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Brain transcriptome analysis of a familial Alzheimer’s disease-like mutation in the zebrafish presenilin 1 gene implies effects on energy production

Contributed equally
Molecular Brain201912:43

https://doi.org/10.1186/s13041-019-0467-y

  • Received: 4 February 2019
  • Accepted: 24 April 2019
  • Published:

Abstract

To prevent or ameliorate Alzheimer’s disease (AD) we must understand its molecular basis. AD develops over decades but detailed molecular analysis of AD brains is limited to postmortem tissue where the stresses initiating the disease may be obscured by compensatory responses and neurodegenerative processes. Rare, dominant mutations in a small number of genes, but particularly the gene PRESENILIN 1 (PSEN1), drive early onset of familial AD (EOfAD). Numerous transgenic models of AD have been constructed in mouse and other organisms, but transcriptomic analysis of these models has raised serious doubts regarding their representation of the disease state. Since we lack clarity regarding the molecular mechanism(s) underlying AD, we posit that the most valid approach is to model the human EOfAD genetic state as closely as possible. Therefore, we sought to analyse brains from zebrafish heterozygous for a single, EOfAD-like mutation in their PSEN1-orthologous gene, psen1. We previously introduced an EOfAD-like mutation (Q96_K97del) into the endogenous psen1 gene of zebrafish. Here, we analysed transcriptomes of young adult (6-month-old) entire brains from a family of heterozygous mutant and wild type sibling fish. Gene ontology (GO) analysis implies effects on mitochondria, particularly ATP synthesis, and on ATP-dependent processes including vacuolar acidification.

Keywords

  • Alzheimer’s disease
  • Presenilin 1
  • Mutation
  • Transcriptome
  • Brain
  • ATP synthesis
  • Mitochondria
  • Vacuolar acidification
  • Zebrafish
  • Genome editing

Background

AD is the most common form of dementia with severe personal, social, and economic impacts. Rare, familial forms of AD exist caused by autosomal dominant mutations in single genes (reviewed by [1]). The majority of these mutations occur in the gene PRESENILIN 1 (PSEN1) that encodes a multipass integral membrane protein involved in intra-membrane cleavage of numerous proteins [1].

A wide variety of transgenic models of AD have been created and studied. These are aimed at reproducing histopathologies posited to be central to the disease process, i.e. amyloid plaques and neurofibrillary tangles of the protein MAPT [2]. However, analysis of the effects on the brain transcriptome of the transgenes driving a number of these mouse models showed little concordance with transcriptomic differences between human AD brains and age-matched controls [3] (although a recent study asserts that this lack of concordance for the popular “5XFAD” transgenic mouse model is due to previous failure to analyse the effects of its transgenes in a variety of genetic backgrounds [4]). We posit that, in the absence of an understanding of the molecular mechanism(s) underlying AD, the most objective approach to modeling this disease (or, at least, modeling its genetic form, EOfAD) is to create a genetic state as similar as possible to the EOfAD state in humans. Mouse “knock-in” models of EOfAD mutations were created over a decade ago and showed subtle phenotypic effects but not the desired histopathologies (e.g. [5, 6]). However, at that time, researchers did not have access to RNA-Seq technology. To the best of our knowledge, transcriptome analysis of the EOfAD mutation knock-in mouse models was never performed.

In humans, AD is thought to develop over decades and the median survival to onset age for EOfAD mutations in human PSEN1 considered collectively is 45 years [7]. Functional MRI of human children carrying EOfAD mutations in PSEN1 has revealed differences in brain activity compared to non-carriers in individuals as young as 9 years of age [8]. Presumably therefore, heterozygosity for EOfAD mutations in PSEN1 causes early molecular changes/stresses that eventually lead to AD.

Transcriptome analysis is currently the most detailed molecular phenotypic analysis possible on cells or tissues. Here we present an initial analysis of the transcriptomic differences caused in young adult (6-month-old) zebrafish brains by the presence of an EOfAD-like mutation in the gene psen1 that is orthologous to the human PSEN1 gene. GO analysis supports very significant effects on mitochondrial function, especially synthesis of ATP, and on ATP-dependent functions such as the acidification of lysosomes that are critical for autophagy.

Materials and methods

The mutant allele, Q96_K97del, of psen1 was a byproduct identified during our introduction of the K97fs mutation into psen1 (that models the K115fs mutation of human PSEN2 – see [9] for an explanation).

Q96_K97del is a deletion of 6 nucleotides from the coding sequence of the psen1 gene. This is predicted to distort the first lumenal loop of the Psen1 protein. In this sense, it is similar to a number of EOfAD mutations of human PSEN1 [10]. Also, in common with all the widely distributed EOfAD mutations in PSEN1, (and consistent with the PRESENILIN EOfAD mutation “reading frame preservation rule” [1]), the Q96_K97del allele is predicted to encode a transcript that includes the C-terminal sequences of the wild type protein. Therefore, as a model of an EOfAD mutation, it is superior to the K97fs mutation in psen1 [9].

To generate a family of heterozygous Q96_K97del allele (i.e. psen1Q96_K97del/+) and wild type (+/+) sibling fish, we mated a psen1Q96_K97del/+ individual with a +/+ individual and raised the progeny from a single spawning event together in one tank. Zebrafish can live for up to 5 years but, in our laboratory, typically show greatly reduced fertility after 18 months. The fish become fertile after around 3 months of age, so we regard 6-month-old fish as equivalent to young adult humans. Therefore we analysed the transcriptomes of entire young adult, 6-month-old fish brains using poly-A enriched RNA-seq technology, and estimated gene expression from the resulting single-end 75 bp reads using the reference GRCz11 zebrafish assembly transcriptome [11, 12]. Each zebrafish brain has a mass of approximately 7 mg. Since AD is more prevalent in human females than males, and to further reduce gene expression “noise” in our analyses, we obtained brain transcriptome data from four female wild type fish and four female heterozygous mutant fish. This data has been made publicly available at the Gene Expression Omnibus (GEO, see under Availability of data and materials below).

Results

Differentially expressed genes (DE genes)

Genes differentially expressed between wild type and heterozygous mutant sibling fish were identified using moderated t-tests and a false discovery rate (FDR)-adjusted p-value cutoff of 0.05 as previously described [9, 13, 14]. In total, 251 genes were identified as differentially expressed (see Additional file 1). Of these, 105 genes showed increased expression in heterozygous mutant brains relative to wild type sibling brains while 146 genes showed decreased expression.

GO analysis

To understand the significance for brain cellular function of the differential gene expression identified in young adult heterozygous mutant brains we used the goana function [15] of the limma package of Bioconductor software [14] to identify GOs in which the DE genes were enriched at an FDR-corrected p-value of less than 0.05. Seventy-eight GOs were identified (Table 1) of which 20 addressed cellular components (CC). Remarkably, most of these CCs concerned the mitochondrion, membranes, or ATPases. Seventeen GOs addressed molecular functions (MF) and largely involved membrane transporter activity, particularly ion transport and ATPase activity coupled to such transport. Forty-one GOs addressed biological processes (BP) and involved ATP metabolism, ribonucleoside metabolism, and transmembrane transport processes including vacuolar acidification (that has previously been identified as affected by EOfAD mutations in PSEN1 [16]). Overall, our GO analysis indicates that this EOfAD-like mutation of zebrafish psen1 has very significant impacts on cellular energy metabolism and transmembrane transport processes.
Table 1

GOs enriched for genes differentially expressed between heterozygous mutant and wild type sibling fish brains

Gene Ontology Term

Ontology

Total Genes

DE Genes

p-value

FDR p-value

ATP biosynthetic process

BP

29

7

3.48987E-08

0.00041

ribonucleoside triphosphate biosynthetic process

BP

49

8

9.41317E-08

0.00045

nucleoside triphosphate biosynthetic process

BP

54

8

2.06555E-07

0.00060

purine nucleoside triphosphate biosynthetic process

BP

41

7

4.46237E-07

0.00060

purine ribonucleoside triphosphate biosynthetic process

BP

41

7

4.46237E-07

0.00060

hydrogen transport

BP

60

8

4.783E-07

0.00060

proton transport

BP

60

8

4.783E-07

0.00060

energy coupled proton transport, down electrochemical gradient

BP

27

6

5.89038E-07

0.00060

ATP synthesis coupled proton transport

BP

27

6

5.89038E-07

0.00060

transport

BP

2072

48

2.11748E-06

0.00165

purine nucleoside monophosphate biosynthetic process

BP

54

7

3.09019E-06

0.00172

purine ribonucleoside monophosphate biosynthetic process

BP

54

7

3.09019E-06

0.00172

hydrogen ion transmembrane transport

BP

54

7

3.09019E-06

0.00172

ribonucleoside triphosphate metabolic process

BP

133

10

3.8448E-06

0.00178

establishment of localization

BP

2123

48

4.20295E-06

0.00182

ATP metabolic process

BP

109

9

5.50772E-06

0.00230

nucleoside triphosphate metabolic process

BP

140

10

6.08925E-06

0.00245

cation transport

BP

452

18

6.61154E-06

0.00258

monovalent inorganic cation transport

BP

219

12

1.10729E-05

0.00392

ribonucleoside monophosphate biosynthetic process

BP

65

7

1.08944E-05

0.00392

nucleoside monophosphate biosynthetic process

BP

68

7

1.47269E-05

0.00492

purine ribonucleoside triphosphate metabolic process

BP

125

9

1.68142E-05

0.00546

purine nucleoside triphosphate metabolic process

BP

126

9

1.79263E-05

0.00552

transmembrane transport

BP

654

21

2.93288E-05

0.00797

purine nucleoside monophosphate metabolic process

BP

136

9

3.2951E-05

0.00837

purine ribonucleoside monophosphate metabolic process

BP

136

9

3.2951E-05

0.00837

energy coupled proton transmembrane transport, against electrochemical gradient

BP

35

5

5.20342E-05

0.01106

ATP hydrolysis coupled proton transport

BP

35

5

5.20342E-05

0.01106

ATP hydrolysis coupled transmembrane transport

BP

35

5

5.20342E-05

0.01106

ATP hydrolysis coupled ion transmembrane transport

BP

35

5

5.20342E-05

0.01106

ATP hydrolysis coupled cation transmembrane transport

BP

35

5

5.20342E-05

0.01106

ion transport

BP

737

22

5.61478E-05

0.01152

localization

BP

2621

52

6.0913E-05

0.01207

ribonucleoside monophosphate metabolic process

BP

147

9

6.06496E-05

0.01207

nucleoside monophosphate metabolic process

BP

150

9

7.09445E-05

0.01360

single-organism localization

BP

819

23

9.51294E-05

0.01738

single-organism transport

BP

776

22

0.000119082

0.02109

ribonucleotide biosynthetic process

BP

129

8

0.000143028

0.02423

ribose phosphate biosynthetic process

BP

129

8

0.000143028

0.02423

vacuolar acidification

BP

11

3

0.000246582

0.04101

ribonucleotide metabolic process

BP

220

10

0.000281352

0.04506

proton-transporting two-sector ATPase complex, proton-transporting domain

CC

25

6

3.59375E-07

0.00060

proton-transporting two-sector ATPase complex

CC

45

7

8.65692E-07

0.00078

mitochondrial membrane

CC

285

15

1.42199E-06

0.00119

mitochondrial envelope

CC

303

15

3.0322E-06

0.00172

membrane part

CC

4868

85

1.1722E-05

0.00403

organelle membrane

CC

789

24

1.84982E-05

0.00555

mitochondrial inner membrane

CC

195

11

1.97958E-05

0.00579

integral component of membrane

CC

4419

78

2.52479E-05

0.00720

intrinsic component of membrane

CC

4453

78

3.37749E-05

0.00840

organelle envelope

CC

420

16

3.76291E-05

0.00917

envelope

CC

422

16

3.98337E-05

0.00950

organelle inner membrane

CC

215

11

4.86028E-05

0.01106

Cul2-RING ubiquitin ligase complex

CC

7

3

5.4156E-05

0.01131

proton-transporting ATP synthase complex

CC

19

4

6.25883E-05

0.01220

mitochondrial membrane part

CC

117

8

7.21148E-05

0.01360

mitochondrial part

CC

404

15

8.83156E-05

0.01639

membrane

CC

5379

88

0.000106964

0.01924

vacuolar proton-transporting V-type ATPase, V0 domain

CC

9

3

0.000127733

0.02229

mitochondrial proton-transporting ATP synthase complex, coupling factor F(o)

CC

12

3

0.000325933

0.04885

proton-transporting V-type ATPase, V0 domain

CC

12

3

0.000325933

0.04885

ATPase activity, coupled to transmembrane movement of ions, rotational mechanism

MF

34

7

1.1446E-07

0.00045

hydrogen ion transmembrane transporter activity

MF

84

9

6.11883E-07

0.00060

ATPase activity, coupled to transmembrane movement of substances

MF

98

9

2.27123E-06

0.00166

hydrolase activity, acting on acid anhydrides, catalyzing transmembrane movement of substances

MF

101

9

2.92425E-06

0.00172

primary active transmembrane transporter activity

MF

104

9

3.73269E-06

0.00178

P-P-bond-hydrolysis-driven transmembrane transporter activity

MF

104

9

3.73269E-06

0.00178

cation-transporting ATPase activity

MF

56

7

3.96731E-06

0.00178

ATPase coupled ion transmembrane transporter activity

MF

56

7

3.96731E-06

0.00178

ATPase activity, coupled to movement of substances

MF

112

9

6.88692E-06

0.00260

active ion transmembrane transporter activity

MF

96

8

1.72916E-05

0.00546

active transmembrane transporter activity

MF

281

13

2.87859E-05

0.00797

proton-transporting ATP synthase activity, rotational mechanism

MF

16

4

3.02121E-05

0.00803

transporter activity

MF

991

25

0.000249051

0.04101

substrate-specific transmembrane transporter activity

MF

709

20

0.000263528

0.04279

ion transmembrane transporter activity

MF

660

19

0.000293184

0.04572

substrate-specific transporter activity

MF

828

22

0.000297009

0.04572

monovalent inorganic cation transmembrane transporter activity

MF

264

11

0.000297217

0.04572

GOs are grouped by ontology (BP, CC or MF) and ranked by FDR-corrected p-value

Notes

Abbreviations

AD: 

Alzheimer’s disease

ATP: 

Adenosine triphosphate

BP: 

Biological process (GO term)

CC: 

Cellular component (GO term)

DE genes: 

Differentially expressed genes

EOfAD: 

Early onset familial Alzheimer’s disease

FDR: 

False discovery rate

GEO: 

Gene Expression Omnibus

GO: 

Gene ontology

MAPT: 

MICROTUBULE-ASSOCIATED PROTEIN TAU (human protein)

MF: 

Molecular function (GO term)

mg: 

Milligrams

MRI: 

Magnetic resonance imaging

PSEN1

PRESENILIN 1 (human gene)

PSEN1: 

PRESENILIN 1 (human protein)

psen1

presenilin 1 (zebrafish gene)

Psen1: 

Presenilin 1 (zebrafish protein)

Declarations

Acknowledgements

The authors wish to thank the Carthew Family Foundation and Prof. David Adelson for their encouragement and support.

Funding

This work was supported by grants from Australia’s National Health and Medical Research Council, GNT1061006 and GNT1126422, and from the Carthew Family Foundation.

Availability of data and materials

The datasets generated and/or analysed during the current study are available in the GEO repository (https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE126096.

psen1Q96_K97del mutant zebrafish are available upon request. However, due to Australia’s strict quarantine and export regulations, export of fish involves considerable effort and expense and these costs must be borne by the party requesting the fish.

Authors’ contributions

MN conceived the project, sought funding, generated the psen1Q96_K97del mutant zebrafish, identified the genotype of individuals, and isolated mRNA from zebrafish brains. NH processed the RNA-seq data and performed bioinformatics analysis to identify DE genes and GOs. SP supervised the work of NH and performed data quality checks. ML conceived the project, sought funding, coordinated the project, and drafted this research report. All authors contributed to interpretation of data and to reviewing and editing drafts of the submitted manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

This study was conducted under the auspices of the Animal Ethics Committee of the University of Adelaide, under permits S-2014-108 and S-2017-073.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Authors’ Affiliations

(1)
Department of Molecular and Biomedical Science, University of Adelaide, School of Biological Sciences, North Terrace, Adelaide, SA, 5005, Australia

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© The Author(s). 2019

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