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1.
Figure 1

Figure 1. NLK interacts with ATXN1. From: Polyglutamine disease toxicity is regulated by Nemo-like kinase in spinocerebellar ataxia type 1.

(A) Co-AP assay shows that ATXN1(30Q) interacts with both wild-type (WT) and kinase activity mutant (KN) NLK. KN is a lysine to methionine substitution at residue 155 (K155M). Co-AP assay was performed in HEK293T cells. Top panel shows the presence of Flag-NLK after affinity purification on Glutathione-Sepharose 4B beads, demonstrating the ATXN1-NLK interaction. GST-empty vector (−) was used as a control. GFP expression was used as a transfection and loading control.
(B) The N-terminal region (amino acids 1–575) of ATXN1 interacted most strongly with NLK. Schematic representation of the ATXN1 constructs is shown at the bottom.

Hyoungseok Ju, et al. J Neurosci. ;33(22):9328-9336.
2.
Figure 7

Figure 7. NLK relieves repression by the ATXN1/CIC transcription complex in a kinase activity-dependent manner. From: Polyglutamine disease toxicity is regulated by Nemo-like kinase in spinocerebellar ataxia type 1.

HEK293T cells were transfected with the pGL3-luciferase reporter construct containing six copies of CIC binding sites, the pRL-TK control vector, and expression plasmids as indicated. The luciferase data are expressed as mean percentage of luciferase activity relative to the reporter alone. All experiments were performed in triplicate. Error bars in the graph represent standard error of the mean (*P<0.001, ANOVA, Scheffe’s test).

Hyoungseok Ju, et al. J Neurosci. ;33(22):9328-9336.
3.
Figure 2

Figure 2. NLK phosphorylates ATXN1. From: Polyglutamine disease toxicity is regulated by Nemo-like kinase in spinocerebellar ataxia type 1.

(A) NLK increases phosphorylation of ATXN1 at S/T-P sites. (B) The S239 residue in ATXN1 is an NLK phosphorylation site. HEK293T cells were transfected with N-terminal-tagged myc-ATXN1(30Q) and either wild-type (WT), kinase-inactive mutant (KN) Nlk, or a control plasmid (−). The phosphorylation of full-length (arrows) or an N-terminal fragment (about 55kDa in size, arrowheads) of wild-type ATXN1 is strongly increased by wild-type NLK, but not by the KN mutant. The asterisk in (A) marks immunoglobin heavy chain used for immnuoprecipitation (IP). S239A is a serine to alanine substitution at residue 239. Normalized levels of MPM2 signal from the full-length ATXN1 are shown (A, right panel). Mean relative levels (control plasmid (−)=100%) and standard error are shown (n=2, *P<0.05, t-test).

Hyoungseok Ju, et al. J Neurosci. ;33(22):9328-9336.
4.
Figure 6

Figure 6. Expression levels and ATXN1-associated protein complex formation in Atxn1154Q/+; Nlk+/− mice. From: Polyglutamine disease toxicity is regulated by Nemo-like kinase in spinocerebellar ataxia type 1.

(A) Nlk heterozygosity doesn’t affect the expression levels of components in Atxn1 native complexes in cerebellar extracts of SCA1 knock-in mice. Expression of Capicua (CIC-L and CIC-S, long and short isoforms of CIC, respectively), wild-type (Atxn1[2Q]) and polyglutamine-expanded mutant (Atxn1[154Q]) Atxn1, and Rbm17 remained the same in cerebellar extracts from 20-week-old Atxn1154Q/+ or Atxn1154Q/+; Nlk+/− mice. Nlk and Gapdh were used as controls.
(B) The level of NLK incorporation in toxic large complexes is low in Atxn1154Q/+; Nlk+/− mice compared to Atxn1154Q/+ mice. Elution profiles of CIC, Atxn1 (both wild-type and mutant Atxn1), Rbm17, and Nlk proteins in Atxn1154Q/+ or Atxn1154Q/+; Nlk+/− mouse cerebellum using gel-filtration chromatography. Representative westerns of 1.0ml gel-filtration fractions of Atxn1154Q/+ or Atxn1154Q/+; Nlk+/− mouse cerebellar extracts analyzed for CIC, Atxn1, Rbm17, and Nlk. The column void volume (Vo), gel-filtration standards thyroglobulin (669kDa) and ADH (150kDa), and elution volume (ml) of each collected fraction are indicated. Ex, extract.

Hyoungseok Ju, et al. J Neurosci. ;33(22):9328-9336.
5.
Figure 4

Figure 4. Generation and evaluation of Nlk gene trapped mice. From: Polyglutamine disease toxicity is regulated by Nemo-like kinase in spinocerebellar ataxia type 1.

(A) Schematic representation of the gene trapped Nlk locus. The Nlk locus is disrupted by insertion of the gene trap within the first or the second intron. Exons are depicted as numbered boxes. Primers used for genotyping are shown as lettered arrows (P1, P2, and P3).
(B) PCR genotyping of tail tip genomic DNA using the primer set shown in (A). Primers ‘P1’ and ‘P2’ create a product in the absence of the gene trap while primers ‘P1’ and ‘P3’ create a product in the presence of the gene trap.
(C) Western blot analysis of cerebellar extracts from wild-type (Nlk+/+), heterozygous (NlkRRJ297/+ and NlkXN619/+), and compound heterozygous mutant (NlkRRJ297/XN619) mice. NLK expression level relative to Gapdh is shown (%).
(D) Strong LacZ expression of the Nlk gene trap is observed within the Purkinje cell layer from 6-week-old NlkXN619/+ mouse cerebellum. MCL, molecular cell layer. PCL, Purkinje cell layer. GCL, granular cell layer.

Hyoungseok Ju, et al. J Neurosci. ;33(22):9328-9336.
6.
Figure 5

Figure 5. Deletion of one Nlk allele rescues SCA1 cerebellar phenotypes in mice. From: Polyglutamine disease toxicity is regulated by Nemo-like kinase in spinocerebellar ataxia type 1.

(A) In the dowel rod walking test, Atxn1154Q/+ mice showed increased latency to reach the sides of the dowel compared to wild-type and Nlk+/− littermates (left). They also walked off fewer times in the 120-second interval (right). In contrast, Atxn1154Q/+; Nlk+/− mice exhibited marked reduction in the time for the first side touch and increased number of side touches compared to Atxn1154Q/+ littermates. Genotypes are color-coded as indicated. Error bars in graphs represent standard error of the mean.
(B–D) Cerebellar neuropathology is partially rescued in Purkinje cells of Atxn1154Q/+ mice lacking one allele of Nlk. (B) Representative confocal images showing Purkinje cell morphology stained with anti-calbindin antibody from age-matched littermates. (C) Quantitative analysis of calbindin immunofluorescence is an indirect measure of cerebellar Purkinje cell soma and dendritic integrity. Mean fluorescence intensity of optical rectangular subsections from the same folia of 38- to 39-week-old animals was plotted as the distance from the perikaryon center. Nlk heterozygosity partially rescues the Atxn1154Q/+ loss of dendritic arborization phenotype. Error bars in graphs represent standard error of the mean. (D) Quantification of the cerebellar molecular layer thickness at the primary fissure from 38- to 39-week-old mice (shown by arrow in B). Atxn1154Q/+ mice (four sections per animal; n=3, *P=0.015, ANOVA) showed a significantly decreased thickness compared to wild-type littermates. Loss of one Nlk allele (four sections per animal; n=2, *P=0.004, ANOVA) rescued defects of the molecular layer thickness in Atxn1154Q/+ mice. Error bars represent standard error of the mean.

Hyoungseok Ju, et al. J Neurosci. ;33(22):9328-9336.
7.
Figure 3

Figure 3. NLK modulates polyglutamine-expanded ATXN1 disease phenotypes in a Drosophila model of SCA1. From: Polyglutamine disease toxicity is regulated by Nemo-like kinase in spinocerebellar ataxia type 1.

(A–H) Loss of one nmo allele suppressed ATXN1(82Q)-mediated retinal phenotypes. Both light microscopy (A–D) and scanning electron microscopy (E–H) of adult Drosophila eyes are shown and magnified images are on the bottom of each panel. Two independent mutant alleles (adk1 and adk2) of nmo showed the same results. Light microscopic analysis clearly shows the depigmentation and necrotic death (marked by arrows) phenotypes of the external eyes. Scanning electron microscopy shows the retinal degeneration phenotypes of SCA1 with better resolution. Flies were raised at 27.5°C and genotypes are: (A,E) GMR-Gal4/+; UAS-EGFP/+, (B,F) UAS-ATXN182Q/+; GMR-Gal4/+, (C,G) UAS-ATXN182Q/+; GMR-Gal4/+; nmoadk1/+, and (D,H) UAS-ATXN182Q/+; GMR-Gal4/+; nmoadk2/+.
(I–L) Coexpression of wild-type NLK (NLK-WT) but not kinase-inactive mutant NLK (NLK-KN) worsened retinal degenerative phenotypes induced by polyglutamine-expanded mutant ATXN1(82Q). Flies were raised at 27.5°C and genotypes are: (I) GMR-Gal4/UAS-EGFP; UAS-EGFP/+, (J) UAS-ATXN182Q/+; GMR-Gal4/UAS-EGFP, (K) UAS-ATXN182Q/+; GMR-Gal4/UAS-NLKWT, and (L) UAS-ATXN182Q/+; GMR-Gal4/UAS-NLKKN.
(M) Quantification of the genetic interaction study between loss-of-nmo-function and SCA1 (A–D). One hundred percent of SCA1 flies showed a yellow eye color (thus, 0% showed a red color) and a heterozygous loss of one nmo allele strongly suppressed SCA1 phenotypes, resulting in a red eye color. For this genetic interaction study, flies were raised at 27.5°C and more than 100 adult eyes per genotype were examined at day 2 after eclosion. Four independent experiments were performed (P<0.005, t-test).
(N) Quantification of the genetic interaction study between gain-of-NLK-function and SCA1 (I–L). SCA1 flies show patches of necrotic death on the external surface of the eyes and coexpression of the wild-type NLK strongly enhances this phenotype. For this genetic interaction study, flies were raised at 27.5°C, collected at day 1 and kept separately at 27.5°C. At day 5 after eclosion, eyes with more than 50% necrotic black spots were scored. More than 100 adult eyes per genotype were examined and five independent experiments were performed (P<0.005, t-test).

Hyoungseok Ju, et al. J Neurosci. ;33(22):9328-9336.

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