Compartmentalized citrullination in Muller glial endfeet during retinal degeneration.

Muller glia (MG) play a central role in reactive gliosis, a stress response associated with rare and common retinal degenerative diseases, including age-related macular degeneration (AMD). The posttranslational modification citrullination​ targeting glial fibrillary acidic protein (GFAP) in MG was initially discovered in a panocular chemical injury model. Here, we report in the paradigms of retinal laser injury, a genetic model of spontaneous retinal degeneration (JR5558 mice) and human wet-AMD tissues that MG citrullination is broadly conserved. After laser injury, GFAP polymers that accumulate in reactive MG are citrullinated in MG endfeet and glial cell processes. The enzyme responsible for citrullination, peptidyl arginine deiminase-4 (PAD4), localizes to endfeet and associates with GFAP polymers. Glial cell-specific PAD4 deficiency attenuates retinal hypercitrullination in injured retinas, indicating PAD4 requirement for MG citrullination. In retinas of 1-mo-old JR5558 mice, hypercitrullinated GFAP and PAD4 accumulate in MG endfeet/cell processes in a lesion-specific manner. Finally, we show that human donor maculae from patients with wet-AMD also feature the canonical endfeet localization of hypercitrullinated GFAP. Thus, we propose that endfeet are a "citrullination bunker" that initiates and sustains citrullination in retinal degeneration.

Identification of fluoxetine as a direct NLRP3 inhibitor to treat atrophic macular degeneration.

The atrophic form of age-related macular degeneration (dry AMD) affects nearly 200 million people worldwide. There is no Food and Drug Administration (FDA)-approved therapy for this disease, which is the leading cause of irreversible blindness among people over 50 y of age. Vision loss in dry AMD results from degeneration of the retinal pigmented epithelium (RPE). RPE cell death is driven in part by accumulation of Alu RNAs, which are noncoding transcripts of a human retrotransposon. Alu RNA induces RPE degeneration by activating the NLRP3-ASC inflammasome. We report that fluoxetine, an FDA-approved drug for treating clinical depression, binds NLRP3 in silico, in vitro, and in vivo and inhibits activation of the NLRP3-ASC inflammasome and inflammatory cytokine release in RPE cells and macrophages, two critical cell types in dry AMD. We also demonstrate that fluoxetine, unlike several other antidepressant drugs, reduces Alu RNA-induced RPE degeneration in mice. Finally, by analyzing two health insurance databases comprising more than 100 million Americans, we report a reduced hazard of developing dry AMD among patients with depression who were treated with fluoxetine. Collectively, these studies identify fluoxetine as a potential drug-repurposing candidate for dry AMD.

Cytoplasmic synthesis of endogenous Alu complementary DNA via reverse transcription and implications in age-related macular degeneration.

Alu retroelements propagate via retrotransposition by hijacking long interspersed nuclear element-1 (L1) reverse transcriptase (RT) and endonuclease activities. Reverse transcription of Alu RNA into complementary DNA (cDNA) is presumed to occur exclusively in the nucleus at the genomic integration site. Whether Alu cDNA is synthesized independently of genomic integration is unknown. Alu RNA promotes retinal pigmented epithelium (RPE) death in geographic atrophy, an untreatable type of age-related macular degeneration. We report that Alu RNA-induced RPE degeneration is mediated via cytoplasmic L1-reverse-transcribed Alu cDNA independently of retrotransposition. Alu RNA did not induce cDNA production or RPE degeneration in L1-inhibited animals or human cells. Alu reverse transcription can be initiated in the cytoplasm via self-priming of Alu RNA. In four health insurance databases, use of nucleoside RT inhibitors was associated with reduced risk of developing atrophic macular degeneration (pooled adjusted hazard ratio, 0.616; 95% confidence interval, 0.493-0.770), thus identifying inhibitors of this Alu replication cycle shunt as potential therapies for a major cause of blindness.

Chronic Dicer1 deficiency promotes atrophic and neovascular outer retinal pathologies in mice.

Degeneration of the retinal pigmented epithelium (RPE) and aberrant blood vessel growth in the eye are advanced-stage processes in blinding diseases such as age-related macular degeneration (AMD), which affect hundreds of millions of people worldwide. Loss of the RNase DICER1, an essential factor in micro-RNA biogenesis, is implicated in RPE atrophy. However, the functional implications of DICER1 loss in choroidal and retinal neovascularization are unknown. Here, we report that two independent hypomorphic mouse strains, as well as a separate model of postnatal RPE-specific DICER1 ablation, all presented with spontaneous RPE degeneration and choroidal and retinal neovascularization. DICER1 hypomorphic mice lacking critical inflammasome components or the innate immune adaptor MyD88 developed less severe RPE atrophy and pathological neovascularization. DICER1 abundance was also reduced in retinas of the JR5558 mouse model of spontaneous choroidal neovascularization. Finally, adenoassociated vector-mediated gene delivery of a truncated DICER1 variant (OptiDicer) reduced spontaneous choroidal neovascularization in JR5558 mice. Collectively, these findings significantly expand the repertoire of DICER1 in preserving retinal homeostasis by preventing both RPE degeneration and pathological neovascularization.

Expression of Vascular Endothelial Growth Factor by Retinal Pigment Epithelial Cells Induced by Amyloid-ß Is Depressed by an Endoplasmic Reticulum Stress Inhibitor.

PURPOSE: Amyloid-ß (Aß) is a 36- to 43-amino-acid peptide that is a constituent of drusen, and it has been demonstrated to upregulate vascular endothelial growth factor (VEGF) expression by retinal pigment epithelial (RPE) cells. This study aimed to determine whether 4-phenylbutyl phosphonylacetate (PBA), a known endoplasmic reticulum (ER) stress inhibitor, can reduce Aß-induced expression of VEGF in RPE cells. METHODS: Aß was added to the medium of regularly cultured or polarized ARPE-19 cells, a human RPE cell line, with or without PBA. The levels of VEGF and ER stress markers, namely GRP78/Bip, cleaved caspases 4 and 12 and GADD153/C-EBP homologous protein, were determined by enzyme-linked immunoassay, immunocytochemistry and Western blotting. RESULTS: Exposure of ARPE-19 cells to Aß induced GRP78/Bip expression and activated caspases 4 and 12; however, their expression was decreased by simultaneous exposure to PBA. Aß increased the expression of VEGF both in regularly cultured and polarized ARPE-19 cells, but it was suppressed by PBA. PBA did not cause RPE cell apoptosis. CONCLUSION: Aß has been suggested to be involved in the development of age-related macular degeneration; therefore, our findings suggest that drugs that target ER stress should be considered for the treatment of age-related macular degeneration.

Therapeutic targeting of telomerase ameliorates experimental choroidal neovascularization.

Choroidal neovascularization (CNV) is the principal driver of blindness in neovascular age-related macular degeneration (nvAMD). Increased activity of telomerase, has been associated with endothelial cell proliferation, survival, migration, and invasion in the context of tumor angiogenesis. Expanding on this knowledge, we investigated the role of telomerase in the development of CNV in mouse model. We observed increased gene expression and activity of telomerase in mouse CNV. Genetic deficiency of the telomerase components, telomerase reverse transcriptase (Tert) and telomerase RNA component (Terc) suppressed laser-induced CNV in mice. Similarly, a small molecule inhibitor of TERT (BIBR 1532), and antisense oligonucleotides (ASOs) targeting Tert and Terc reduced CNV growth. Bone marrow chimera studies suggested that telomerase activity in non-bone marrow-derived cells is crucial for the development of CNV. Comparison of BIBR 1532 with VEGF neutralizing therapeutic strategy in mouse revealed a comparable level of angiosuppressive activity. However, when BIBR and anti-VEGF antibodies were administered as a combination at sub-therapeutic doses, a statistically significant suppression of CNV was observed. These findings underscore the potential benefits of combining sub-therapeutic doses of BIBR and anti-VEGF antibodies for developing newer therapeutic strategies for NV-AMD. Telomerase inhibition with BIBR 1532 suppressed induction of multiple cytokines and growth factors critical for neovascularization. In conclusion, our study identifies telomerase as a promising therapeutic target for treating neovascular disease of the eye and thus provides a proof of principle for further exploration of telomerase inhibition as a novel treatment strategy for nvAMD.

Phagocytosis in the retina promotes local insulin production in the eye.

The retina is highly metabolically active, relying on glucose uptake and aerobic glycolysis. Situated in close contact to photoreceptors, a key function of cells in the retinal pigment epithelium (RPE) is phagocytosis of damaged photoreceptor outer segments (POS). Here we identify RPE as a local source of insulin in the eye that is stimulated by POS phagocytosis. We show that Ins2 messenger RNA and insulin protein are produced by RPE cells and that this production correlates with RPE phagocytosis of POS. Genetic deletion of phagocytic receptors ('loss of function') reduces Ins2, whereas increasing the levels of the phagocytic receptor MerTK ('gain of function') increases Ins2 production in male mice. Contrary to pancreas-derived systemic insulin, RPE-derived local insulin is stimulated during starvation, which also increases RPE phagocytosis. Global or RPE-specific Ins2 gene deletion decreases retinal glucose uptake in starved male mice, dysregulates retinal physiology, causes defects in phototransduction and exacerbates photoreceptor loss in a mouse model of retinitis pigmentosa. Collectively, these data identify RPE cells as a phagocytosis-induced local source of insulin in the retina, with the potential to influence retinal physiology and disease.

Kamuvudine-9 Protects Retinal Structure and Function in a Novel Model of Experimental Rhegmatogenous Retinal Detachment.

Purpose: Rhegmatogenous retinal detachment (RRD) is a vision-threatening event that benefits from surgical intervention. While awaiting surgical reattachment, irreversible hypoxic and inflammatory damage to the retina often occurs. An interim therapy protecting photoreceptors could improve functional outcomes. We sought to determine whether Kamuvudine-9 (K-9), a derivative of nucleoside reverse transcriptase inhibitors (NRTIs) that inhibits inflammasome activation, and the NRTIs lamivudine (3TC) and azidothymidine (AZT) could protect the retina following RRD. Methods: RRD was induced in mice via subretinal injection (SRI) of 1% carboxymethylcellulose (CMC). To simulate outcomes following the clinical management of RRD, we determined the optimal conditions by which SRI of CMC induced spontaneous retinal reattachment (SRR) occurs over 10 days (RRD/SRR). K-9, 3TC, or AZT was administered via intraperitoneal injection. Inflammasome activation pathways were monitored by abundance of cleaved caspase-1, IL-18, and cleaved caspase-8, and photoreceptor death was assessed by TUNEL staining. Retinal function was assessed by full-field scotopic electroretinography. Results: RRD induced retinal inflammasome activation and photoreceptor death in mice. Systemic administration of K-9, 3TC, or AZT inhibited retinal inflammasome activation and photoreceptor death. In the RRD/SRR model, K-9 protected retinal electrical function during the time of RRD and induced an improvement following retinal reattachment. Conclusions: K-9 and NRTIs exhibit anti-inflammatory and neuroprotective activities in experimental RRD. Given its capacity to protect photoreceptor function during the period of RRD and enhance retinal function following reattachment, K-9 shows promise as a retinal neuroprotectant and warrants study in RRD. Further, this novel RRD/SRR model may facilitate experimental evaluation of functional outcomes relevant to RRD.

Reduction of human Alzheimer's disease risk and reversal of mouse model cognitive deficit with nucleoside analog use.

Innate immune signaling through the NLRP3 inflammasome has been implicated in the pathogenesis of Alzheimer's disease (AD), the most prevalent form of dementia. We previously demonstrated that nucleoside reverse transcriptase inhibitors (NRTIs), drugs approved to treat HIV and hepatitis B infections, also inhibit inflammasome activation. Here we report that in humans, NRTI exposure was associated with a significantly lower incidence of AD in two of the largest health insurance databases in the United States. Treatment of aged 5xFAD mice (a mouse model of amyloid-ß deposition that expresses five mutations found in familial AD) with Kamuvudine-9 (K-9), an NRTI-derivative with enhanced safety profile, reduced Aß deposition and reversed their cognitive deficit by improving their spatial memory and learning performance to that of young wild-type mice. These findings support the concept that inflammasome inhibition could benefit AD and provide a rationale for prospective clinical testing of NRTIs or K-9 in AD.

The Learning Curve of Murine Subretinal Injection Among Clinically Trained Ophthalmic Surgeons.

Purpose: Subretinal injection (SRI) in mice is widely used in retinal research, yet the learning curve (LC) of this surgically challenging technique is unknown. Methods: To evaluate the LC for SRI in a murine model, we analyzed training data from three clinically trained ophthalmic surgeons from 2018 to 2020. Successful SRI was defined as either the absence of retinal pigment epithelium (RPE) degeneration after phosphate buffered saline injection or the presence of RPE degeneration after Alu RNA injection. Multivariable survival-time regression models were used to evaluate the association between surgeon experience and success rate, with adjustment for injection agents, and to calculate an approximate case number to achieve a 95% success rate. Cumulative sum (CUSUM) analyses were performed and plotted individually to monitor each surgeon's simultaneous performance. Results: Despite prior microsurgery experience, the combined average success rate of the first 50 cases in mice was only 27%. The predicted SRI success rate did not reach a plateau above 95% until approximately 364 prior cases. Using the 364 training cases as a cutoff point, the predicted probability of success for cases 1 to 364 was 65.38%, and for cases 365 to 455 it was 99.32% (P < 0.0001). CUSUM analysis showed an initial upward slope and then remained within the decision intervals with an acceptable success rate set at 95% in the late stage. Conclusions: This study demonstrates the complexity and substantial LC for successful SRI in mice with high confidence. A systematic training system could improve the reliability and reproducibility of SRI-related experiments and improve the interpretation of experimental results using this technique. Translational Relevance: Our prediction model and monitor system allow objective quantification of technical proficiency in the field of subretinal drug delivery and gene therapy for the first time, to the best of our knowledge.

Subretinal injection in mice to study retinal physiology and disease.

Subretinal injection (SRI) is a widely used technique in retinal research and can be used to deliver nucleic acids, small molecules, macromolecules, viruses, cells or biomaterials such as nanobeads. Here we describe how to undertake SRI of mice. This protocol was adapted from a technique initially described for larger animals. Although SRI is a common procedure in eye research laboratories, there is no published guidance on the best practices for determining what constitutes a 'successful' SRI. Optimal injections are required for reproducibility of the procedure and, when carried out suboptimally, can lead to erroneous conclusions. To address this issue, we propose a standardized protocol for SRI with 'procedure success' defined by follow-up examination of the retina and the retinal pigmented epithelium rather than solely via intraoperative endpoints. This protocol takes 7-14 d to complete, depending on the reagent delivered. We have found, by instituting a standardized training program, that trained ophthalmologists achieve reliable proficiency in this technique after ~350 practice injections. This technique can be used to gain insights into retinal physiology and disease pathogenesis and to test the efficacy of experimental compounds in the retina or retinal pigmented epithelium.

Nucleoside reverse transcriptase inhibitors and Kamuvudines inhibit amyloid-ß induced retinal pigmented epithelium degeneration.

Nonfibrillar amyloid-ß oligomers (AßOs) are a major component of drusen, the sub-retinal pigmented epithelium (RPE) extracellular deposits characteristic of age-related macular degeneration (AMD), a common cause of global blindness. We report that AßOs induce RPE degeneration, a clinical hallmark of geographic atrophy (GA), a vision-threatening late stage of AMD that is currently untreatable. We demonstrate that AßOs induce activation of the NLRP3 inflammasome in the mouse RPE in vivo and that RPE expression of the purinergic ATP receptor P2RX7, an upstream mediator of NLRP3 inflammasome activation, is required for AßO-induced RPE degeneration. Two classes of small molecule inflammasome inhibitors-nucleoside reverse transcriptase inhibitors (NRTIs) and their antiretrovirally inert modified analog Kamuvudines-both inhibit AßOs-induced RPE degeneration. These findings crystallize the importance of P2RX7 and NLRP3 in a disease-relevant model of AMD and identify inflammasome inhibitors as potential treatments for GA.

DDX17 is an essential mediator of sterile NLRC4 inflammasome activation by retrotransposon RNAs.

Detection of microbial products by multiprotein complexes known as inflammasomes is pivotal to host defense against pathogens. Nucleotide-binding domain leucine-rich repeat (NLR) CARD domain containing 4 (NLRC4) forms an inflammasome in response to bacterial products; this requires their detection by NLR family apoptosis inhibitory proteins (NAIPs), with which NLRC4 physically associates. However, the mechanisms underlying sterile NLRC4 inflammasome activation, which is implicated in chronic noninfectious diseases, remain unknown. Here, we report that endogenous short interspersed nuclear element (SINE) RNAs, which promote atrophic macular degeneration (AMD) and systemic lupus erythematosus (SLE), induce NLRC4 inflammasome activation independent of NAIPs. We identify DDX17, a DExD/H box RNA helicase, as the sensor of SINE RNAs that licenses assembly of an inflammasome comprising NLRC4, NLR pyrin domain­containing protein 3, and apoptosis-associated speck-like protein­containing CARD and induces caspase-1 activation and cytokine release. Inhibiting DDX17-mediated NLRC4 inflammasome activation decreased interleukin-18 release in peripheral blood mononuclear cells of patients with SLE and prevented retinal degeneration in an animal model of AMD. Our findings uncover a previously unrecognized noncanonical NLRC4 inflammasome activated by endogenous retrotransposons and provide potential therapeutic targets for SINE RNA­driven diseases.

Alu complementary DNA is enriched in atrophic macular degeneration and triggers retinal pigmented epithelium toxicity via cytosolic innate immunity.

Long interspersed nuclear element-1 (L1)­mediated reverse transcription (RT) of Alu RNA into cytoplasmic Alu complementary DNA (cDNA) has been implicated in retinal pigmented epithelium (RPE) degeneration. The mechanism of Alu cDNA­induced cytotoxicity and its relevance to human disease are unknown. Here we report that Alu cDNA is highly enriched in the RPE of human eyes with geographic atrophy, an untreatable form of age-related macular degeneration. We demonstrate that the DNA sensor cGAS engages Alu cDNA to induce cytosolic mitochondrial DNA escape, which amplifies cGAS activation, triggering RPE degeneration via the inflammasome. The L1-extinct rice rat was resistant to Alu RNA­induced Alu cDNA synthesis and RPE degeneration, which were enabled upon L1-RT overexpression. Nucleoside RT inhibitors (NRTIs), which inhibit both L1-RT and inflammasome activity, and NRTI derivatives (Kamuvudines) that inhibit inflammasome, but not RT, both block Alu cDNA toxicity, identifying inflammasome activation as the terminal effector of RPE degeneration.

Voluntary Exercise Suppresses Choroidal Neovascularization in Mice.

Purpose: To determine the effect of voluntary exercise on choroidal neovascularization (CNV) in mice. Methods: Age-matched wild-type C57BL/6J mice were housed in cages equipped with or without running wheels. After four weeks of voluntary running or sedentariness, mice were subjected to laser injury to induce CNV. After surgical recovery, mice were placed back in cages with or without exercise wheels for seven days. CNV lesion volumes were measured by confocal microscopy. The effect of wheel running only in the seven days after injury was also evaluated. Macrophage abundance and cytokine expression were quantified. Results: In the first study, exercise-trained mice exhibited a 45% reduction in CNV volume compared to sedentary mice. In the replication study, a 32% reduction in CNV volume in exercise-trained mice was observed (P = 0.029). Combining these two studies, voluntary exercise was found to reduce CNV by 41% (P = 0.0005). Exercise-trained male and female mice had similar CNV volumes (P = 0.99). The daily running distance did not correlate with CNV lesion size. Exercise only after the laser injury without a preconditioning period did not reduce CNV size (P = 0.41). CNV lesions of exercise-trained mice also exhibited significantly lower F4/80+ macrophage staining and Vegfa and Ccl2 mRNA expression. Conclusions: These findings provide the first experimental evidence that voluntary exercise improves CNV outcomes. These studies indicate that exercise before laser treatment is required to improve CNV outcomes.

Number of Hyperreflective Foci in the Outer Retina Correlates with Inflammation and Photoreceptor Degeneration in Retinitis Pigmentosa.

PURPOSE: Hyperreflective foci (HRF) in the outer retina are associated with the progression of multiple retinal diseases, but their pathogenic significance in retinitis pigmentosa (RP) remains obscure. We studied outer retinal HRF number and distribution in RP using spectral-domain (SD) OCT and assessed associations with visual field loss, retinal inflammation, and photoreceptor degeneration. DESIGN: Retrospective cross-sectional study. PARTICIPANTS: Medical records of 128 eyes of 66 RP patients (mean age, 45.7±16.1 years; range, 10-85 years). METHODS: All participants underwent high-resolution SD-OCT. Outer retinal HRF were counted on vertical and horizontal SD-OCT images centered at the fovea of 55 eyes of 29 patients. In addition, SD-OCT macular volume scans of 92 eyes of 48 patients were reviewed and compared with fundus autofluorescence images of the same area to investigate outer retinal HRF distribution and the spatial relations to areas of low autofluorescence and photoreceptor inner segment and outer segment junction (IS/OS) preservation. MAIN OUTCOME MEASURES: The number and distribution of outer retinal HRF, aqueous flare value, thickness of outer layers, visual field area, and fundus autofluorescence. RESULTS: In the HRF counting analysis, the mean number of outer retinal HRF per scan was 24.0±18.6, mean aqueous flare value was 7.7±3.2 photon count/ms, visual field area was 25.2±26.6%, and thickness of the outer layers was 95.2±39.6 µm. Generalized estimating equations revealed that the number of outer retinal HRF was associated positively with aqueous flare value (P = 0.01) and associated negatively with visual field area and outer layer thickness (P = 0.016 and P < 0.001, respectively). In the distribution analysis, macular areas exhibiting outer retinal HRF (HRF areas) spatially overlapped with areas of low autofluorescence. In contrast, HRF areas did not overlap with areas showing preserved IS/OS. CONCLUSIONS: Outer retinal HRF number is associated with intraocular inflammation and photoreceptor degeneration in RP. Their distribution in areas with IS/OS disruption and low autofluorescence suggests that outer retinal HRF reflect defects in the retinal pigment epithelium (RPE) layer caused by RPE cell or microglial migration in response to photoreceptor degeneration.

RF/6A Chorioretinal Cells Do Not Display Key Endothelial Phenotypes.

Purpose: The misuse of inauthentic cell lines is widely recognized as a major threat to the integrity of biomedical science. Whereas the majority of efforts to address this have focused on DNA profiling, we sought to anatomically, transcriptionally, and functionally authenticate the RF/6A chorioretinal cell line, which is widely used as an endothelial cell line to model retinal and choroidal angiogenesis. Methods: Multiple vials of RF/6A cells obtained from different commercial distributors were studied to validate their genetic, transcriptomic, anatomic, and functional fidelity to bona fide endothelial cells. Results: Transcriptomic profiles of RF/6A cells obtained either de novo or from a public data repository did not correspond to endothelial gene expression signatures. Expression of established endothelial markers were very low or undetectable in RF/6A compared to primary human endothelial cells. Importantly, RF/6A cells also did not display functional characteristics of endothelial cells such as uptake of acetylated LDL, expression of E-selectin in response to TNF-α exposure, alignment in the direction of shear stress, and AKT and ERK1/2 phosphorylation following VEGFA stimulation. Conclusions: Multiple independent sources of RF/6A do not exhibit key endothelial cell phenotypes. Therefore, these cells appear unsuitable as surrogates for choroidal or retinal endothelial cells. Further, cell line authentication methods should extend beyond genomic profiling to include anatomic, transcriptional, and functional assessments.

Nutritional Supplementation Inhibits the Increase in Serum Malondialdehyde in Patients with Wet Age-Related Macular Degeneration.

Purpose. To compare serum levels of malondialdehyde (MDA) in patients with wet age-related macular degeneration (wAMD), patients with dry AMD (dAMD), and patients without AMD and to evaluate the efficacy of nutritional supplementation for treating elevated serum MDA in patients with wAMD. Methods. MDA levels were measured in sera from 20 patients with wAMD, 20 with dAMD, and 24 without AMD. Patients with wAMD were randomized to receive or not receive nutritional supplementation (10 patients in each group), and MDA levels were measured after 3 months of treatment. Results. MDA levels in patients with wAMD were significantly greater compared with patients without AMD. In eyes with wAMD, there was a significant correlation between MDA levels and choroidal neovascularization lesion area. Serum MDA levels decreased in most patients that received supplementation and significantly increased in those who did not. Conclusion. Baseline serum MDA levels were elevated in patients with wAMD, and MDA levels were directly correlated with choroidal neovascularization lesion area. In addition, nutritional supplementation appeared to exert a protective effect against oxidative stress in patients with wAMD.

Total Synthesis of TAN1251C via Diastereoselective Construction of the Azaspiro Skeleton.

An efficient total synthesis of TAN1251C was accomplished by employing a Ugi four-component accumulation reaction and a Dieckmann condensation to construct the spiro-fused cyclohexanone and γ-lactam ring. Diastereoselective reduction by side-chain-controlled hydrogenation of enamide 15 or Zn reduction of oxime 23 enabled construction of the amino group with the desired stereochemistry.