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. 2023 Mar 31;24(7):6559.
doi: 10.3390/ijms24076559.

Deregulated Transcription and Proteostasis in Adult mapt Knockout Mouse

Pol Andrés-Benito  1   2 , África Flores  3 , Sara Busquet-Areny  4 , Margarita Carmona  2   4 , Karina Ausín  5 , Paz Cartas-Cejudo  5 , Mercedes Lachén-Montes  5 , José Antonio Del Rio  2   6 , Joaquín Fernández-Irigoyen  5 , Enrique Santamaría  5 , Isidro Ferrer  2   4   7   8
Affiliations

Deregulated Transcription and Proteostasis in Adult mapt Knockout Mouse

Pol Andrés-Benito et al. Int J Mol Sci. .

Abstract

Transcriptomics and phosphoproteomics were carried out in the cerebral cortex of B6.Cg-Mapttm1(EGFP)Klt (tau knockout: tau-KO) and wild-type (WT) 12 month-old mice to learn about the effects of tau ablation. Compared with WT mice, tau-KO mice displayed reduced anxiety-like behavior and lower fear expression induced by aversive conditioning, whereas recognition memory remained unaltered. Cortical transcriptomic analysis revealed 69 downregulated and 105 upregulated genes in tau-KO mice, corresponding to synaptic structures, neuron cytoskeleton and transport, and extracellular matrix components. RT-qPCR validated increased mRNA levels of col6a4, gabrq, gad1, grm5, grip2, map2, rab8a, tubb3, wnt16, and an absence of map1a in tau-KO mice compared with WT mice. A few proteins were assessed with Western blotting to compare mRNA expression with corresponding protein levels. Map1a mRNA and protein levels decreased. However, β-tubulin III and GAD1 protein levels were reduced in tau-KO mice. Cortical phosphoproteomics revealed 121 hypophosphorylated and 98 hyperphosphorylated proteins in tau-KO mice. Deregulated phosphoproteins were categorized into cytoskeletal (n = 45) and membrane proteins, including proteins of the synapses and vesicles, myelin proteins, and proteins linked to membrane transport and ion channels (n = 84), proteins related to DNA and RNA metabolism (n = 36), proteins connected to the ubiquitin-proteasome system (UPS) (n = 7), proteins with kinase or phosphatase activity (n = 21), and 22 other proteins related to variegated pathways such as metabolic pathways, growth factors, or mitochondrial function or structure. The present observations reveal a complex altered brain transcriptome and phosphoproteome in tau-KO mice with only mild behavioral alterations.

Keywords: cytoskeleton; phosphoproteomics; synapse; tau-KO; transcriptomics.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(A) Western blots of brain homogenates of WT mice show immunoreactive bands at the appropriate molecular weight for antibodies tau 5, 4R tau, and PHF1; Western blots for 3R tau are negative. As expected, Western blots to tau 5, 4R tau, and PHF1 (and 3R tau) of brain homogenates from tau-KO mice are negative. Molecular weights pointed with arrows indicate the theoretical positions of all tau isoform bands in an immunoblotting membrane. A densitometric study of the bands confirms significant differences between WT and tau-KO mice, *** p < 0.001. (B) A two-object recognition test reveals no differences between WT and tau-KO mice. (C) The results of the elevated plus maze reveal a reduced anxiety-like behavior in tau-KO mice, spending more time in the open arms compared to WT (p < 0.05). (D) No differences are seen in the training trial of the contextual fear conditioning test. (E) However, transient but significantly less freezing occurs in the first 3 min of re-exposure to the aversively conditioned context in tau-KO mice (* p < 0.05).
Figure 2
Figure 2
(A) Transcriptomic analysis shows differentially expressed (DE) genes in tau-KO mice compared with WT. After filtering, 69 were downregulated (purple tones), and 105 were upregulated (red tones). Deregulated transcripts are graphically illustrated in the heat map representation at a probability of an adjusted p-value < 0.05. Sample legend in the heat-map plot: white boxes correspond to wild-type animals, and black boxes correspond to tau-KO animals. (B) Enrichment analysis against the Gene Ontology database (p < 0.05) identifies primary altered genes corresponding to (i) synaptic structures, (ii) neuron cytoskeleton and transport, and (iii) extracellular matrix components. The blue dot size beneath each gene cellular component category is proportional to the enrichment FDR score number (between dot and category name) obtained from the differentially expressed genes involved in the functional group.
Figure 3
Figure 3
RT-qPCR of the cerebral cortex of tau-KO and WT mice aged twelve months. Tau-KO mice show increased mRNA levels of col6a4, gabrq, gad, grm5, grip2, map1a, map2, rab8a, tubb3, and wnt16 compared with WT mice. However, the expression of gabrb2, nlgn3, rab37, and utrn is similar in tau-KO and WT mice. As expected, mapt mRNA expression is 0 in tau-KO mice. * p < 0.05, ** p < 0.01, and *** p < 0.001.
Figure 4
Figure 4
(A) A heatmap of the cerebral cortex’s phosphoproteome shows 121 hypophosphorylated and 98 hyperphosphorylated proteins. (B) The altered phosphoproteome in tau-KO mice mainly affects synapses, axons, dendrites, and the neuronal cell body. Sample legend in the heat-map plot: white boxes correspond to wild-type animals, and black boxes correspond to tau-KO animals. The blue dot size beneath each gene cellular component category is proportional to the enrichment FDR score number (between dot and category name) obtained from the differentially expressed proteins involved in the functional group.
Figure 5
Figure 5
Western blotting of the cerebral cortex of tau-KO and WT mice aged twelve months. A significant reduction of MAP1A, β-tubulin III, and GAD1 is found in tau-KO mice. Phosphorylated neurofilament heavy chain (pNFL-H) is increased, and phosphorylated neurofilament light chain (pNFL-L) is decreased in tau-KO mice. * p < 0.05, ** p < 0.01, and *** p < 0.001.
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