Imbalanced unfolded protein response signaling contributes to 1-deoxysphingolipid retinal toxicity.

The accumulation of atypical, cytotoxic 1-deoxysphingolipids (1-dSLs) has been linked to retinal diseases such as diabetic retinopathy and Macular Telangiectasia Type 2. However, the molecular mechanisms by which 1-dSLs induce toxicity in retinal cells remain poorly understood. Here, we integrate bulk and single-nucleus RNA-sequencing to define biological pathways that modulate 1-dSL toxicity in human retinal organoids. Our results demonstrate that 1-dSLs differentially activate signaling arms of the unfolded protein response (UPR) in photoreceptor cells and Müller glia. Using a combination of pharmacologic activators and inhibitors, we show that sustained PERK signaling through the integrated stress response (ISR) and deficiencies in signaling through the protective ATF6 arm of the UPR are implicated in 1-dSL-induced photoreceptor toxicity. Further, we demonstrate that pharmacologic activation of ATF6 mitigates 1-dSL toxicity without impacting PERK/ISR signaling. Collectively, our results identify new opportunities to intervene in 1-dSL linked diseases through targeting different arms of the UPR.

Integrating human iPSC-derived macrophage progenitors into retinal organoids to generate a mature retinal microglial niche.

In the retina, microglia are resident immune cells that are essential for development and function. Retinal microglia play a central role in mediating pathological degeneration in diseases such as glaucoma, retinitis pigmentosa, age-related neurodegeneration, ischemic retinopathy, and diabetic retinopathy. Current models of mature human retinal organoids (ROs) derived from iPS cell (hiPSC) do not contain resident microglia integrated into retinal layers. Increasing cellular diversity in ROs by including resident microglia would more accurately represent the native retina and better model diseases in which microglia play a key role. In this study, we develop a new 3D in vitro tissue model of microglia-containing retinal organoids by co-culturing ROs and hiPSC-derived macrophage precursor cells (MPCs). We optimized the parameters for successful integration of MPCs into retinal organoids. We show that while in the ROs, MPCs migrate to the equivalent of the outer plexiform layer where retinal microglia cells reside in healthy retinal tissue. While there, they develop a mature morphology characterized by small cell bodies and long branching processes which is only observed in vivo. During this maturation process these MPCs cycle through an activated phase followed by a stable mature microglial phase as seen by the down regulation of pro-inflammatory cytokines and upregulation of anti-inflammatory cytokines. Finally, we characterized mature ROs with integrated MPCs using RNAseq showing an enrichment of cell-type specific microglia markers. We propose that this co-culture system may be useful for understanding the pathogenesis of retinal diseases involving retinal microglia and for drug discovery directly in human tissue.

iPSC-derived retinal pigmented epithelial cells from patients with macular telangiectasia show decreased mitochondrial function.

Patient-derived induced pluripotent stem cells (iPSCs) provide a powerful tool for identifying cellular and molecular mechanisms of disease. Macular telangiectasia type 2 (MacTel) is a rare, late-onset degenerative retinal disease with an extremely heterogeneous genetic architecture, lending itself to the use of iPSCs. Whole-exome sequencing screens and pedigree analyses have identified rare causative mutations that account for less than 5% of cases. Metabolomic surveys of patient populations and GWAS have linked MacTel to decreased circulating levels of serine and elevated levels of neurotoxic 1-deoxysphingolipids (1-dSLs). However, retina-specific, disease-contributing factors have yet to be identified. Here, we used iPSC-differentiated retinal pigmented epithelial (iRPE) cells derived from donors with or without MacTel to screen for novel cell-intrinsic pathological mechanisms. We show that MacTel iRPE cells mimicked the low serine levels observed in serum from patients with MacTel. Through RNA-Seq and gene set enrichment pathway analysis, we determined that MacTel iRPE cells are enriched in cellular stress pathways and dysregulation of central carbon metabolism. Using respirometry and mitochondrial stress testing, we functionally validated that MacTel iRPE cells had a reduction in mitochondrial function that was independent of defects in serine biosynthesis and 1-dSL accumulation. Thus, we identified phenotypes that may constitute alternative disease mechanisms beyond the known serine/sphingolipid pathway.

Deletion of Tgfß signal in activated microglia prolongs hypoxia-induced retinal neovascularization enhancing Igf1 expression and retinal leukostasis.

Retinal neovascularization (NV) is the major cause of severe visual impairment in patients with ischemic eye diseases. While it is known that retinal microglia contribute to both physiological and pathological angiogenesis, the molecular mechanisms by which these glia regulate pathological NV have not been fully elucidated. In this study, we utilized a retinal microglia-specific Transforming Growth Factor-ß (Tgfß) receptor knock out mouse model and human iPSC-derived microglia to examine the role of Tgfß signaling in activated microglia during retinal NV. Using a tamoxifen-inducible, microglia-specific Tgfß receptor type 2 (Tgfßr2) knockout mouse [Tgfßr2 KO (ΔMG)] we show that Tgfß signaling in microglia actively represses leukostasis in retinal vessels. Furthermore, we show that Tgfß signaling represses expression of the pro-angiogenic factor, Insulin-like growth factor 1 (Igf1), independent of Vegf regulation. Using the mouse model of oxygen-induced retinopathy (OIR) we show that Tgfß signaling in activated microglia plays a role in hypoxia-induced NV where a loss in Tgfß signaling microglia exacerbates and prolongs retinal NV in OIR. Using human iPSC-derived microglia cells in an in vitro assay, we validate the role of Transforming Growth Factor-ß1 (Tgfß1) in regulating Igf1 expression in hypoxic conditions. Finally, we show that Tgfß signaling in microglia is essential for microglial homeostasis and that the disruption of Tgfß signaling in microglia exacerbates retinal NV in OIR by promoting leukostasis and Igf1 expression.

Cell-specific cis-regulatory elements and mechanisms of non-coding genetic disease in human retina and retinal organoids.

Cis-regulatory elements (CREs) play a critical role in the development and disease-states of all human cell types. In the retina, CREs have been implicated in several inherited disorders. To better characterize human retinal CREs, we performed single-nucleus assay for transposase-accessible chromatin sequencing (snATAC-seq) and single-nucleus RNA sequencing (snRNA-seq) on the developing and adult human retina and on induced pluripotent stem cell (iPSC)-derived retinal organoids. These analyses identified developmentally dynamic, cell-class-specific CREs, enriched transcription-factor-binding motifs, and putative target genes. CREs in the retina and organoids are highly correlated at the single-cell level, and this supports the use of organoids as a model for studying disease-associated CREs. As a proof of concept, we disrupted a disease-associated CRE at 5q14.3, confirming its principal target gene as the miR-9-2 primary transcript and demonstrating its role in neurogenesis and gene regulation in mature glia. This study provides a resource for characterizing human retinal CREs and showcases organoids as a model to study the function of CREs that influence development and disease.

Serine biosynthesis defect due to haploinsufficiency of PHGDH causes retinal disease.

Macular telangiectasia type 2 (MacTel) is a progressive, late-onset retinal degenerative disease linked to decreased serum levels of serine that elevate circulating levels of a toxic ceramide species, deoxysphingolipids (deoxySLs); however, causal genetic variants that reduce serine levels in patients have not been identified. Here we identify rare, functional variants in the gene encoding the rate-limiting serine biosynthetic enzyme, phosphoglycerate dehydrogenase (PHGDH), as the single locus accounting for a significant fraction of MacTel. Under a dominant collapsing analysis model of a genome-wide enrichment analysis of rare variants predicted to impact protein function in 793 MacTel cases and 17,610 matched controls, the PHGDH gene achieves genome-wide significance (P = 1.2 × 10-13) with variants explaining ~3.2% of affected individuals. We further show that the resulting functional defects in PHGDH cause decreased serine biosynthesis and accumulation of deoxySLs in retinal pigmented epithelial cells. PHGDH is a significant locus for MacTel that explains the typical disease phenotype and suggests a number of potential treatment options.

Toxicity Screens in Human Retinal Organoids for Pharmaceutical Discovery.

Organoids provide a promising platform to study disease mechanism and treatments, directly in the context of human tissue with the versatility and throughput of cell culture. Mature human retinal organoids are utilized to screen potential pharmaceutical treatments for the age-related retinal degenerative disease macular telangiectasia type 2 (MacTel). We have recently shown that MacTel can be caused by elevated levels of an atypical lipid species, deoxysphingolipids (deoxySLs). These lipids are toxic to the retina and may drive the photoreceptor loss that occurs in MacTel patients. To screen drugs for their ability to prevent deoxySL photoreceptor toxicity, we generated human retinal organoids from a non-MacTel induced pluripotent stem cell (iPSC) line and matured them to a post-mitotic age where they develop all of the neuronal lineage-derived cells of the retina, including functionally mature photoreceptors. The retinal organoids were treated with a deoxySL metabolite and apoptosis was measured within the photoreceptor layer using immunohistochemistry. Using this toxicity model, pharmacological compounds that prevent deoxySL-induced photoreceptor death were screened. Using a targeted candidate approach, we determined that fenofibrate, a drug commonly prescribed for the treatment of high cholesterol and triglycerides, can also prevent deoxySL toxicity in the cells of the retina. The toxicity screen successfully identified an FDA-approved drug that can prevent photoreceptor death. This is a directly actionable finding owing to the highly disease-relevant model tested. This platform can be easily modified to test any number of metabolic stressors and potential pharmacological interventions for future treatment discovery in retinal diseases.

Serine and Lipid Metabolism in Macular Disease and Peripheral Neuropathy.

BACKGROUND: Identifying mechanisms of diseases with complex inheritance patterns, such as macular telangiectasia type 2, is challenging. A link between macular telangiectasia type 2 and altered serine metabolism has been established previously. METHODS: Through exome sequence analysis of a patient with macular telangiectasia type 2 and his family members, we identified a variant in SPTLC1 encoding a subunit of serine palmitoyltransferase (SPT). Because mutations affecting SPT are known to cause hereditary sensory and autonomic neuropathy type 1 (HSAN1), we examined 10 additional persons with HSAN1 for ophthalmologic disease. We assayed serum amino acid and sphingoid base levels, including levels of deoxysphingolipids, in patients who had macular telangiectasia type 2 but did not have HSAN1 or pathogenic variants affecting SPT. We characterized mice with low serine levels and tested the effects of deoxysphingolipids on human retinal organoids. RESULTS: Two variants known to cause HSAN1 were identified as causal for macular telangiectasia type 2: of 11 patients with HSAN1, 9 also had macular telangiectasia type 2. Circulating deoxysphingolipid levels were 84.2% higher among 125 patients with macular telangiectasia type 2 who did not have pathogenic variants affecting SPT than among 94 unaffected controls. Deoxysphingolipid levels were negatively correlated with serine levels, which were 20.6% lower than among controls. Reduction of serine levels in mice led to increases in levels of retinal deoxysphingolipids and compromised visual function. Deoxysphingolipids caused photoreceptor-cell death in retinal organoids, but not in the presence of regulators of lipid metabolism. CONCLUSIONS: Elevated levels of atypical deoxysphingolipids, caused by variant SPTLC1 or SPTLC2 or by low serine levels, were risk factors for macular telangiectasia type 2, as well as for peripheral neuropathy. (Funded by the Lowy Medical Research Institute and others.).

Diverse reprogramming codes for neuronal identity.

The transcriptional programs that establish neuronal identity evolved to produce the rich diversity of neuronal cell types that arise sequentially during development. Remarkably, transient expression of certain transcription factors can also endow non-neural cells with neuronal properties. The relationship between reprogramming factors and the transcriptional networks that produce neuronal identity and diversity remains largely unknown. Here, from a screen of 598 pairs of transcription factors, we identify 76 pairs of transcription factors that induce mouse fibroblasts to differentiate into cells with neuronal features. By comparing the transcriptomes of these induced neuronal cells (iN cells) with those of endogenous neurons, we define a 'core' cell-autonomous neuronal signature. The iN cells also exhibit diversity; each transcription factor pair produces iN cells with unique transcriptional patterns that can predict their pharmacological responses. By linking distinct transcription factor input 'codes' to defined transcriptional outputs, this study delineates cell-autonomous features of neuronal identity and diversity and expands the reprogramming toolbox to facilitate engineering of induced neurons with desired patterns of gene expression and related functional properties.

Forward engineering neuronal diversity using direct reprogramming.

The nervous system is comprised of a vast diversity of distinct neural cell types. Differences between neuronal subtypes drive the assembly of neuronal circuits and underlie the subtype specificity of many neurological diseases. Yet, because neurons are irreversibly post-mitotic and not readily available from patients, it has not been feasible to study specific subtypes of human neurons in larger numbers. A powerful means to study neuronal diversity and neurological disease is to establish methods to produce desired neuronal subtypes in vitro. Traditionally this has been accomplished by treating pluripotent or neural stem cells with growth factors and morphogens that recapitulate exogenous developmental signals. These approaches often require extended periods of culture, which can limit their utility. However, more recently, it has become possible to produce neurons directly from fibroblasts using transcription factors and/or microRNAs. This technique referred to as direct reprogramming or transdifferentiation has proven to be a rapid, robust, and reproducible method to generate mature neurons of many different subtypes from multiple cell sources. Here, we highlight recent advances in generating neurons of specific subtypes using direct reprogramming and outline various scenarios in which induced neurons may be applied to studies of neuronal function and neurological disease.

Selective conversion of fibroblasts into peripheral sensory neurons.

Humans and mice detect pain, itch, temperature, pressure, stretch and limb position via signaling from peripheral sensory neurons. These neurons are divided into three functional classes (nociceptors/pruritoceptors, mechanoreceptors and proprioceptors) that are distinguished by their selective expression of TrkA, TrkB or TrkC receptors, respectively. We found that transiently coexpressing Brn3a with either Ngn1 or Ngn2 selectively reprogrammed human and mouse fibroblasts to acquire key properties of these three classes of sensory neurons. These induced sensory neurons (iSNs) were electrically active, exhibited distinct sensory neuron morphologies and matched the characteristic gene expression patterns of endogenous sensory neurons, including selective expression of Trk receptors. In addition, we found that calcium-imaging assays could identify subsets of iSNs that selectively responded to diverse ligands known to activate itch- and pain-sensing neurons. These results offer a simple and rapid means for producing genetically diverse human sensory neurons suitable for drug screening and mechanistic studies.

Developmental transcriptional networks are required to maintain neuronal subtype identity in the mature nervous system.

During neurogenesis, transcription factors combinatorially specify neuronal fates and then differentiate subtype identities by inducing subtype-specific gene expression profiles. But how is neuronal subtype identity maintained in mature neurons? Modeling this question in two Drosophila neuronal subtypes (Tv1 and Tv4), we test whether the subtype transcription factor networks that direct differentiation during development are required persistently for long-term maintenance of subtype identity. By conditional transcription factor knockdown in adult Tv neurons after normal development, we find that most transcription factors within the Tv1/Tv4 subtype transcription networks are indeed required to maintain Tv1/Tv4 subtype-specific gene expression in adults. Thus, gene expression profiles are not simply "locked-in," but must be actively maintained by persistent developmental transcription factor networks. We also examined the cross-regulatory relationships between all transcription factors that persisted in adult Tv1/Tv4 neurons. We show that certain critical cross-regulatory relationships that had existed between these transcription factors during development were no longer present in the mature adult neuron. This points to key differences between developmental and maintenance transcriptional regulatory networks in individual neurons. Together, our results provide novel insight showing that the maintenance of subtype identity is an active process underpinned by persistently active, combinatorially-acting, developmental transcription factors. These findings have implications for understanding the maintenance of all long-lived cell types and the functional degeneration of neurons in the aging brain.

Neuronal phenotype in the mature nervous system is maintained by persistent retrograde bone morphogenetic protein signaling.

The terminal differentiation of many developing neurons occurs after they innervate their target cells and is triggered by secreted target-derived signals that are transduced by presynaptic cognate receptors. Such retrograde signaling induces the expression of genes that are often distinctive markers of neuronal phenotype and function. However, whether long-term maintenance of neuronal phenotype requires persistent retrograde signaling remains poorly understood. Previously, we demonstrated that retrograde bone morphogenetic protein (BMP) signaling induces expression of a phenotypic marker of Drosophila Tv neurons, the neuropeptide FMRFamide (FMRFa). Here, we used a genetic technique that spatiotemporally targets transgene expression in Drosophila to test the role of persistent BMP signaling in the maintenance of Tv phenotype. We show that expression of dominant blockers of BMP signaling selectively in adult Tv neurons dramatically downregulated FMRFa expression. Moreover, adult-onset expression of mutant Glued, which blocks dynein/dynactin-mediated retrograde axonal transport, eliminated retrograde BMP signaling and dramatically downregulated FMRFa expression. Finally, we found that BMP deprivation did not affect Tv neuron survival and that FMRFa expression fully recovered to control levels after the termination of BMP blockade or Glued expression. Our results show that persistent retrograde BMP signaling is required to induce and to subsequently maintain the expression of a stably expressed phenotypic marker in a subset of mature Drosophila neurons. We postulate that retrograde maintenance of neuronal phenotype is conserved in vertebrates, and as a consequence, neuronal phenotype is likely vulnerable to neurodegenerative disease pathologies that disrupt neuronal connectivity or axonal transport.