Nuclear lamina invaginations are not a pathological feature of C9orf72 ALS/FTD

Abstract

The most common genetic cause of familial and sporadic amyotrophic lateral sclerosis (ALS) is a GGGGCC hexanucleotide repeat expansion (HRE) in the C9orf72 gene. While direct molecular hallmarks of the C9orf72 HRE (repeat RNA foci, dipeptide repeat protein pathology) are well characterized, the mechanisms by which the C9orf72 HRE causes ALS and the related neurodegenerative disease frontotemporal dementia (FTD) remain poorly understood. Recently, alterations to the nuclear pore complex and nucleocytoplasmic transport have been accepted as a prominent pathomechanism underlying C9orf72 ALS/FTD. However, global disruptions to nuclear morphology and the nuclear lamina itself remain controversial. Here, we use a large number of induced pluripotent stem cell derived spinal neurons and postmortem human motor cortex sections to thoroughly examine nuclear morphology and nuclear lamina disruptions with light microscopy. In contrast to previous studies in artificial overexpression model systems, endogenous levels of the C9orf72 HRE do not increase the frequency of nuclear lamina invaginations. In addition, the C9orf72 HRE has no impact on overall nuclear shape and size. Notably, the frequency of nuclear Lamin B1 invaginations increases with cellular aging, independent of the C9orf72 HRE. Together, our data suggest that nuclear morphology is unaltered in C9orf72 ALS/FTD.

Keywords: Amyotrophic Lateral Sclerosis; C9orf72; Frontotemporal Dementia; Lamin B1; Nuclear envelope; Nuclear morphology; Nuclear pore complex; Nucleocytoplasmic transport.

Fig. 1

Cellular age increases the frequency of nuclear lamina disruptions independent of the presence of the C9orf72 HRE. a Immunostaining and confocal imaging for Lamin B1 in iPSNs at day 32 of differentiation. Genotype as indicated on left, antibody and iPSC passage number as indicated on top. b, c Quantification of nuclear sphericity (b) and nuclear volume (c). n = 8 control and 8 C9orf72 iPSC lines, at least 100 Map2 + neurons per line and iPSC passage. Two-way ANOVA Tukey’s multiple comparison test was used to calculate statistical significance. *p < 0.05, **p < 0.01. d Quantification of number of Lamin B1 invaginations per nucleus. n = 8 control and 8 C9orf72 iPSC lines, at least 100 Map2 + neurons per line and iPSC passage. Chi-square test was used to calculate statistical significance. ****p < 0.0001. Scale bar = 10 μm

Fig. 2

Isolation of iPSN nuclei does not impact nuclear morphology. a Maximum intensity projections from SIM imaging of Lamin B1 in nuclei isolated from control and C9orf72 iPSNs at day 32 of differentiation from passage 27 iPSCs. Genotype as indicated on top. b, c Quantification of nuclear sphericity (b) and nuclear volume (c). n = 10 control and 10 C9orf72 iPSC lines, at least 50 NeuN + nuclei per line. Student’s t-test was used to calculate statistical significance. d Quantification of number of Lamin B1 invaginations per nucleus. n = 10 control and 10 C9orf72 iPSC lines, at least 50 NeuN + nuclei per line. Chi-square test was used to calculate statistical significance. Scale bar = 5 μm

Fig. 3

Nuclear lamina invaginations are not a pathological feature of C9orf72 ALS/FTD neurons in postmortem patient motor cortex. a Immunostaining for Lamin B1 in postmortem paraffin embedded motor cortex sections. Genotype as indicated on left, antibody as indicated on top. b Quantification of nuclear circularity. n = 8 control and 8 C9orf72 cases, at least 100 Map2 + neurons per case. Student’s t-test was used to calculate statistical significance. c Quantification of number of Lamin B1 invaginations per nucleus. n = 8 control and 8 C9orf72 cases, at least 100 Map2 + neurons per case. Chi-square test was used to calculate statistical significance. Scale bar = 10 μm

Fig. 4

Nuclear lamina invaginations are not a pathological feature in nuclei isolated from C9orf72 ALS/FTD motor cortex. a Maximum intensity projections from confocal imaging of Lamin B1 in nuclei isolated from control and C9orf72 postmortem motor cortex tissue. Genotype as indicated on top. b, c Quantification of nuclear sphericity (b) and nuclear volume (c). n = 4 control and 4 C9orf72 cases, at least 50 NeuN + nuclei per case. Student’s t-test was used to calculate statistical significance. d Quantification of number of Lamin B1 invaginations per nucleus. n = 4 control and 4 C9orf72 iPSC lines, at least 50 NeuN + nuclei per line. Chi-square test was used to calculate statistical significance. Scale bar = 5 μm