No observed effect on brain vasculature of Alzheimer’s disease-related mutations in the zebrafish presenilin 1 gene Barthelson et al. Mol Brain (2021) 14:22 https://doi.org/10.1186/s13041-021-00734-5 M I C R O R E P O R T No observed effect on brain vasculature of Alzheimer’s disease-related mutations in the zebrafish presenilin 1 gene Karissa Barthelson1* , Morgan Newman1, Cameron J. Nowell2 and Michael Lardelli1 Abstract Previously, we found that brains of adult zebrafish heterozygous for Alzheimer’s disease-related mutations in their pre- senilin 1 gene (psen1, orthologous to human PSEN1) show greater basal expression levels of hypoxia responsive genes relative to their wild type siblings under normoxia, suggesting hypoxic stress. In this study, we investigated whether this might be due to changes in brain vasculature. We generated and compared 3D reconstructions of GFP-labelled blood vessels of the zebrafish forebrain from heterozygous psen1 mutant zebrafish and their wild type siblings. We observed no statistically significant differences in vessel density, surface area, overall mean diameter, overall straight- ness, or total vessel length normalised to the volume of the telencephalon. Our findings do not support that changes in vascular morphology are responsible for the increased basal expression of hypoxia responsive genes in psen1 heterozygous mutant brains. Keywords: Zebrafish, Vasculature, Confocal laser scanning microscopy, 3D reconstruction © The Author(s) 2021. 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The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Introduction The dominant hypothesis of Alzheimer’s disease (AD) pathogenesis is the amyloid cascade hypothesis (ACH) [1], which postulates the amyloid β peptide (Aβ) as ini- tiating a pathological process resulting in neurodegen- eration and dementia (reviewed in (2)). An alternative to the ACH is the vascular hypothesis [3], asserting that age-related cerebral vascular abnormalities induce AD pathologies by limiting nutrient and oxygen delivery to produce hypoxic stress, a neural energy crisis and, con- sequently, neurodegeneration. Significant evidence sup- ports the vascular hypothesis of AD (reviewed in [4]). Rare, inherited forms of AD are caused by dominant mutations in a small number of genes (early-onset famil- ial AD, EOfAD). Most EOfAD cases are due to heterozy- gous mutations in the gene presenilin 1 (PSEN1) that obey a “reading-frame preservation rule” [5]. Mutations allowing production of a transcript(s) with an altered coding sequence but, nevertheless, utilising the original stop codon cause EOfAD while mutant alleles coding only for truncated proteins do not. We previously gen- erated knock-in models in zebrafish with each of these types of mutant psen1 allele: K97Gfs, a frameshift muta- tion encoding a truncated protein similar to the human PS2V isoform that is increased in sporadic, late onset AD [6], and Q96_K97del: an EOfAD-like, reading-frame-pre- serving deletion of two codons [7]. We recently observed in normoxic adult zebrafish brains that heterozygosity for either of the above two muta- tions causes increased basal expression levels of hypoxia responsive genes (HRGs, genes with expression regulated by a master regulator of the transcriptional response to hypoxia: hypoxia-inducible factor 1 (HIF1)). This implied that the heterozygous psen1 mutant fish brains were already under some form of hypoxic stress [8], possibly due to changes in vasculature, as have been observed in Open Access *Correspondence: karissa.barthelson@adelaide.edu.au 1 Alzheimer’s Disease Genetics Laboratory, School of Biological Sciences, University of Adelaide, North Terrace, Adelaide, SA 5005, Australia Full list of author information is available at the end of the article http://orcid.org/0000-0002-4693-8833 http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/publicdomain/zero/1.0/ http://creativecommons.org/publicdomain/zero/1.0/ http://crossmark.crossref.org/dialog/?doi=10.1186/s13041-021-00734-5&domain=pdf Page 2 of 4Barthelson et al. Mol Brain (2021) 14:22 transgenic mice expressing human PSEN1 EOfAD muta- tion-bearing transgenes in neurons [9]. Therefore, we examined the effects on forebrain vasculature with age of heterozygosity for the K97Gfs and Q96_K97del mutations of psen1 by exploiting the fli1:GFP transgene that labels zebrafish endothelial cells [10]. e d c b a Page 3 of 4Barthelson et al. Mol Brain (2021) 14:22 Methods Single zebrafish heterozygous for either psen1 muta- tion were mated with single fish bearing the fli1::GFP [10] transgene. GFP-fluorescent progeny were selected to form families of siblings either wild type or heterozy- gous for the psen1 mutant alleles (Fig. 1a). We used n = 4 brains of each sibling genotype at 6 months (young adult) and 24 months (aged) of age for tissue clearing using the PACT method [11]. Briefly, PACT involves infusing and crosslinking the brain with an acrylamide-based hydro- gel. Then, light scattering lipids are passively removed by incubating the brain with a detergent, allowing light to penetrate deep into the tissue [11, 12]. We imaged the telencephalons (thought to be the region loosely equiva- lent of the prefrontal cortex in humans) using an Olym- pus FV3000 confocal microscope, and performed 3D image analysis using Imaris v9.1 (Bitplane) (Fig.  1b). For a detailed description of methods, see Additional File 1. Results and conclusion No statistically significant differences between sibling genotypes at each age were observed for any of the meas- ured parameters (see Fig.  1). This does not support that the increased basal levels of HRGs observed previously in our zebrafish psen1 mutants are due to vascular changes. However, subtle changes to vasculature due to psen1 gen- otype may be too small to detect using this method and further experimentation using a larger number of bio- logical replicates may increase statistical power to detect changes to these measured parameters. Alternatively, other factors such as altered γ-secretase activity [13] and/ or cellular ferrous iron levels [14] may influence HIF1-⍺ activity to affect basal HRG expression. Supplementary Information The online version contains supplementary material available at https ://doi. org/10.1186/s1304 1-021-00734 -5. Additional file 1. Detailed description of sample preparation, imaging and 3D image analysis. Additional file 2. Quantified values used to produce the graphs in Fig. 1. Abbreviations ACH: Amyloid cascade hypothesis; AD: Alzheimer’s disease; Aβ: Amyloid beta; EOfAD: Early-onset familial Alzheimer’s disease; HIF1: Hypoxia-inducible factor 1; HIF1-⍺: Hypoxia-inducible factor 1, alpha subunit; HRG: Hypoxia response gene; PSEN1: Presenilin 1. Acknowledgements Confocal imaging data were generated at Adelaide Microscopy (The Univer- sity of Adelaide). Authors’ contributions KB performed the experiments, MN and ML conceived the project, CN provided advice and access for 3D image analysis. All authors read and con- tributed to the final manuscript. Funding Grants GNT1061006 and GNT1126422 from the National Health and Medical Research Council of Australia (NHMRC) funded this work, and supported MN and ML. KB is supported by an Australian Government Research Training Pro- gram Scholarship. ML is an academic employee of the University of Adelaide. Funding bodies did not play a role in the design of the study, data collection, analysis, interpretation or in writing the manuscript. Availability of data and materials The quantified values used to produce the graphs in Fig. 1 can be found in Additional File 2. Raw microscopy images from the current study are available from the corresponding author upon reasonable request. Ethics approval and consent to participate Work with zebrafish was conducted under the auspices of the University of Adelaide Animal Ethics Committee (permit numbers: S-2017-073 and S-2017- 089) and Institutional Biosafety Committee (permit number 15037). (See figure on previous page.) Fig. 1 No statistically significant changes to brain vascular network parameters due to heterozygosity for the Q96_K97del or K97Gfs mutations of psen1. a Experimental design flow diagram. Genome-edited psen1 heterozygous mutant fish were pair-mated with transgenic zebrafish expressing green fluorescent protein (GFP) under the control of the fli1 promotor (fli1:GFP transgene). GFP-fluorescent larvae were selected to give a family of transgenic siblings either wild type or heterozygous for a psen1 mutation. Analysis of the brain vascular network was performed at 6 and 24 months of age. b 3D image analysis pipeline. The telencephalon was manually segmented from the optic tectum using contour lines to generate a masked surface channel containing only GFP signals from the telencephalon. Then, an additional surface was generated over the vessels to remove background fluorescence. A masked surface channel was generated from this surface as input for the filament trace algorithm. c Measured values from the surface and filament trace algorithms for the 6 month old zebrafish and d the 24 month old female zebrafish for (left to right) the volume of the telencephalon, the density of fli1:GFP positive vessels per telencephalon, the surface area of vessels normalised to the volume of the telencephalon, the overall mean diameter of vessels, the overall straightness of the vessels, and the total length of the vessels normalised to the volume of the telencephalon. Data are presented as the mean ± standard deviation. Colours of the bars represent the two families of fish used in this analysis. P-values were determined by Student’s t-test assuming unequal variance. e Representative images of a 200 µm section of the right hemisphere of the telencephalon from fish of each age and genotype. Scale bars indicate 100 µm. Vessels appeared morphologically similar in each age and genotype https://doi.org/10.1186/s13041-021-00734-5 https://doi.org/10.1186/s13041-021-00734-5 Page 4 of 4Barthelson et al. Mol Brain (2021) 14:22 • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your researchReady to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Author details 1 Alzheimer’s Disease Genetics Laboratory, School of Biological Sciences, University of Adelaide, North Terrace, Adelaide, SA 5005, Australia. 2 Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3058, Australia. Received: 19 November 2020 Accepted: 13 January 2021 References 1. Hardy JA, Higgins GA. Alzheimer’s disease: the amyloid cascade hypoth- esis. Science. 1992;256(5054):184–5. 2. Morris GP, Clark IA, Vissel B. Questions concerning the role of amyloid-β in the definition, aetiology and diagnosis of Alzheimer’s disease. Acta Neuropathol. 2018;136(5):663–89. 3. de la Torre JC, Mussivan T. Can disturbed brain microcirculation cause Alzheimer’s disease? Neurol Res. 1993;15(3):146–53. 4. Rius-Pérez S, Tormos AM, Pérez S, Taléns-Visconti R. 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