Single-Molecule Sequencing of the C9orf72 Repeat Expansion in Patient iPSCs.

A hexanucleotide GGGGCC repeat expansion in the C9orf72 gene is the most frequent genetic cause of amyotrophic lateral sclerosis (ALS) and frontal temporal dementia (FTD). C9orf72 repeat expansions are currently identified with long-range PCR or Southern blot for clinical and research purposes, but these methods lack accuracy and sensitivity. The GC-rich and repetitive content of the region cannot be amplified by PCR, which leads traditional sequencing approaches to fail. We turned instead to PacBio single-molecule sequencing to detect and size the C9orf72 repeat expansion without amplification. We isolated high molecular weight genomic DNA from patient-derived iPSCs of varying repeat lengths and then excised the region containing the C9orf72 repeat expansion from naked DNA with a CRISPR/Cas9 system. We added adapters to the cut ends, capturing the target region for sequencing on PacBio's Sequel, Sequel II, or Sequel IIe. This approach enriches the C9orf72 repeat region without amplification and allows the repeat expansion to be consistently and accurately sized, even for repeats in the thousands. Key features • This protocol is adapted from PacBio's previous "no-amp targeted sequencing utilizing the CRISPR-Cas9 system." • Optimized for sizing C9orf72 repeat expansions in patient-derived iPSCs and applicable to DNA from any cell type, blood, or tissue. • Requires high molecular weight naked DNA. • Compatible with Sequel I and II but not Revio.

Zfp106 binds to G-quadruplex RNAs and inhibits RAN translation and formation of RNA foci caused by G4C2 repeats.

Expansion of intronic GGGGCC repeats in the C9orf72 gene causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Transcription of the expanded repeats results in the formation of RNA-containing nuclear foci and altered RNA metabolism. In addition, repeat-associated non-AUG (RAN) translation of the expanded GGGGCC-repeat sequence results in the production of highly toxic dipeptide-repeat (DPR) proteins. GGGGCC repeat-containing transcripts form G-quadruplexes, which are associated with formation of RNA foci and RAN translation. Zfp106, an RNA-binding protein essential for motor neuron survival in mice, suppresses neurotoxicity in a Drosophila model of C9orf72 ALS. Here, we show that Zfp106 inhibits formation of RNA foci and significantly reduces RAN translation caused by GGGGCC repeats in cultured mammalian cells, and we demonstrate that Zfp106 coexpression reduces the levels of DPRs in C9orf72 patient-derived cells. Further, we show that Zfp106 binds to RNA G-quadruplexes and causes a conformational change in the G-quadruplex structure formed by GGGGCC repeats. Together, these data demonstrate that Zfp106 suppresses the formation of RNA foci and DPRs caused by GGGGCC repeats and suggest that the G-quadruplex RNA-binding function of Zfp106 contributes to its suppression of GGGGCC repeat-mediated cytotoxicity.

Novel avenues of tau research.

INTRODUCTION: The pace of innovation has accelerated in virtually every area of tau research in just the past few years. METHODS: In February 2022, leading international tau experts convened to share selected highlights of this work during Tau 2022, the second international tau conference co-organized and co-sponsored by the Alzheimer's Association, CurePSP, and the Rainwater Charitable Foundation. RESULTS: Representing academia, industry, and the philanthropic sector, presenters joined more than 1700 registered attendees from 59 countries, spanning six continents, to share recent advances and exciting new directions in tau research. DISCUSSION: The virtual meeting provided an opportunity to foster cross-sector collaboration and partnerships as well as a forum for updating colleagues on research-advancing tools and programs that are steadily moving the field forward.

Building CRISPR Gene Therapies for the Central Nervous System: A Review.

Importance: Gene editing using clustered regularly interspaced short palindromic repeats (CRISPR) holds the promise to arrest or cure monogenic disease if it can be determined which genetic change to create without inducing unintended cellular dysfunction and how to deliver this technology to the target organ reliably and safely. Clinical trials for blood and liver disorders, for which delivery of CRISPR is not limiting, show promise, yet no trials have begun for central nervous system (CNS) indications. Observations: The CNS is arguably the most challenging target given its innate exclusion of large molecules and its defenses against bacterial invasion (from which CRISPR originates). Herein, the types of CRISPR editing (DNA cutting, base editing, and templated repair) and how these are applied to different genetic variants are summarized. The challenges of delivering genome editors to the CNS, including the viral and nonviral delivery vehicles that may ultimately circumvent these challenges, are discussed. Also, ways to minimize the potential in vivo genotoxic effects of genome editors through delivery vehicle design and preclinical off-target testing are considered. The ethical considerations of germline editing, a potential off-target outcome of any gene editing therapy, are explored. The unique regulatory challenges of a human-specific therapy that cannot be derisked solely in animal models are also discussed. Conclusions and Relevance: An understanding of both the potential benefits and challenges of CRISPR gene therapy better informs the scientific, clinical, regulatory, and timeline considerations of developing CRISPR gene therapy for neurologic diseases.

Functional characterization of Alzheimer's disease genetic variants in microglia.

Candidate cis-regulatory elements (cCREs) in microglia demonstrate the most substantial enrichment for Alzheimer's disease (AD) heritability compared to other brain cell types. However, whether and how these genome-wide association studies (GWAS) variants contribute to AD remain elusive. Here we prioritize 308 previously unreported AD risk variants at 181 cCREs by integrating genetic information with microglia-specific 3D epigenome annotation. We further establish the link between functional variants and target genes by single-cell CRISPRi screening in microglia. In addition, we show that AD variants exhibit allelic imbalance on target gene expression. In particular, rs7922621 is the effective variant in controlling TSPAN14 expression among other nominated variants in the same cCRE and exerts multiple physiological effects including reduced cell surface ADAM10 and altered soluble TREM2 (sTREM2) shedding. Our work represents a systematic approach to prioritize and characterize AD-associated variants and provides a roadmap for advancing genetic association to experimentally validated cell-type-specific phenotypes and mechanisms.

A CRISPRi/a platform in human iPSC-derived microglia uncovers regulators of disease states.

Microglia are emerging as key drivers of neurological diseases. However, we lack a systematic understanding of the underlying mechanisms. Here, we present a screening platform to systematically elucidate functional consequences of genetic perturbations in human induced pluripotent stem cell-derived microglia. We developed an efficient 8-day protocol for the generation of microglia-like cells based on the inducible expression of six transcription factors. We established inducible CRISPR interference and activation in this system and conducted three screens targeting the 'druggable genome'. These screens uncovered genes controlling microglia survival, activation and phagocytosis, including neurodegeneration-associated genes. A screen with single-cell RNA sequencing as the readout revealed that these microglia adopt a spectrum of states mirroring those observed in human brains and identified regulators of these states. A disease-associated state characterized by osteopontin (SPP1) expression was selectively depleted by colony-stimulating factor-1 (CSF1R) inhibition. Thus, our platform can systematically uncover regulators of microglial states, enabling their functional characterization and therapeutic targeting.

Astrocyte-derived extracellular vesicles enhance the survival and electrophysiological function of human cortical neurons in vitro.

Neurons derived from human induced pluripotent stem cells (hiPSCs) are powerful tools for modeling neural pathophysiology and preclinical efficacy/toxicity screening of novel therapeutic compounds. However, human neurons cultured in vitro typically do not fully recapitulate the physiology of the human nervous system, especially in terms of exhibiting morphological maturation, longevity, and electrochemical signaling ability comparable to that of adult human neurons. In this study, we investigated the potential for astrocyte-derived extracellular vesicles (EVs) to modulate survival and electrophysiological function of human neurons in vitro. Specifically, we demonstrate that EVs obtained from human astrocytes promote enhanced single cell electrophysiological function and anti-apoptotic behavior in a homogeneous population of human iPSC-derived cortical neurons. Furthermore, EV-proteomic analysis was performed to identify cargo proteins with the potential to promote the physiological enhancement observed. EV cargos were found to include neuroprotective proteins such as heat shock proteins, alpha-synuclein, and lipoprotein receptor-related protein 1 (LRP1), as well as apolipoprotein E (APOE), which negatively regulates neuronal apoptosis, and a peroxidasin homolog that supports neuronal oxidative stress management. Proteins that positively regulate neuronal excitability and synaptic development were also detected, such as potassium channel tetramerization domain containing 12 (KCTD12), glucose-6- phosphate dehydrogenase (G6PD), kinesin family member 5B (KIF5B), spectrin-alpha non-erythrocytic1 (SPTAN1). The remarkable improvements in electrophysiological function and evident inhibition of apoptotic signaling in cultured neurons exposed to these cargos may hold significance for improving preclinical in vitro screening modalities. In addition, our collected data highlight the potential for EV-based therapeutics as a potential class of future clinical treatment for tackling inveterate central and peripheral neuropathies.

Microglial microRNAs mediate sex-specific responses to tau pathology.

Sex is a key modifier of neurological disease outcomes. Microglia are implicated in neurological diseases and modulated by microRNAs, but it is unknown whether microglial microRNAs have sex-specific influences on disease. We show in mice that microglial microRNA expression differs in males and females and that loss of microRNAs leads to sex-specific changes in the microglial transcriptome and tau pathology. These findings suggest that microglial microRNAs influence tau pathogenesis in a sex-specific manner.

Common cerebral networks associated with distinct deep brain stimulation targets for cluster headache.

BACKGROUND: Several centers have reported efficacious cluster headache suppression with deep brain stimulation (DBS) of the hypothalamic region using a variety of targets. While the connectivity of some of these targets has individually been studied, commonalities across these targets, especially with respect to network-level connectivity, have not previously been explored. METHODS: We examined the anatomic connectivity of the four distinct DBS targets reported in the literature using probabilistic diffusion tensor tractography in normal subjects. RESULTS: Despite being described as hypothalamic, the DBS targets localized in the midbrain tegmentum posterior to the hypothalamus. Common tracts across DBS targets and subjects included projections to the ipsilateral hypothalamus, reticular formation, and cerebellum. DISCUSSION: Although DBS target coordinates are not located within the hypothalamus, a strong connection between DBS targets and the hypothalamus likely exists. Moreover, a common projection to the medial ipsilateral cerebellum was identified. Understanding the common connectivity of DBS-targeted regions may elucidate anatomic pathways that are involved in modulating cluster headache attacks and facilitate more precise patient-specific targeting of DBS.

Cell therapy in Huntington disease.

Huntington disease (HD), caused by polyglutamate expansions in the huntingtin protein, is a progressive neurodegenerative disease resulting in cognitive and motor impairments and death. Neuronal dysfunction and degeneration contribute to progressive physiological, motor, cognitive, and emotional disturbances characteristic of HD. A major impetus for research into the treatment of HD has centered on cell therapy strategies to protect vulnerable neuronal cell populations or to replace dysfunctional or dying cells. The work underlying 3 approaches to HD cell therapy includes the potential for self-repair through the manipulation of endogenous stem cells and/or neurogenesis, the use of fetal or stem cell transplantation as a cell replacement strategy, and the administration of neurotrophic factors to protect susceptible neuronal populations. These approaches have shown some promising results in animal models of HD. Although striatal transplantation of fetal-derived cells has undergone clinical assessment since the 1990s, many cell therapy strategies have yet to be applied in the clinic environment. A more thorough understanding of the pathophysiologies underlying HD as well as the response of both endogenous and exogenous cells to the degenerating brain will inform their merit as potential therapeutic agents and enhance the framework by which the success of such strategies are determined.