The autophagy gene Wdr45/Wipi4 regulates learning and memory function and axonal homeostasis

Abstract

WDR45/WIPI4, encoding a WD40 repeat-containing PtdIns(3)P binding protein, is essential for the basal autophagy pathway. Mutations in WDR45 cause the neurodegenerative disease β-propeller protein-associated neurodegeneration (BPAN), a subtype of NBIA. We generated CNS-specific Wdr45 knockout mice, which exhibit poor motor coordination, greatly impaired learning and memory, and extensive axon swelling with numerous axon spheroids. Autophagic flux is defective and SQSTM1 (sequestosome-1)/p62 and ubiquitin-positive protein aggregates accumulate in neurons and swollen axons. Nes-Wdr45(fl/Y) mice recapitulate some hallmarks of BPAN, including cognitive impairment and defective axonal homeostasis, providing a model for revealing the disease pathogenesis of BPAN and also for investigating the possible role of autophagy in axon maintenance.

Keywords: ACTB, β-actin; AMC, aminomethylcoumarin; Atg, autophagy-related; BPAN, β-propeller protein-associated neurodegeneration; CALB, calbindin; CNS, central nervous system; DCN, deep cerebellar nuclei; Ei24, etoposide-induced gene 24; GFAP, glial fibrillary acid protein; H&E, hematoxylin and eosin; KO, knockout; LC3, microtubule-associated protein 1 light chain 3; LTP, long-term potentiation; MBP, myelin basic protein; NBIA, neurodegeneration with brain iron accumulation; RBFOX3, RNA binding protein, fox-1 homolog (C. elegans) 3; SENDA, static encephalopathy of childhood with neurodegeneration in adulthood; SQSTM1, sequestosome-1; WDR5/WIPI4, WD repeat domain 45; WT, wild type.; Wdr45/Wipi4; autophagy; axon swelling; epg, ectopic P granule; fEPSP, field excitatory postsynaptic potential; learning and memory; neurodegeneration; rpm, rotations per min.

Figure 1.

For figure legend, see page 884.Figure 1. (See previous page) Nes-Wdr45^fl/Y mice show cognitive impairment and LTP attenuation. (A) Scheme for generating Wdr45 conditional knockout mice. (B) Southern blot analysis of genomic DNA from +/+ and F/+ ES clones. After digestion with SpeI, the WT and flox alleles were detected as 12- and 7.7-kb bands with a 5′ probe, respectively. After digestion with NdeI, the WT and flox alleles were detected as 14- and 8.4-kb bands with 3′ probe, respectively. (C) Wdr45 mRNA level in different tissues of WT and Nes-Wdr45^fl/Y mice at 13 mo. Results are representative of at least 3 experiments. Compared to WT mice, the Wdr45 mRNA level is slightly decreased in the heart of Nes-Wdr45^fl/Ymice, probably due to nonspecific expression of Nes-Cre (http://cre.jax.org/Nes/Nes-CreNano.html). (D) Rotarod performance of Nes-Wdr45^fl/Y and Nes-Wdr45^fl/+ mice (7 to 8 mo) at rolling speed of 20 rpm. Mean±SEM of 11 mice is shown. (E) Rotarod performance of Nes-Wdr45^fl/Y and Nes-Wdr45^fl/+ mice at rolling speeds of 5, 10, 20, and 40 rpm. *, P < 0.05; **, P < 0.01. (F–H) In the Morris water maze test, Nes-Wdr45^fl/Ymice show decreased learning and memory ability in the learning and probe test, respectively. The swimming speeds show no obvious difference between Nes-Wdr45^+/Y and Nes-Wdr45^fl/Y mice. (I) Nes-Wdr45^fl/Y mice display a reduced percentage of correct alternations, compared with Nes-Wdr45^+/Y mice in a Y-maze test. No obvious difference in the total number of alternations was detected between WT and Nes-Wdr45^fl/Y mice. (J) In a contextual fear conditioning test, the percentage of the test time taken up by a freezing response is significantly lower in Nes-Wdr45^fl/Y mice. In a cued test, the freezing time is slightly reduced in Nes-Wdr45^fl/Ymice. Mean±SEM of 11 mice (11 to 13 mo) is shown (C–H). (K and L) Impaired LTP in Nes-Wdr45^fl/Y mice. The upper part in (K) shows averaged fEPSPs comparing baseline and the last 5 min in Nes-Wdr45^+/Y and Nes-Wdr45^fl/Y mice. LTP was not induced in 7 out of 11 hippocampal slices from 3 Nes-Wdr45^fl/Y mice, while LTP was normally induced in all of 5 slices from 2 WT mice. Only slices with LTP induction were analyzed here. Averaged responses from the last 5 min in control and Nes-Wdr45^fl/Y mice (L).

Figure 2.

Nes-Wdr45^fl/Ymice show axon swelling. (A) The number of Purkinje cells was quantified and divided by the total length of the lobules III, IV and V. (B) The number of CA1 hippocampal pyramidal cells of Nes-Wdr45^+/Y and Nes-Wdr45^fl/Y mice at 13 mo was quantified and divided by the length of the layer. Mean ± SEM of 3 mice is shown (A and B). (C–E) Compared to control mice, the GFAP signals (red) in sections of hippocampus (C) and cortex (D) are stronger in Nes-Wdr45^fl/Y mice. Quantification data are shown as mean±SEM of 3 mice (E). Bar: 50µm. (F) H&E staining reveals the presence of eosinophilic spheroids (arrow) in the cortex. (G) H&E staining shows vacuolated structures (arrows) in thalamus. (H and I) H&E staining reveals the presence of eosinophilic spheroids (arrows) in the DCN of Nes-Wdr45^fl/Y mice at 13 mo. Bar: 20 µm (F–I). (J and K) Costaining of CALB (green) and MBP (red) shows that Nes-Wdr45^fl/Y mice at 13 mo exhibit dilated CALB-positive bulbs (arrows) in the DCN region. Bar: 10 µm. (L to Q) EM pictures of DCN in Nes-Wdr45^+/Y (L and N) and Nes-Wdr45^fl/Y (M, O, P and Q) mice at 13 mo. Arrows in (M) indicate swollen axons. Swollen mitochondria accumulate in these axons (arrows in O).The arrow in (P) indicates a demyelinated axon. Degenerated axons were also occasionally detected (Q). Bar: 2 µm (L and M), 500 nm (N–Q). (R and S) EM pictures of medulla in Nes-Wdr45^+/Y (R) and Nes-Wdr45^fl/Y (S) mice at 13 mo. Arrows in (S) indicate degenerated axons. Bar: 500 nm. (T–X) H&E staining of the DCN from Wdr45 mutant mice and controls at 6 wk and 4 mo. Arrows indicate eosinophilic spheroids. Quantification data are shown as mean±SEM of 3 mice (X). Bar: 20µm.

Figure 3.

Nes-Wdr45^fl/Y mice exhibit defective autophagic flux. (A–D) Coimmunostaining with anti-SQSTM1 (red) and anti-ubiquitin (green) antibodies in DCN (A), thalamus (B), hippocampus (C) and caudate nucleus (D) of Nes-Wdr45^fl/Y mice at 13 mo. (E) No increase in the Sqstm1 mRNA level is detected in Nes-Wdr45^fl/Y mice. (F) Compared to control littermates, proteasome activity is not altered in brain extracts of Nes-Wdr45^fl/Y mice at 13 mo. Proteasome activity was measured using AMC-linked substrate peptides. A: Ac-Gly-Pro-Leu-Asp-AMC; B: Suc-Leu-Leu-Val-Tyr-AMC; C: Ac-Arg-Leu-Arg-AMC; D: Boc-Leu-Arg-Arg-AMC. Results are representative of 2 experiments. (G and H) Anti-ATG9 staining shows that ATG9 puncta accumulate in the DCN of Nes-Wdr45^fl/Y mice at 13 months. Quantification data are shown as mean±SEM of 3 mice (1 unit = 10⁴ μm²) (H). (I and J) Anti-RBFOX3 (green) and anti-SQSTM1 (red) costaining shows that SQSTM1 aggregates are located in pyramidal neurons in the cortex (I) and hippocampus (J) of Nes-Wdr45^fl/Y mice at 13 mo. (K) Anti-ubiquitin (green) and anti-CALB (red) costaining in the DCN of Nes-Wdr45^fl/Y mice at 13 mo (arrows indicate dilated axonal terminals). Bar: 10 µm (A–D, G, and I–K). (L and M) Immunoblotting assays show that levels of LC3-I, LC3-II and SQSTM1 are increased in Wdr45-deficient neurons. Data are relative to ACTB level and representative of 3 independent experiments. (N and O) Immunoblotting assays show that levels of LC3-I and LC3-II are comparable between WT and Wdr45-deficient neurons after 6 h treatment with 20 μM bafilomycin A₁. Data are relative to ACTB level and representative of 3 independent experiments.

Figure 4.

Axon swelling and autophagy defects in Nes-Wdr45^fl/fl and Nes-Wdr45^fl/+ female mice. (A) H&E staining of cerebellar sections shows accumulation of eosinophilic spheroids (arrows) in both Nes-Wdr45^fl/+ and Nes-Wdr45^fl/fl female mice at 13 mo. Bar: 20 µm. (B) The Wdr45 mRNA level in the brain of WT, Nes-Wdr45^fl/+ mice and Nes-Wdr45^fl/fl mice. Results are representative of at least 3 experiments. (C and D) Costaining of CALB (green) and MBP (red) in DCN of Nes-Wdr45^fl/+ and Nes-Wdr45^fl/fl female mice at 13 mo. Quantification data are shown as mean±SEM of 3 mice (1 unit = 10⁴ μm²) (D). (E and F) SQSTM1 (red) and Ubiquitin (green) costaining in DCN of Nes-Wdr45^fl/+ and Nes-Wdr45^fl/fl female mice at 13 mo. Quantification data are shown as mean ± SEM of 3 mice (1 unit = 10⁴ μm²) (F). Bar: 10 µm (C and E).