Premature lethality, hyperactivity, and aberrant phosphorylation in transgenic mice expressing a constitutively active form of Fyn

Abstract

The kinase Fyn, the microtubule-associated protein tau and the peptide amyloid-β (Aβ) constitute a toxic triad in Alzheimer's disease (AD). Tau's subcellular localization is mainly regulated by phosphorylation whereas Fyn's localization is dictated by palmitoylation targeting it to the plasma membrane in a reversible manner. We have previously shown that tau is required for Fyn to be targeted to the dendritic spine. We had also shown that a truncated form of tau (Δtau) that accumulates in the cell soma is capable of trapping Fyn and preventing it from entering the spine. Here we determined that palmitoylation is required for Fyn's membrane and spine localization. We further evaluated the functional consequences of neuronal over-expression of the constitutively active Y531F mutant form of Fyn (FynCA) in transgenic mice. We found that the FynCA transgenic mice displayed a reduced weight, a massively reduced lifespan and a high level of hyperactivity. The lifespan of the FynCA mice was only slightly extended by crossing them with Δtau transgenic mice, possibly reflecting differences in expression patterns of the transgenes and high levels of transgenic FynCA compared to endogenous Fyn. Analysis of synaptosomes revealed that FynCA accumulated at high levels in the spine, resulting in increased levels of the NMDA receptor subunit NR2b phosphorylated at residue Y1472. Tau was strongly phosphorylated at the AT8 epitope S202/T205 as shown by Western blot and immunohistochemistry indicating that an increased tyrosine kinase activity of Fyn has down-stream consequences for serine/threonine-directed phosphorylation.

Keywords: Alzheimer; Fyn kinase; dendrite; palmitoylation; phosphorylation; spine; tau.

Figure 1

Structure of Fyn, its membrane localization, and constructs used for expression of Fyn, tau and ΔTau. (A) Fyn is a 59 kDa protein that contains an amino-terminal Src-homology (SH) region with acylation sites, a unique domain, an SH3 domain (with which it interacts with PXXP motifs), an SH2 domain (with which it interacts with phospho-tyrosine), an SH1/kinase domain, and a carboxy-terminal regulatory tail. The most amino-terminal glycine residue can be myristoylated, which occurs co-translationally on free ribosomes. In addition, Fyn's amino-terminal cysteines C3 and C6 can be palmitoylated, and it is this acylation reaction that anchors the kinase to the plasma membrane. (B) Expression constructs for human Fyn were either wild-type, C3S/C6S to prevent palmitoylation, or Y531F to generate a constitutively active form of Fyn (FynCA). Tau was expressed either as full-length [with the longest human tau isoform, hTau40, containing four microtubule-binding domains (MBDs) indicated in black] or a truncated form (Δtau) lacking the MBDs. For detection of tau and Fyn, myc, GFP and V5 were used as tags as indicated.

Figure 2

Palmitoylation and hence, membrane association, is required for the localization of Fyn into dendritic spines. Transfection of Fyn expression constructs in HEK293 cells (A,B) and primary hippocampal cultures (C–H) reveals that wild-type Fyn associates with the plasma membrane in HEK293 cells (A), whereas mutating Fyn's two cysteines (C3/C6) that are acetylated in a palmitoylation reaction to cysteines causes Fyn's accumulation intracellularly in endosomes (B). In the neuronal cultures, wild-type Fyn is localized to spines (C–E) whereas Fyn(C3S/C6S)-GFP is excluded from entering the spines (F–H). RFP (D,G) was used to visualize all neuronal structures including the spines. Scale bar: 10 μm (A,B); 20 μm (C–H).

Figure 3

Subcellular localization of Fyn and Δtau. Transfection of tau knock-out primary neuronal cultures with a full-length tau construct equipped with a V5 tag (Tau-V5) (A) together with RFP (B) shows distribution of full-length tau throughout the neuron, but spines are excluded. (Merged image: C). Transfection of Δtau-V5 (D) together with RFP (E) reveals that Δtau is dramatically reduced from the dendritic compartment, and that levels are below detection past the secondary branch-point (as indicated by the arrows) (Merged image: F). When Fyn-EGFP (G) was over-expressed together with Δtau-V5 (H) and RFP (I) in wild-type primary neurons, different from Δtau74 mice in which transgenic over-expression of Δtau prevents endogenous Fyn from entering the spines, transfecting Δtau into primary neurons does not prevent transfected Fyn from entering the spine, possibly reflecting differences in expression levels (Merged image: K). Scale bar: 100 μm (A–K).

Figure 4

FynCA mice are characterized by early mortality, reduced weight, and hyperactivity. Transgene-positive offspring derived from FynCA founders #14, 35, and 55 is characterized by early lethality (A). Crossing FynCA mice with Δtau mice results in a slight extension of the lifespan of the FynCA mice. The transgene-positive offspring derived from these lines tends to be smaller as shown for #55 offspring (B), and to show less weight gain than the non-transgenic littermates such that by 2–3 weeks of age they weigh only half of their non-transgenic littermates (Student's t-test, P < 0.0001) (C). Tracking of four FynCA transgenic mice (top) and four non-transgenic littermates (bottom) at 18 days of age. The FynCA mice run in circles and keep this hyperactivity up for hours unless being put back to their litter (D).

Figure 5

Fyn expression in FynCA mice and tau phosphorylation. A myc-specific antibody was used to reveal Fyn expression in FynCA-transgenic mice (A), using non-transgenic littermates as controls (B). Phosphorylation of endogenous tau was revealed using the Ser202/Thr205-specific antibody AT8, analyzing FynCA-transgenic mice (C) and non-transgenic littermate controls (D).

Figure 6

Western blot of total brain extract and synaptosomal fractions. We compared wild-type mice (1,2), FynCA mice (3,4), and FynCA mice crossed onto a Δtau background (5,6). (A) Total extracts and synaptosomal PSD (SDS) fractions (B) were analyzed with antibodies for GAPDH, actin, Fyn (short and long exposure), Myc, PSD-95, NR2b, NR2b phosphorylated at Y1472, total tau, human tau (HT7) detecting Δtau, and phospho-tau (AT8, 12E8).