A tailored mouse model of CLN2 disease: A nonsense mutant for testing personalized therapies - PubMed (original) (raw)

A tailored mouse model of CLN2 disease: A nonsense mutant for testing personalized therapies

Ryan D Geraets et al. PLoS One. 2017.

Abstract

The Neuronal Ceroid Lipofuscinoses (NCLs), also known as Batten disease, result from mutations in over a dozen genes. Although, adults are susceptible, the NCLs are frequently classified as pediatric neurodegenerative diseases due to their greater pediatric prevalence. Initial clinical presentation usually consists of either seizures or retinopathy but develops to encompass both in conjunction with declining motor and cognitive function. The NCLs result in premature death due to the absence of curative therapies. Nevertheless, preclinical and clinical trials exist for various therapies. However, the genotypes of NCL animal models determine which therapeutic approaches can be assessed. Mutations of the CLN2 gene encoding a soluble lysosomal enzyme, tripeptidyl peptidase 1 (TPP1), cause late infantile NCL/CLN2 disease. The genotype of the original mouse model of CLN2 disease, Cln2-/-, excludes mutation guided therapies like antisense oligonucleotides and nonsense suppression. Therefore, the purpose of this study was to develop a model of CLN2 disease that allows for the assessment of all therapeutic approaches. Nonsense mutations in CLN2 disease are frequent, the most common being CLN2R208X. Thus, we created a mouse model that carries a mutation equivalent to the human p.R208X mutation. Molecular assessment of Cln2R207X/R207X tissues determined significant reduction in Cln2 transcript abundance and TPP1 enzyme activity. This reduction leads to the development of neurological impairment (e.g. tremors) and neuropathology (e.g. astrocytosis). Collectively, these assessments indicate that the Cln2R207X/R207X mouse is a valid CLN2 disease model which can be used for the preclinical evaluation of all therapeutic approaches including mutation guided therapies.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1

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

Fig 2. Genetic design of the CLN2 disease mouse model, Cln2 R207X/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 Cln2 Neo;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 Cln2 R207X allele (Line 4) resulting in Cln2 +/R207X mice. Cln2 +/R207X mice were bred to create Cln2 R207X/R207X mice. (B) Cln2 sequencing results confirm the retention of the mutation following breeding.

Fig 3

Fig 3. Ubiquitously reduced Cln2 transcript abundance in Cln2 R207X/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 Cln2 R207X/R207X (n = 3) mice. Three technical replicates were performed using the three biological samples obtained from Cln2 +/+ and Cln2 R207X/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

Fig 4. Decreased TPP1 activity in various Cln2 R207X/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 Cln2 R207X/R207X (n = 3) mice. Four technical replicates were performed using the three biological samples obtained from Cln2 +/+ and Cln2 R207X/R207X mice. Cln2 R207X/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

Fig 5. Significantly reduced lifespan in Cln2 R207X/R207X mice.

Cln2 +/+ (n = 14) and Cln2 R207X/R207X (n = 26) mice were plotted on a survival curve and tracked overtime. Cln2 R207X/R207X mice died between 3 and 6 months of age.

Fig 6

Fig 6. Altered motor skills in 3-month-old Cln2 R207X/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 Cln2 R207X/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 Cln2 R207X/R207X but at 3 months of age Cln2 R207X/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 Cln2 R207X/R207X. By 3 months of age however, Cln2 R207X/R207X mice demonstrated significant difficulties turning around. (C) Cln2 +/+ and Cln2 R207X/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 Cln2 R207X/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

Fig 7. Cln2 R207X/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 Cln2 R207X/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 Cln2 R207X/R207X at either time points. (B) Cln2 +/+ and Cln2 R207X/R207X mice had similar bouts of low mobility at both 1 and 3 months. (C,D) By 3 months of age, Cln2 R207X/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 Cln2 R207X/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

Fig 8. Lysosomal accumulation of mitochondrial ATP synthase subunit c in Cln2 R207X/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 Cln2 R207X/R207X mice. (B) Images from 3-month-old Cln2 +/+ (n = 4) and Cln2 R207X/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. Cln2 R207X/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

Fig 9. Diffuse cortical astrocytosis in Cln2 R207X/R207X mice.

(A) Images of superficial and deep cortical layers from 3-month-old Cln2 +/+ and Cln2 R207X/R207X mice demonstrate increased GFAP immunostaining and enlarged astrocytes. (B) Images from 3-month-old Cln2 +/+ (n = 4) and Cln2 R207X/R207X (n = 5) mice were blindly collected and analyzed for GFAP immunostaining intensity, GFAP staining area, and astrocyte area. Cln2 R207X/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).

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