Fig 1.
Frequency of CLN2 disease-associated mutations.
(A) Allele frequency of CLN2 disease mutations demonstrates a predominance of nonsense (29%) and missense (22%) mutations. (B) The most common CLN2 disease mutations consists of either the intronic transversion c.509-1G>C that results in altered transcript splicing or the exonic transition c.622C>T that results in the p.R208X nonsense mutation.
Fig 2.
Genetic design of the CLN2 disease mouse model, Cln2R207X/R207X.
(A) A Cln2 targeting vector carrying the c.619C>T mutation (Line 2), also known as p.R207X mutation, was electroporated into C57BL/6x129S6/SvEv embryonic stem cells. Homologous recombination between wildtype Cln2 (Line 1) and the targeting vector (Line 2) resulted in the generation of a Cln2Neo;R207X allele (Line 3). Removal of the Neo cassette via FRT-flippase mediated excision (FRT sites represented by grey arrows flanking the Neo cassette) generated the Cln2R207X allele (Line 4) resulting in Cln2+/R207X mice. Cln2+/R207X mice were bred to create Cln2R207X/R207X mice. (B) Cln2 sequencing results confirm the retention of the mutation following breeding.
Fig 3.
Ubiquitously reduced Cln2 transcript abundance in Cln2R207X/R207X mice.
Quantitative real-time PCR was used to measure endogenous levels of the Cln2 transcript from five different tissues obtained from 1-month-old Cln2+/+ (n = 3) and Cln2R207X/R207X (n = 3) mice. Three technical replicates were performed using the three biological samples obtained from Cln2+/+ and Cln2R207X/R207X mice. Cln2 transcript levels were normalized to Gapdh expression. Columns and bars represent mean ± SEM. Statistical significance was determined using an unpaired t-test (**p < 0.01 and **** p < 0.0001).
Fig 4.
Decreased TPP1 activity in various Cln2R207X/R207X mouse tissues.
Fluorogenic TPP1 enzyme activity assays were used to measure endogenous levels of TPP1 activity from five different tissues obtained from 1-month-old Cln2+/+ (n = 3) and Cln2R207X/R207X (n = 3) mice. Four technical replicates were performed using the three biological samples obtained from Cln2+/+ and Cln2R207X/R207X mice. Cln2R207X/R207X TPP1 activity was normalized to Cln2+/+ TPP1 activity. Columns and bars represent mean ± SEM. Statistical significance was determined using an unpaired t-test (** p < 0.01, ***p < 0.001, and **** p < 0.0001).
Fig 5.
Significantly reduced lifespan in Cln2R207X/R207X mice.
Cln2+/+ (n = 14) and Cln2R207X/R207X (n = 26) mice were plotted on a survival curve and tracked overtime. Cln2R207X/R207X mice died between 3 and 6 months of age.
Fig 6.
Altered motor skills in 3-month-old Cln2R207X/R207X mice.
A panel of behavioral tests including modified vertical pole (A and B), rotarod (C), and modified hanging wire (D) were used to assess motor skills in 1 and 3-month-old Cln2+/+ (n = 14) and Cln2R207X/R207X (n = 20) mice. The modified hanging wire was only evaluated at 3 months of age. (A) Climb down time as assessed by the vertical pole showed no differences at 1 month of age between Cln2+/+ and Cln2R207X/R207X but at 3 months of age Cln2R207X/R207X mice trended towards an increased time. (B) Time to turn downward, also assessed by the vertical pole, showed no differences at 1 month of age between Cln2+/+ and Cln2R207X/R207X. By 3 months of age however, Cln2R207X/R207X mice demonstrated significant difficulties turning around. (C) Cln2+/+ and Cln2R207X/R207X mice exhibited no differences in their latency to fall from an accelerating rotarod at both 1 and 3 months of age. (D) 3-month-old Cln2R207X/R207X mice have significant difficulties hanging from a wire rack. Columns and bars represent mean ± SEM. Statistical significance was determined using an unpaired t-test (***p < 0.001).
Fig 7.
Cln2R207X/R207X mice display characteristics of hyperactivity and develop tremors by 3 months of age.
Various behavioral characteristics of 1 and 3-month-old Cln2+/+ (n = 14) and Cln2R207X/R207X (n = 20) mice were evaluated using a force-plate actimeter. (A) Average force increased from 1 to 3 months of age but did not differ between Cln2+/+ and Cln2R207X/R207X at either time points. (B) Cln2+/+ and Cln2R207X/R207X mice had similar bouts of low mobility at both 1 and 3 months. (C,D) By 3 months of age, Cln2R207X/R207X mice display signs of hyperactivity such as significantly increased distance traveled and area coverage. (E-H) Software analysis of the force-plate actimeter data permits the evaluation of tremors by calculating a tremor index score. Tremor index scores are separated according to tremor frequencies such as, 5–10 Hz (E), 10–15 Hz (F), 15–20 Hz (G) and 20–25 Hz (H). As Cln2R207X/R207X mice aged from 1 to 3 months, tremor index scores increased significantly. Columns and bars represent mean ± SEM. Statistical significance was determined using an unpaired t-test (*p < 0.05, **p < 0.01, and ***p < 0.001).
Fig 8.
Lysosomal accumulation of mitochondrial ATP synthase subunit c in Cln2R207X/R207X mice.
Subunit c accumulation was detected by immunofluorescent staining. (A) Images of superficial and deep cortical layers demonstrate diffuse and pronounced accumulation of mitochondrial ATP synthase subunit c in 3-month-old Cln2R207X/R207X mice. (B) Images from 3-month-old Cln2+/+ (n = 4) and Cln2R207X/R207X (n = 5) mice were blindly collected and analyzed for differences in cell number, total number of immunoreactive puncta, number of puncta per cell, and average punctum area. Cln2R207X/R207X mice have significantly increased total number of puncta, puncta per cell, and punctum size. Columns and bars represent mean ± SEM. Statistical significance was determined using an unpaired t-test (**p < 0.01, ***p < 0.001, and **** p < 0.0001).
Fig 9.
Diffuse cortical astrocytosis in Cln2R207X/R207X mice.
(A) Images of superficial and deep cortical layers from 3-month-old Cln2+/+ and Cln2R207X/R207X mice demonstrate increased GFAP immunostaining and enlarged astrocytes. (B) Images from 3-month-old Cln2+/+ (n = 4) and Cln2R207X/R207X (n = 5) mice were blindly collected and analyzed for GFAP immunostaining intensity, GFAP staining area, and astrocyte area. Cln2R207X/R207X mice have significantly increased GFAP immunostaining and astrocyte size relative to Cln2+/+. Columns and bars represent mean ± SEM. Statistical significance was determined using an unpaired t-test (*p < 0.05, **p < 0.01, ***p < 0.001, and **** p < 0.0001).