Clinicopathological analysis of NEK1 variants in amyotrophic lateral sclerosis

Abstract

Many genes have been linked to amyotrophic lateral sclerosis (ALS), including never in mitosis A (NIMA)-related kinase 1 (NEK1), a serine/threonine kinase that plays a key role in several cellular functions, such as DNA damage response and cell cycle regulation. Whole-exome sequencing studies have shown that NEK1 mutations are associated with an increased risk for ALS, where a significant enrichment of NEK1 loss-of-function (LOF) variants were found in individuals with ALS compared to controls. In particular, the p.Arg261His missense variant was associated with significantly increased disease susceptibility. This case series aims to understand the neuropathological phenotypes resulting from NEK1 mutations in ALS. We examined a cohort of three Scottish patients with a mutation in the NEK1 gene and evaluated the distribution and cellular expression of NEK1, as well as the abundance of phosphorylated TDP-43 (pTDP-43) aggregates, in the motor cortex compared to age- and sex-matched control tissue. We show pathological, cytoplasmic TDP-43 aggregates in all three NEK1-ALS cases. NEK1 protein staining revealed no immunoreactivity in two of the NEK1-ALS cases, indicating a LOF and corresponding to a reduction in NEK1 mRNA as detected by in situ hybridisation. However, the p.Arg261His missense mutation resulted in an increase in NEK1 mRNA molecules and abundant NEK1-positive cytoplasmic aggregates, with the same morphologic appearance, and within the same cells as co-occurring TDP-43 aggregates. Here we show the first neuropathological assessment of a series of ALS cases carrying mutations in the NEK1 gene. Specifically, we show that TDP-43 pathology is present in these cases and that potential NEK1 LOF can either be mediated through loss of NEK1 translation or through aggregation of NEK1 protein as in the case with p.Arg261His mutation, a potential novel pathological feature of NEK1-ALS.

Keywords: ALS; NEK1; genetics; neuropathology; post‐mortem.

FIGURE 1

Visualisation of NEK1 mutations in the NEK1 transcript sequence and NEK1 protein structure. (A) Sequences flanking the junction between protein‐coding exon 1 and exon 2 of NEK1. The c.214 + 1G > A mutation is indicated in red, and snRNP recognition sites are underlined. (B) Domains of the NEK1 protein with the location of p.Arg261His shown in red. NLS, nuclear localisation signal; NES, nuclear export signal. (C) Crystal structure of NEK1 visualised in AlphaFold. Predicted hydrogen bonding of Arg261 to neighbouring Asp103 and Asp107 is shown in yellow. (D) Sequences flanking the junction between protein‐coding exon 15 and exon 16 of NEK1. The c.1911 + 1 > TATA insertion mutation is indicated in red, and snRNP recognition sites are underlined.

FIGURE 2

Histological staining reveals distinct NEK1 mutation associated pathology. (A) upper panel ( blue outline ) Immunohistochemistry staining of motor cortex demonstrating mild pTDP‐43 aggregation in Patients 1 and 3 but extensive aggregation in Patient 2 (top row); absent NEK1 protein in Patients 1 and 3 and NEK1 aggregates in Patient 2 (second row). Blue arrowhead indicates absence of staining; brown arrowhead indicates increased immunostaining (protein). (A) lower panel ( red outline ). BaseScope™ in situ hybridisation in motor cortex reveals absent NEK1 mRNA transcripts in Patients 1 and 3, consistent with LOF through loss of expression, and increased NEK1 mRNA transcripts in Patient 2 (red box), consistent with compensatory increased expression secondary to NEK1 protein aggregation seen in the upper panel. Red arrowhead indicates increased mRNA transcripts (i.e., each red dot is a single mRNA transcript of NEK1). (B) Quantification of immunohistochemical staining using digital burden scoring employing a superpixel analysis and detection classifier in QuPath. Graphs demonstrate superpixel count based on detection intensity based on a modified Allred score analysis. (C) Quantification of mRNA burden as mRNA counts per cell across 10 cells per randomly generated region of interest.

FIGURE 3

Dual chromogenic staining reveals cells containing both TDP‐43 and NEK1 aggregates. Photomicrographs of motor cortex from Patient 2 stained with TDP‐43^APT (red chromogen) and NEK1 (brown chromogen) demonstrating examples of: (A) mutually exclusive aggregation events where red arrowheads indicate cells with only TDP‐43 aggregation and orange arrowheads indicate cells with only NEK1 aggregation. (B) Aggregation of TDP‐43 and NEK1 in the same cells where red arrowheads indicate TDP‐43 positive staining and orange arrowheads indicte NEK1 positive staining. (C) Co‐localisation of TDP‐43 and NEK1, where white arrowheads indicate dual (red and brown) positive staining and orange arrowheads indicate NEK1 (brown)‐only staining for comparison. (D) Staining in neuropil threads, which can be dual positive, solely NEK1 positive, or solely TDP‐43 positve.