Biology subjects

Vacano, G. N.

Publications and source records attributed to Vacano, G. N..

4 recordsLinked to original sources

bioRxiv2023

APOE3 Christchurch modulates tau phosphorylation and regulates β-catenin/Wnt/Cadherin signaling in induced pluripotent stem cell-derived cerebral organoids from Alzheimer's cases

Alzheimers disease (AD) is the most common cause of dementia among older adults. APOE3 Christchurch (R136S, APOE3Ch) variant homozygosity was reported in an individual with extreme resistance to autosomal dominant AD due to the PSEN1 E280A mutation. This subject had a delayed clinical age at onset and resistance to tauopathy and neurodegeneration despite extremely high amyloid plaque burden. We established induced pluripotent stem (iPS) cell-derived cerebral organoids from this resistant case and from a non-protected kindred control (with PSEN1 E280A and APOE3/3). We used CRISPR/Cas9 gene editing to successfully remove the APOE3Ch to wild type in iPS cells from the protected case and to introduce the APOE3Ch as homozygote in iPS cells from the non-protected case to examine causality. We found significant reduction of tau phosphorylation (pTau 202/205 and pTau396) in cerebral organoids with the APOE3Ch variant, consistent with the strikingly reduced tau pathology found in the resistant case. We identified Cadherin and Wnt pathways as signaling mechanisms regulated by the APOE3Ch variant through single cell RNA sequencing in cerebral organoids. We also identified elevated {beta}-catenin protein, a regulator of tau phosphorylation, as a candidate mediator of APOE3Ch resistance to tauopathy. Our findings show that APOE3Ch is necessary and sufficient to confer resistance to tauopathy in an experimental ex-vivo model establishing a foundation for the development of novel, protected case-inspired therapeutics for tauopathies, including Alzheimers.

neuroscience↗

bioRxiv2020

The CRISPR-Cas9 crGART HeLa transcriptome: A novel cell model of de novo purine synthesis deficiency

In humans, GART [phosphoribosylglycinamide formyltransferase (EC 2.1.2.2) / phosphoribosylglycinamide synthetase (EC 6.3.4.13) / phosphoribosylaminoimidazole synthetase (EC 6.3.3.1)] is a trifunctional protein which catalyzes the second, third, and fifth reactions of the ten step de novo purine synthesis (DNPS) pathway. The second step of DNPS is conversion of phosphoribosylamine (5-PRA) to glycineamide ribonucleotide (GAR). 5-PRA is extremely unstable under physiological conditions and is unlikely to accumulate in the absence of GART activity. Recently, a HeLa cell line null mutant for GART was constructed via CRISPR-Cas9 mutagenesis. This cell line, crGART, is an important cellular model of DNPS inactivation that does not accumulate DNPS pathway intermediates. In the current study, we characterized the crGART versus HeLa transcriptomes in purine-supplemented and purine-depleted growth conditions. We observed multiple transcriptome changes and discuss pathways and ontologies particularly relevant to Alzheimer disease and Down syndrome. We also report initial analysis of the Cluster of Differentiation (CD36) gene, which is highly expressed in crGART versus HeLa.

cell biology↗

bioRxiv2020

Reduced purine biosynthesis in humans after their divergence from Neandertals

We analyze the metabolomes of humans, chimpanzees and macaques in muscle, kidney and three different regions of the brain. Whereas several compounds in amino acid metabolism occur at either higher or lower concentrations in humans than in the other primates, metabolites in oxidative phosphorylation and purine biosynthesis are consistently present in lower concentrations in the brains of humans. In particular, metabolites downstream of adenylosuccinate lyase, which catalyzes two reactions in purine synthesis, occur at lower concentrations in humans. This enzyme carries an amino acid substitution that is present in all humans today but absent in Neandertals. By introducing the modern human substitution into the genomes of mice, as well as the ancestral, Neandertal-like substitution into the genomes of human cells, we show that this amino acid substitution is responsible for much or all of the reduction of de novo synthesis of purines in humans.

evolutionary biology↗

bioRxiv2020

The CRISPR-Cas9 crATIC HeLa transcriptome: Characterization of a novel cellular model for ATIC deficiency and ZMP accumulation

In de novo purine biosynthesis (DNPS), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (2.1.2.3) / inosine monophosphate cyclohydrolase (3.5.4.10) (ATIC) catalyzes the last two reactions of the pathway: conversion of 5-aminoimidazole-4-carboxamide ribonucleotide [aka Z-nucleotide monophosphate (ZMP)] to 5-formamido-4-imidazolecarboxamide ribonucleotide (FAICAR) then to inosine monophosphate (IMP). ZMP is an adenosine monophosphate (AMP) mimetic and a known activator of AMP-activated protein kinase (AMPK). Recently, a HeLa cell line null mutant for ATIC was constructed via CRISPR-Cas9 mutagenesis. This mutant, crATIC, accumulates ZMP during purine starvation. Given that the mutant can accumulate ZMP in the absence of treatment with exogenous compounds, crATIC is likely an important cellular model of DNPS inactivation and ZMP accumulation. In the current study, we characterize the crATIC transcriptome versus the HeLa transcriptome in purine-supplemented and purine-depleted growth conditions. We report and discuss transcriptome changes with particular relevance to Alzheimers disease and in genes relevant to lipid and fatty acid synthesis, neurodevelopment, embryogenesis, cell cycle maintenance and progression, extracellular matrix, immune function, TGF{beta} and other cellular processes.

cell biology↗