Skip to main content

No observed effect on brain vasculature of Alzheimer’s disease-related mutations in the zebrafish presenilin 1 gene


Previously, we found that brains of adult zebrafish heterozygous for Alzheimer’s disease-related mutations in their presenilin 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 straightness, 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.


The dominant hypothesis of Alzheimer’s disease (AD) pathogenesis is the amyloid cascade hypothesis (ACH) [1], which postulates the amyloid β peptide (Aβ) as initiating a pathological process resulting in neurodegeneration 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, consequently, neurodegeneration. Significant evidence supports 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 familial AD, EOfAD). Most EOfAD cases are due to heterozygous 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 generated knock-in models in zebrafish with each of these types of mutant psen1 allele: K97Gfs, a frameshift mutation 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-preserving deletion of two codons [7].

We recently observed in normoxic adult zebrafish brains that heterozygosity for either of the above two mutations 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 transgenic mice expressing human PSEN1 EOfAD mutation-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].


Single zebrafish heterozygous for either psen1 mutation 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 heterozygous 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 hydrogel. 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 equivalent of the prefrontal cortex in humans) using an Olympus 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.

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

Results and conclusion

No statistically significant differences between sibling genotypes at each age were observed for any of the measured 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 genotype may be too small to detect using this method and further experimentation using a larger number of biological 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.

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.



Amyloid cascade hypothesis


Alzheimer’s disease


Amyloid beta


Early-onset familial Alzheimer’s disease


Hypoxia-inducible factor 1


Hypoxia-inducible factor 1, alpha subunit


Hypoxia response gene


Presenilin 1


  1. 1.

    Hardy JA, Higgins GA. Alzheimer’s disease: the amyloid cascade hypothesis. Science. 1992;256(5054):184–5.

    CAS  Article  Google Scholar 

  2. 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.

    CAS  Article  Google Scholar 

  3. 3.

    de la Torre JC, Mussivan T. Can disturbed brain microcirculation cause Alzheimer’s disease? Neurol Res. 1993;15(3):146–53.

    Article  Google Scholar 

  4. 4.

    Rius-Pérez S, Tormos AM, Pérez S, Taléns-Visconti R. Vascular pathology: cause or effect in alzheimer disease? Neurología (English Edition). 2017.

    Article  Google Scholar 

  5. 5.

    Jayne T, Newman M, Verdile G, Sutherland G, Munch G, Musgrave I, et al. Evidence for and against a pathogenic role of reduced gamma-secretase activity in familial Alzheimer’s disease. JAD. 2016.

    Article  PubMed  Google Scholar 

  6. 6.

    Hin N, Newman M, Kaslin J, Douek AM, Lumsden A, Nik SHM, et al. Accelerated brain aging towards transcriptional inversion in a zebrafish model of the K115fs mutation of human PSEN2. PLoS ONE. 2020;15(1):e0227258.

    CAS  Article  Google Scholar 

  7. 7.

    Newman M, Hin N, Pederson S, Lardelli M. Brain transcriptome analysis of a familial Alzheimer’s disease-like mutation in the zebrafish presenilin 1 gene implies effects on energy production. Mol Brain. 2019.

    Article  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Newman M, Nik HM, Sutherland GT, Hin N, Kim WS, Halliday GM, et al. Accelerated loss of hypoxia response in zebrafish with familial Alzheimer’s disease-like mutation of presenilin 1. Hum Mol Genet. 2020;29(14):2379–94.

    CAS  Article  Google Scholar 

  9. 9.

    Gama Sosa MA, Gasperi RD, Rocher AB, Wang AC, Janssen WG, Flores T, et al. Age-related vascular pathology in transgenic mice expressing presenilin 1-associated familial Alzheimer’s disease mutations. Am J Pathol. 2010;176(1):353–68.

    Article  Google Scholar 

  10. 10.

    Lawson ND, Weinstein BM. In vivo imaging of embryonic vascular development using transgenic zebrafish. Developmental Biology. 2002;248(2):307–18.

    CAS  Article  Google Scholar 

  11. 11.

    Yang B, Treweek Jennifer B, Kulkarni Rajan P, Deverman Benjamin E, Chen C-K, Lubeck E, et al. Single-cell phenotyping within transparent intact tissue through whole-body clearing. Cell. 2014;158(4):945–58.

    CAS  Article  Google Scholar 

  12. 12.

    Chung K, Wallace J, Kim S-Y, Kalyanasundaram S, Andalman AS, Davidson TJ, et al. Structural and molecular interrogation of intact biological systems. Nature. 2013;497:332.

    CAS  Article  Google Scholar 

  13. 13.

    Le Moan N, Houslay DM, Christian F, Houslay MD, Akassoglou K. Oxygen-dependent cleavage of the p75 neurotrophin receptor triggers stabilization of HIF-1α. Mol Cell. 2011;44(3):476–90.

    Article  Google Scholar 

  14. 14.

    Yambire KF, Rostosky C, Watanabe T, Pacheu-Grau D, Torres-Odio S, Sanchez-Guerrero A, et al. Impaired lysosomal acidification triggers iron deficiency and inflammation in vivo. Elife. 2019.

    Article  PubMed  PubMed Central  Google Scholar 

Download references


Confocal imaging data were generated at Adelaide Microscopy (The University of Adelaide).


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 Program 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.

Author information




KB performed the experiments, MN and ML conceived the project, CN provided advice and access for 3D image analysis. All authors read and contributed to the final manuscript.

Corresponding author

Correspondence to Karissa Barthelson.

Ethics declarations

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).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

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.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Barthelson, K., Newman, M., Nowell, C.J. et al. No observed effect on brain vasculature of Alzheimer’s disease-related mutations in the zebrafish presenilin 1 gene. Mol Brain 14, 22 (2021).

Download citation


  • Zebrafish
  • Vasculature
  • Confocal laser scanning microscopy
  • 3D reconstruction