are suitable for drug testing and co-culture studies. For complete details on the use and execution of this protocol, please refer to Paquet et al.1 and Weisheit et al..2.

Cell type-specific loops linked to RNA polymerase II elongation in human neural differentiation.

DNA is folded into higher-order structures that shape and are shaped by genome function. The role for long-range loops in the establishment of new gene expression patterns during cell fate transitions remains poorly understood. Here, we investigate the link between cell-specific loops and RNA polymerase II (RNAPolII) during neural lineage commitment. We find thousands of loops decommissioned or gained de novo upon differentiation of human induced pluripotent stem cells (hiPSCs) to neural progenitors (NPCs) and post-mitotic neurons. During hiPSC-to-NPC and NPC-to-neuron transitions, genes changing from RNAPolII initiation to elongation are >4-fold more likely to anchor cell-specific loops than repressed genes. Elongated genes exhibit significant mRNA upregulation when connected in cell-specific promoter-enhancer loops but not invariant promoter-enhancer loops, promoter-promoter loops, or unlooped. Genes transitioning from repression to RNAPolII initiation exhibit slight mRNA increase independent of loop status. Our data link cell-specific loops and robust RNAPolII-mediated elongation during neural cell fate transitions.

A MICROGLIAL ACTIVITY STATE BIOMARKER PANEL DIFFERENTIATES FTD-GRANULIN AND ALZHEIMER'S DISEASE PATIENTS FROM CONTROLS.

Background: With the emergence of microglia-modulating therapies there is an urgent need for reliable biomarkers to evaluate microglial activation states. Methods: Using mouse models and human induced pluripotent stem cell-derived microglia (hiMGL), which were genetically modified to yield the most opposite homeostatic ( TREM2- knockout) and disease-associated ( GRN -knockout) states, we identified microglia activity-dependent markers. Non-targeted mass spectrometry was used to identify changes in microglial and cerebrospinal (CSF) proteome of Grn - and Trem2 -knockout mice. Additionally, we analyzed the proteome of GRN - and TREM2 -knockout hiMGL and their conditioned media. Candidate marker proteins were tested in two independent patient cohorts, the ALLFTD cohort with 11 GRN mutation carriers and 12 non-carriers, as well as the proteomic data set available from the European Medical Information Framework Alzheimer's Disease Multimodal Biomarker Discovery (EMIF-AD MBD). Findings: We identified proteomic changes between the opposite activation states in mouse microglia and cerebrospinal fluid (CSF), as well as in hiMGL cell lysates and conditioned media. For further verification, we analyzed the CSF proteome of heterozygous GRN mutation carriers suffering from frontotemporal dementia (FTD). We identified a panel of six proteins (FABP3, MDH1, GDI1, CAPG, CD44, GPNMB) as potential indicators for microglial activation. Moreover, we confirmed three of these proteins (FABP3, GDI1, MDH1) to be significantly elevated in the CSF of AD patients. In AD, these markers differentiated amyloid-positive cases with mild cognitive impairment (MCI) from amyloid-negative individuals. Interpretation: The identified candidate proteins reflect microglia activity and may be relevant for monitoring the microglial response in clinical practice and clinical trials modulating microglial activity and amyloid deposition. Moreover, the finding that three of these markers differentiate amyloid-positive from amyloid-negative MCI cases in the AD cohort suggests that these marker proteins associate with a very early immune response to seeded amyloid. This is consistent with our previous findings in the DIAN (Dominantly Inherited Alzheimer's Disease Network) cohort, where soluble TREM2 increases as early as 21 years before symptom onset. Moreover, in mouse models for amyloidogenesis, seeding of amyloid is limited by physiologically active microglia further supporting their early protective role. The biological functions of some of our main candidates (FABP3, CD44, GPNMB) also further emphasize that lipid dysmetabolism may be a common feature of neurodegenerative disorders. Funding: This work was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany's Excellence Strategy within the framework of the Munich Cluster for Systems Neurology (EXC 2145 SyNergy - ID 390857198 to CH, SFL and DP) and a Koselleck Project HA1737/16-1 (to CH).

A microglial activity state biomarker panel differentiates FTD-granulin and Alzheimer's disease patients from controls.

BACKGROUND: With the emergence of microglia-modulating therapies there is an urgent need for reliable biomarkers to evaluate microglial activation states. METHODS: Using mouse models and human induced pluripotent stem cell-derived microglia (hiMGL), genetically modified to yield the most opposite homeostatic (TREM2-knockout) and disease-associated (GRN-knockout) states, we identified microglia activity-dependent markers. Non-targeted mass spectrometry was used to identify proteomic changes in microglia and cerebrospinal fluid (CSF) of Grn- and Trem2-knockout mice. Additionally, we analyzed the proteome of GRN- and TREM2-knockout hiMGL and their conditioned media. Candidate marker proteins were tested in two independent patient cohorts, the ALLFTD cohort (GRN mutation carriers versus non-carriers), as well as the proteomic data set available from the EMIF-AD MBD study. RESULTS: We identified proteomic changes between the opposite activation states in mouse microglia and CSF, as well as in hiMGL cell lysates and conditioned media. For further verification, we analyzed the CSF proteome of heterozygous GRN mutation carriers suffering from frontotemporal dementia (FTD). We identified a panel of six proteins (FABP3, MDH1, GDI1, CAPG, CD44, GPNMB) as potential indicators for microglial activation. Moreover, we confirmed three of these proteins (FABP3, GDI1, MDH1) to be significantly elevated in the CSF of Alzheimer's (AD) patients. Remarkably, each of these markers differentiated amyloid-positive cases with mild cognitive impairment (MCI) from amyloid-negative individuals. CONCLUSIONS: The identified candidate proteins reflect microglia activity and may be relevant for monitoring the microglial response in clinical practice and clinical trials modulating microglial activity and amyloid deposition. Moreover, the finding that three of these markers differentiate amyloid-positive from amyloid-negative MCI cases in the AD cohort suggests that these proteins associate with a very early immune response to seeded amyloid. This is consistent with our previous findings in the Dominantly Inherited Alzheimer's Disease Network (DIAN) cohort, where soluble TREM2 increases as early as 21 years before symptom onset. Moreover, in mouse models for amyloidogenesis, seeding of amyloid is limited by physiologically active microglia further supporting their early protective role. The biological functions of some of our main candidates (FABP3, CD44, GPNMB) also further emphasize that lipid dysmetabolism may be a common feature of neurodegenerative disorders.

Single-cell transcriptomic atlas-guided development of CAR-T cells for the treatment of acute myeloid leukemia.

Chimeric antigen receptor T cells (CAR-T cells) have emerged as a powerful treatment option for individuals with B cell malignancies but have yet to achieve success in treating acute myeloid leukemia (AML) due to a lack of safe targets. Here we leveraged an atlas of publicly available RNA-sequencing data of over 500,000 single cells from 15 individuals with AML and tissue from 9 healthy individuals for prediction of target antigens that are expressed on malignant cells but lacking on healthy cells, including T cells. Aided by this high-resolution, single-cell expression approach, we computationally identify colony-stimulating factor 1 receptor and cluster of differentiation 86 as targets for CAR-T cell therapy in AML. Functional validation of these established CAR-T cells shows robust in vitro and in vivo efficacy in cell line- and human-derived AML models with minimal off-target toxicity toward relevant healthy human tissues. This provides a strong rationale for further clinical development.

TPC2 rescues lysosomal storage in mucolipidosis type IV, Niemann-Pick type C1, and Batten disease.

Lysosomes are cell organelles that degrade macromolecules to recycle their components. If lysosomal degradative function is impaired, e.g., due to mutations in lysosomal enzymes or membrane proteins, lysosomal storage diseases (LSDs) can develop. LSDs manifest often with neurodegenerative symptoms, typically starting in early childhood, and going along with a strongly reduced life expectancy and quality of life. We show here that small molecule activation of the Ca2+ -permeable endolysosomal two-pore channel 2 (TPC2) results in an amelioration of cellular phenotypes associated with LSDs such as cholesterol or lipofuscin accumulation, or the formation of abnormal vacuoles seen by electron microscopy. Rescue effects by TPC2 activation, which promotes lysosomal exocytosis and autophagy, were assessed in mucolipidosis type IV (MLIV), Niemann-Pick type C1, and Batten disease patient fibroblasts, and in neurons derived from newly generated isogenic human iPSC models for MLIV and Batten disease. For in vivo proof of concept, we tested TPC2 activation in the MLIV mouse model. In sum, our data suggest that TPC2 is a promising target for the treatment of different types of LSDs, both in vitro and in-vivo.

A reference human induced pluripotent stem cell line for large-scale collaborative studies.

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate human iPSC lines and deeply characterized their genetic properties using whole genome sequencing, their genomic stability upon CRISPR-Cas9-based gene editing, and their phenotypic properties including differentiation to commonly used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field.

Simple and reliable detection of CRISPR-induced on-target effects by qgPCR and SNP genotyping.

The recent CRISPR revolution has provided researchers with powerful tools to perform genome editing in a variety of organisms. However, recent reports indicate widespread occurrence of unintended CRISPR-induced on-target effects (OnTEs) at the edited site in mice and human induced pluripotent stem cells (iPSCs) that escape standard quality controls. By altering gene expression of targeted or neighbouring genes, OnTEs can severely affect phenotypes of CRISPR-edited cells and organisms and thus lead to data misinterpretation, which can undermine the reliability of CRISPR-based studies. Here we describe a broadly applicable framework for detecting OnTEs in genome-edited cells and organisms after non-homologous end joining-mediated and homology-directed repair-mediated editing. Our protocol enables identification of OnTEs such as large deletions, large insertions, rearrangements or loss of heterozygosity (LOH). This is achieved by subjecting genomic DNA first to quantitative genotyping PCR (qgPCR), which determines the number of intact alleles at the target site using the same PCR amplicon that has been optimized for genotyping. This combination of genotyping and quantitation makes it possible to exclude clones with monoallelic OnTEs and hemizygous editing, which are often mischaracterized as correctly edited in standard Sanger sequencing. Second, occurrence of LOH around the edited locus is detected by genotyping neighbouring single-nucleotide polymorphisms (SNPs), using either a Sanger sequencing-based method or SNP microarrays. All steps are optimized to maximize simplicity and minimize cost to promote wide dissemination and applicability across the field. The entire protocol from genomic DNA extraction to OnTE exclusion can be performed in 6-9 d.

Repurposing of tamoxifen ameliorates CLN3 and CLN7 disease phenotype.

Batten diseases (BDs) are a group of lysosomal storage disorders characterized by seizure, visual loss, and cognitive and motor deterioration. We discovered increased levels of globotriaosylceramide (Gb3) in cellular and murine models of CLN3 and CLN7 diseases and used fluorescent-conjugated bacterial toxins to label Gb3 to develop a cell-based high content imaging (HCI) screening assay for the repurposing of FDA-approved compounds able to reduce this accumulation within BD cells. We found that tamoxifen reduced the lysosomal accumulation of Gb3 in CLN3 and CLN7 cell models, including neuronal progenitor cells (NPCs) from CLN7 patient-derived induced pluripotent stem cells (iPSC). Here, tamoxifen exerts its action through a mechanism that involves activation of the transcription factor EB (TFEB), a master gene of lysosomal function and autophagy. In vivo administration of tamoxifen to the CLN7Δex2 mouse model reduced the accumulation of Gb3 and SCMAS, decreased neuroinflammation, and improved motor coordination. These data strongly suggest that tamoxifen may be a suitable drug to treat some types of Batten disease.

Methylene blue fails to inhibit Tau and polyglutamine protein dependent toxicity in zebrafish.

Methylene blue is an FDA approved compound with a variety of pharmacologic activities. It inhibits aggregation of several amyloidogenic proteins known to be deposited in neurodegenerative diseases. Recently, it has been proposed that methylene blue shows significant beneficial effects in a phase 2 clinical trial by slowing cognitive decline in Alzheimer's disease patients. To analyze its therapeutic potential, we investigated the effect of methylene blue on neurotoxicity in a zebrafish model for tauopathies. Transgenic expression of the frontotemporal dementia associated Tau-P301L mutation recapitulates a number of the pathological features observed in humans including abnormal phosphorylation and folding of Tau, tangle formation and Tau dependent neuronal loss. Upon incubation of zebrafish larvae with methylene blue, neither abnormal phosphorylation nor neuronal cell loss, reduced neurite outgrowth or a swimming defect were rescued. Methylene blue is biologically active in zebrafish since it reduced aggregation of a huntingtin variant containing a stretch of 102 glutamine residues. However, although huntingtin aggregation was largely prevented by methylene blue, huntingtin-dependent toxicity was unaffected. Our findings are consistent with the hypothesis that toxicity is not necessarily associated with deposition of insoluble amyloid proteins.