The ESCRT-III protein VPS4, but not CHMP4B or CHMP2B, is pathologically increased in familial and sporadic ALS neuronal nuclei

Abstract

Nuclear pore complex injury has recently emerged as an early and significant contributor to familial and sporadic ALS disease pathogenesis. However, the molecular events leading to this pathological phenomenon characterized by the reduction of specific nucleoporins from neuronal nuclear pore complexes remain largely unknown. This is due in part to a lack of knowledge regarding the biological pathways and proteins underlying nuclear pore complex homeostasis specifically in human neurons. We have recently uncovered that aberrant nuclear accumulation of the ESCRT-III protein CHMP7 initiates nuclear pore complex in familial and sporadic ALS neurons. In yeast and non-neuronal mammalian cells, nuclear relocalization of CHMP7 has been shown to recruit the ESCRT-III proteins CHMP4B, CHMP2B, and VPS4 to facilitate nuclear pore complex and nuclear envelope repair and homeostasis. Here, using super resolution structured illumination microscopy, we find that neither CHMP4B nor CHMP2B are increased in ALS neuronal nuclei. In contrast, VPS4 expression is significantly increased in ALS neuronal nuclei prior to the emergence of nuclear pore injury in a CHMP7 dependent manner. However, unlike our prior CHMP7 knockdown studies, impaired VPS4 function does not mitigate alterations to the NPC and the integral transmembrane nucleoporin POM121. Collectively our data suggest that while alterations in VPS4 subcellular localization appear to be coincident with nuclear pore complex injury, therapeutic efforts to mitigate this pathogenic cascade should be targeted towards upstream events such as the nuclear accumulation of CHMP7 as we have previously described.

Keywords: ALS; CHMP2B; CHMP4B; CHMP7; ESCRT-III; FTD; Nuclear pore complex; Nucleoporins; POM121; VPS4.

Fig. 1

VPS4, but not CHMP4B nor CHMP2B, is increased in C9orf72 and sALS iPSN nuclei. a, b Maximum intensity projections from SIM imaging of CHMP4B, CHMP2B, and VPS4 in nuclei isolated from control, C9orf72, and sALS iPSNs at day 18 (a) and 32 (b) of differentiation. Genotype as indicated on left, time point and antibody as indicated on top. c–e Quantification of CHMP4B (c), CHMP2B (d), and VPS4 (e) spots. n = 10 control, 10 C9orf72, and 10 sALS iPSC lines, 50 NeuN + nuclei per line/time point. One-way ANOVA with Tukey’s multiple comparison test was used to calculate statistical significance. **p < 0.01, ****p < 0.0001. Scale bar = 5 μm

Fig. 2

VPS4, but not CHMP4B nor CHMP2B, is increased in C9orf72 and sALS postmortem motor cortex neuronal nuclei. a, b Maximum intensity projections from SIM imaging of CHMP4B, CHMP2B, and VPS4 in nuclei isolated from postmortem control, C9orf72, and sALS motor (a) and occipital (b) cortex tissue. Genotype as indicated on left, brain region and antibody as indicated on top. c–e Quantification of CHMP4B (c), CHMP2B (d), and VPS4 (e) spots. n = 6 control, 6 C9orf72, and 9 sALS cases, 50 NeuN + nuclei per line/brain region. One-way ANOVA with Tukey’s multiple comparison test was used to calculate statistical significance. ****p < 0.0001. Scale bar = 5 μm

Fig. 3

Nuclear expression of VPS4 is dependent upon CHMP7 in C9orf72 and sALS iPSNs. a, c, e Maximum intensity projections from SIM imaging of CHMP4B (a), CHMP2B (c), and VPS4 (e) in nuclei isolated from control, C9orf72, and sALS iPSNs following 2 week exposure to 5 μM scrambled control or CHMP7 ASO. Treatment as indicated on left, genotype and antibody as indicated on top. b, d, f Quantification of CHMP4B (b), CHMP2B (d), and VPS4 (f) spots. n = 5 control, 5 C9orf72, and 5 sALS iPSC lines, 50 NeuN + nuclei per line/treatment. Two-way ANOVA with Tukey’s multiple comparison test was used to calculate statistical significance. **p < 0.01, ****p < 0.0001. Scale bar = 5 μm

Fig. 4

Overexpression of a dominant negative VPS4 increases nuclear POM121 spots but does not restore their distribution in C9orf72 and sALS iPSNs. a, c Maximum intensity projections from SIM imaging of VPS4 (a) and POM121 (c) in nuclei isolated from control, C9orf72, and sALS iPSNs overexpressing GFP or GFP tagged VPS4 variants. Overexpression as indicated on left, genotype and antibody as indicated on top. Arrows (c) indicate uneven distribution of POM121 observed following overexpression of dominant negative VPS4 (VPS4^E228Q) in ALS nuclei. b, d Quantification of VPS4 (b) and POM121 (d) spots. n = 4 control, 4 C9orf72, and 4 sALS iPSC lines, 50 GFP + nuclei per line/overexpression. Two-way ANOVA with Tukey’s multiple comparison test was used to calculate statistical significance. *p < 0.05, **p < 0.01, ****p < 0.0001. Scale bar = 5 μm