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TaubCONNECT Research Perspective:
July 2023

Caspase-9 Inhibition Confers Stronger Neuronal and Vascular Protection Compared to VEGF Neutralization in a Mouse Model of Retinal Vein Occlusion

Carol M. Troy, MD, PhD

Retinal vein occlusion (RVO) is a leading cause of blindness throughout the world. The current first line therapy is the intravitreal injection of drugs which neutralize vascular endothelial growth factor (VEGF). This is an invasive therapy and moreover, more than 50% of patients with RVO are not helped by this treatment. Previous work from our lab identified caspase-9, a member of the caspase family of cell death proteases, as a critical mediator of pathology in RVO (Avrutsky et al., 2020; Colon Ortiz et al., 2022). In our prior work, our lab developed a cell-permeant inhibitor of caspase-9, Pen1-XBir3, which can be applied topically as eyedrops. The eyedrops are therapeutically effective in the mouse model of RVO (Avrutsky et al., 2020). In the current study, we compared the therapeutic efficacy of the Pen1-XBir3 eyedrops targeting caspase-9 with intravitreal injection of a VEGF neutralizing antibody. We used an integrated panel of ophthalmic imaging readouts to follow the progression of retinal ischemia, edema, and neurodegeneration in a well-defined mouse model of RVO (Colon Ortiz et al., 2021; Chen et al., 2022).

Adult male C57Bl/6 J mice were randomized to induction of RVO and treated using either a control substance, a VEGF-neutralizing antibody, a topical caspase-9 inhibitor (Pen1-XBir3), or a combination of treatments. As recently reported in Frontiers in Neuroscience, we found that both VEGF inhibition and caspase-9 blockade significantly shielded the retina from RVO damage in comparison to the control. However, as featured on CUIMC Newsroom, the topical caspase-9 blocker notably sped up the healing of blocked veins, cut down edema roughly twice as effectively as VEGF inhibition, preserved electroretinogram (ERG) responses, and noticeably reduced retinal detachment and retinal thinning. VEGF inhibition did not stop nerve tissue damage post-RVO.

Figure 1. Mouse model of retinal vein occlusion. (B) Representative image of an OCT retinal scan labeling retinal layers in an uninjured mouse. Diagram depicts retinal neuronal and vascular layers in uninjured animals and in acute and late phases of RVO. GCL; ganglion cell layer, IPL; inner plexiform layer, INL; inner nuclear layer, OPL; outer plexiform layer, ONL; outer nuclear layer, IS/OS; inner segment/outer segment, RPE; retinal pigment endothelium. Figure made with Biorender.com

Overall, our study suggests that the topical caspase-9 blocker offers better protection for both neurons and blood vessels than VEGF inhibition. A non-invasive topical therapy for RVO and other related retinal diseases would provide therapeutic intervention for those who are not helped by VEGF inhibition. It could also potentially be used in areas of the world that lack sufficient medical infrastructure to support administering anti-VEGF injections to patients. Our goal is to complete the pre-clinical studies to support an investigational new drug application to the FDA so that a clinical trial can begin.

Carol M. Troy, MD, PhD
Professor of Pathology & Cell Biology and Neurology (in the Taub Institute)
cmt2@cumc.columbia.edu

Christiane Reitz, MD, PhD

The Early-Onset Alzheimer's Disease Whole-Genome Sequencing Project: Study Design and Methodology

Sequencing efforts to identify genetic variants and mechanistic pathways underlying Alzheimer’s Disease (AD) have largely focused on late-onset AD although early-onset AD (EOAD) accounting for ~10% of cases is largely unexplained by known mutations, resulting in a lack of understanding of its molecular etiology. Early-onset Alzheimer’s Disease tends to be more aggressive in its course and shows a higher prevalence of atypical clinical features and impairment in other cognitive domains including impairment in executive dysfunction, apraxia, dyscalculia, visual dysfunction and aphasia, resulting in particularly detrimental medical, emotional, social, and financial consequences for patients and their families.

Figure 1. Project flow and primary aims of the Early-Onset Alzheimer’s Disease Whole-genome Sequencing Project. The 5206 samples (4097 cases and 1109 controls) sequenced by the Early-Onset Alzheimer’s Disease Whole-genome Sequencing Project will be integrated with over 50,000 whole-genomes collected by the Alzheimer’s Disease Sequencing Project (ADSP) and 200 multiplex families loaded for early-onset Alzheimer’s Disease (EOAD). Phenotype information will be harmonized using AD biomarkers, brain imaging, cognitive, neuropath, and multi-omics data. First-pass analyses will be conducted to identify novel loci associated with EOAD, cognitive decline, and age-at-onset modulation. Follow-up analyses will be conducted to assess polygenic risk, mechanistic pathways, comparison of the genetic architecture between EOAD and late-onset Alzheimer’s Disease (LOAD) and vascular traits, and to examine local ancestry. eQTL, expression quantitative trait loci.

As recently reported in Alzheimer’s & Dementia, we have established the Early-Onset Alzheimer's Disease Whole-genome Sequencing Project, a large-scale genomics resource to identify genetic variants underlying EOAD. This collaborative project involves integrating harmonized phenotype and biomarker data with whole-genome sequencing (WGS) data from over 5,000 EOAD samples. The primary goals of the project include identifying novel loci associated with EOAD, cognitive decline, and age-of-onset (AAO). The project will also compare the genetic architecture between EOAD and late-onset Alzheimer's disease (LOAD), examine local genetic ancestry at identified risk loci, and assess polygenic risk and mechanistic pathways. Various computational and statistical approaches will be employed to infer causality, identify potential druggable targets, and understand the role of polygenic effects in EOAD. We anticipate that the findings from this project will contribute to personalized preventive and therapeutic measures and help unravel observed health disparities.

Christiane Reitz, MD, PhD
Associate Professor of Neurology and Epidemiology
cr2101@cumc.columbia.edu

Heart Failure-Induced Cognitive Dysfunction is Mediated by Intracellular Ca2+ Leak Through Ryanodine Receptor Type 2

Elentina K. Argyrousi, PhD		Ottavio Arancio, MD, PhD		Andrew R. Marks, MD

Heart failure (HF) is a rising global concern with severe implications on mortality, quality of life, and economic costs. Increasing evidence suggests HF may directly cause cardiogenic dementia, affecting up to 80% of HF patients. Symptoms of cognitive dysfunction (CD), such as forgetfulness, affect self-care and medication adherence in most HF patients, leading to life-threatening risks. HF is also linked to brain structural abnormalities and persistent inflammatory reactions. The continuous activation of the sympathetic nervous system in HF patients influences Ca2+ balance in cells, crucial for cellular function. Dysregulated Ca2+ is also a feature in neurodegenerative conditions like Alzheimer's and Parkinson's disease.

Intraneuronal upregulation of Ca2+ leads to the activation of the Ca2+-activated intracellular Ca2+ release ryanodine receptor type 2 (RyR2) channel. Proper regulation of Ca2+ homeostasis via RyR2 is essential for plasticity and synaptic transmission underlying memory formation. Previous research by the Columbia laboratory of Dr. Andrew R. Marks (Department of Physiology and Cellular Biophysics) and others have identified abnormalities in RyR2 not only in heart cells of individuals with HF, but also in skeletal muscle, indicating a common dysfunction mechanism across body systems that express different isoforms of RyR channels, such as RyR2 in the brain. Although RyR2 has been linked to cardiac muscle dysfunction, its role in CD in HF remains unclear.

Treatment with the the Rycal drug S107 that stabilizes leaky RyR2 channels attenuates long-term potentiation impairments observed in a mouse model of heart failure. a, Schematic representation of a hippocampal brain slice for LTP experiments and the positioning of the stimulating and recording electrodes. b, fEPSPs in hippocampal slices from each experimental group (SHAM (n = 13), MI (n = 12), MI + ARM036 (n = 12), MI + S107 (n = 11), MI + propranolol (n = 17) and MI + SD208 (n = 16)). c, fEPSPs at 150 min in all the experimental groups.

Here, in a new study led by the Marks lab, we evaluated hippocampal neurons from individuals and mice with HF and found that the RyR2/intracellular Ca2+ release channels were subjected to post-translational modification (PTM) and were leaky. RyR2 PTM included protein kinase A phosphorylation, oxidation, nitrosylation and depletion of the stabilizing subunit calstabin2. As recently reported in Nature Neuroscience and featured in the CUIMC Newsroom, we discovered that heightened adrenergic activity and inflammation in HF trigger a calcium leak in neuronal RyR2, impacting cognition and memory. This calcium imbalance leads to oxidative stress and changes in cognitive genes. Treatment of HF mice with a small-molecule Rycal drug S107 that stabilizes leaky RyR2 channels, effectively mitigated the cognitive impairments caused by HF. These findings highlight the impact of intracellular Ca2+ leak in HF and suggest that HF is a systemic illness leading to cardiogenic dementia.

Ottavio Arancio, MD, PhD
Professor of Pathology and Cell Biology (in the Taub Institute), and Medicine
oa1@cumc.columbia.edu

Elentina K. Argyrousi, PhD
Postdoctoral Research Scientist in the Taub Institute
ea2693@cumc.columbia.edu