Mouse γ-Synuclein Promoter-Mediated Gene Expression and Editing in Mammalian Retinal Ganglion Cells

Optic neuropathies are a group of optic nerve (ON) diseases caused by various insults including glaucoma, inflammation, ischemia, trauma, and genetic deficits, which are characterized by retinal ganglion cell (RGC) death and ON degeneration. An increasing number of genes involved in RGC intrinsic signaling have been found to be promising neural repair targets that can potentially be modulated directly by gene therapy, if we can achieve RGC specific gene targeting. To address this challenge, we first used adeno-associated virus (AAV)-mediated gene transfer to perform a low-throughput in vivo screening in both male and female mouse eyes and identified the mouse γ-synuclein (mSncg) promoter, which specifically and potently sustained transgene expression in mouse RGCs and also works in human RGCs. We further demonstrated that gene therapy that combines AAV-mSncg promoter with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing can knock down pro-degenerative genes in RGCs and provide effective neuroprotection in optic neuropathies.SIGNIFICANCE STATEMENT Here, we present an RGC-specific promoter, mouse γ-synuclein (mSncg) promoter, and perform extensive characterization and proof-of-concept studies of mSncg promoter-mediated gene expression and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing in RGCs in vivo To our knowledge, this is the first report demonstrating in vivo neuroprotection of injured RGCs and optic nerve (ON) by AAV-mediated CRISPR/Cas9 inhibition of genes that are critical for neurodegeneration. It represents a powerful tool to achieve RGC-specific gene modulation, and also opens up a promising gene therapy strategy for optic neuropathies, the most common form of eye diseases that cause irreversible blindness

The Diverse Roles of Mitochondrial Dynamics in Retinal Ganglion Cell Maturation, Disease, and Regeneration

Mitochondria maintain a diversity of roles central to the array of neuronal functions and survival, as implied by the ubiquitous evidence of mitochondrial roles in neuronal developmental, aging, and disease progression. The canonical function of mitochondria is to produce ATP, a critical and dominant molecule used by cells for energy requiring activities. However, since their discovery more than a century ago, mitochondria have been implicated in a vast collection of cellular processes, largely driven by their expanded function beyond metabolism, including secondary messenger signaling, cytoplasmic Ca2+ signaling, fatty acid oxidation, reactive oxygen species (ROS) production, and protein scaffolding. More recently, the diversity of these functions has been shown to be further modulated both globally and within a cellular domain by fluid and dynamic changes in mitochondrial size, number, and movement, collectively termed mitochondrial dynamics. Furthermore, these mitochondrial dynamics have been implicated in the regulation of processes in CNS neuron development and disease. The complexity of these dynamic behaviors and their involvement in the maturation, disease progression, and regenerative capacity of CNS neurons is a centralized theme of the work presented in this thesis, with a specialized focus on the visual system and retinal ganglion cell biology. Appendix studies also present future imaging approaches and therapeutic strategies for restoring mitochondrial function, in mitochondrial genetic diseases and aging.</p

Treatment of Inherited Eye Defects by Systemic Hematopoietic Stem Cell Transplantation.

PurposeCystinosis is caused by a deficiency in the lysosomal cystine transporter, cystinosin (CTNS gene), resulting in cystine crystal accumulation in tissues. In eyes, crystals accumulate in the cornea causing photophobia and eventually blindness. Hematopoietic stem progenitor cells (HSPCs) rescue the kidney in a mouse model of cystinosis. We investigated the potential for HSPC transplantation to treat corneal defects in cystinosis.MethodsWe isolated HSPCs from transgenic DsRed mice and systemically transplanted irradiated Ctns-/- mice. A year posttransplantation, we investigated the fate and function of HSPCs by in vivo confocal and fluorescence microscopy (IVCM), quantitative RT-PCR (RT-qPCR), mass spectrometry, histology, and by measuring the IOP. To determine the mechanism by which HSPCs may rescue disease cells, we transplanted Ctns-/- mice with Ctns-/- DsRed HSPCs virally transduced to express functional CTNS-eGFP fusion protein.ResultsWe found that a single systemic transplantation of wild-type HSPCs prevented ocular pathology in the Ctns-/- mice. Engraftment-derived HSPCs were detected within the cornea, and also in the sclera, ciliary body, retina, choroid, and lens. Transplantation of HSPC led to substantial decreases in corneal cystine crystals, restoration of normal corneal thickness, and lowered IOP in mice with high levels of donor-derived cell engraftment. Finally, we found that HSPC-derived progeny differentiated into macrophages, which displayed tunneling nanotubes capable of transferring cystinosin-bearing lysosomes to diseased cells.ConclusionsTo our knowledge, this is the first demonstration that HSPCs can rescue hereditary corneal defects, and supports a new potential therapeutic strategy for treating ocular pathologies

SARS-CoV-2 Susceptibility and ACE2 Gene Variations Within Diverse Ethnic Backgrounds.

There is considerable variability in the susceptibility and progression for COVID-19 and it appears to be strongly correlated with age, gender, ethnicity and pre-existing health conditions. However, to our knowledge, cohort studies of COVID-19 in clinically vulnerable groups are lacking. Host genetics has also emerged as a major risk factor for COVID-19, and variation in the ACE2 receptor, which facilitates entry of the SARS-CoV-2 virus into the cell, has become a major focus of attention. Thus, we interrogated an ethnically diverse cohort of National Health Service (NHS) patients in the United Kingdom (United Kingdom) to assess the association between variants in the ACE2 locus and COVID-19 risk. We analysed whole-genome sequencing (WGS) data of 1,837 cases who were tested positive for SARS-CoV-2, and 37,207 controls who were not tested, from the UK's 100,000 Genomes Project (100KGP) for the presence of ACE2 coding variants and extract expression quantitative trait loci (eQTLs). We identified a splice site variant (rs2285666) associated with increased ACE2 expression with an overrepresentation in SARS-CoV-2 positive patients relative to 100KGP controls (p = 0.015), and in hospitalised European patients relative to outpatients in intra-ethnic comparisons (p = 0.029). We also compared the prevalence of 288 eQTLs, of which 23 were enriched in SARS-CoV-2 positive patients. The eQTL rs12006793 had the largest effect size (d = 0.91), which decreases ACE2 expression and is more prevalent in controls, thus potentially reducing the risk of COVID-19. We identified three novel nonsynonymous variants predicted to alter ACE2 function, and showed that three variants (p.K26R, p. H378R, p. Y515N) alter receptor affinity for the viral Spike (S) protein. Variant p. N720D, more prevalent in the European population (p < 0.001), potentially increases viral entry by affecting the ACE2-TMPRSS2 complex. The spectrum of genetic variants in ACE2 may inform risk stratification of COVID-19 patients and could partially explain the differences in disease susceptibility and severity among different ethnic groups

A pesticide and iPSC dopaminergic neuron screen identifies and classifies Parkinson-relevant pesticides.

Parkinson's disease (PD) is a complex neurodegenerative disease with etiology rooted in genetic vulnerability and environmental factors. Here we combine quantitative epidemiologic study of pesticide exposures and PD with toxicity screening in dopaminergic neurons derived from PD patient induced pluripotent stem cells (iPSCs) to identify Parkinson's-relevant pesticides. Agricultural records enable investigation of 288 specific pesticides and PD risk in a comprehensive, pesticide-wide association study. We associate long-term exposure to 53 pesticides with PD and identify co-exposure profiles. We then employ a live-cell imaging screening paradigm exposing dopaminergic neurons to 39 PD-associated pesticides. We find that 10 pesticides are directly toxic to these neurons. Further, we analyze pesticides typically used in combinations in cotton farming, demonstrating that co-exposures result in greater toxicity than any single pesticide. We find trifluralin is a driver of toxicity to dopaminergic neurons and leads to mitochondrial dysfunction. Our paradigm may prove useful to mechanistically dissect pesticide exposures implicated in PD risk and guide agricultural policy