Figure 5

Figure 5

Lamin B2 localization shifts from the nuclear envelope to the nucleoplasm in Annexin A11a homozygous larvae at 48 hpf and post-mortem tissue from patients with Annexin A11 mutations. (A) Top: Endogenous Lamin B2 (green) in wild-type (WT) Annexin A11a larvae is localized to the nuclear envelope (and in merge). Large nucleoli are indicated by white arrows (DAPI). Bottom: In Annexin A11a homozygous knockout larvae a pool of Lamin B2 shifts to the nucleoplasm (green and DAPI merge) with a loss of large nucleoli (DAPI). (n = 3) Scale bar = 20 uM. (B) Brightfield Lamin B2 staining of individual anterior horn neurons revealing preserved nuclear membrane staining in the control (CTL) and sporadic ALS (SALS) cases, but diffuse nuclear staining in the G38R and D40G Annexin A11 mutation cases. Scale bar = 50 µm. (C) Violin plot quantifying the mean ratio of the number of neurons containing abnormal nucleoplasmic LMNB2 compared to total number of neuronal nuclei per field (×40 magnification). This included controls (n = 9), G38R, D40G, R191Q (n = 4) non-disease polymorphism, with a minor allele frequency (MAF) of 4.7% in Europeans, R235Q, SALS (n = 5), FUS positive cases (n = 6) and cases with the C9ORF72 GGGGCC expansion (n = 2). Only the G38R and D40G cases showed a significant association with nucleoplasmic LMNB2 (G38R versus Controls, ****P ≤ 0.0001; D40G versus Controls, ***P ≤ 0.005; Kruskal-Wallis test for multiple comparisons). (D) Western blot Lamin B2 expression of post-mortem motor cortex tissue from an Annexin A11 D40G patient (n = 3) and control cases (n = 7) compared to GAPDH expression. (E) Quantification of Lamin B2 expression in D40G tissue is ∼1.8-fold higher compared to controls (****P ≤ 0.0001, unpaired t-test). dpf = days post-fertilization; Hom = homozygous.