Large-Scale Phenotyping of an Accurate Genetic Mouse Model of JNCL Identifies Novel Early Pathology Outside the Central Nervous System

Cln3Δex7/8 mice harbor the most common genetic defect causing juvenile neuronal ceroid lipofuscinosis (JNCL), an autosomal recessive disease involving seizures, visual, motor and cognitive decline, and premature death. Here, to more thoroughly investigate the manifestations of the common JNCL mutation, we performed a broad phenotyping study of Cln3Δex7/8 mice. Homozygous Cln3Δex7/8 mice, congenic on a C57BL/6N background, displayed subtle deficits in sensory and motor tasks at 10–14 weeks of age. Homozygous Cln3Δex7/8 mice also displayed electroretinographic changes reflecting cone function deficits past 5 months of age and a progressive decline of retinal post-receptoral function. Metabolic analysis revealed increases in rectal body temperature and minimum oxygen consumption in 12–13 week old homozygous Cln3Δex7/8mice, which were also seen to a lesser extent in heterozygous Cln3Δex7/8 mice. Heart weight was slightly increased at 20 weeks of age, but no significant differences were observed in cardiac function in young adults. In a comprehensive blood analysis at 15–16 weeks of age, serum ferritin concentrations, mean corpuscular volume of red blood cells (MCV), and reticulocyte counts were reproducibly increased in homozygous Cln3Δex7/8 mice, and male homozygotes had a relative T-cell deficiency, suggesting alterations in hematopoiesis. Finally, consistent with findings in JNCL patients, vacuolated peripheral blood lymphocytes were observed in homozygous Cln3Δex7/8 neonates, and to a greater extent in older animals. Early onset, severe vacuolation in clear cells of the epididymis of male homozygous Cln3Δex7/8 mice was also observed. These data highlight additional organ systems in which to study CLN3 function, and early phenotypes have been established in homozygous Cln3Δex7/8 mice that merit further study for JNCL biomarker development

Metabolic abnormalities in <i>Cln3</i>^Δ

<p>^{<b><i>ex7/8</i></b>}<b> mice.</b> (A) Graphs depicting female (left) and male (right) mean body weight data from wild-type (diamonds), heterozygous (squares), and homozygous (triangles) <i>Cln3</i><b>^Δ</b>^<i>ex7/8</i> mice at ages between 11 and 20-weeks are shown (n = 5–10 mice per genotype/sex/age). No significant genotypic differences were observed. Error bars represent SEM. (B) Mean ± SEM rectal body temperatures are shown for male (black bars) and female (gray bars) wild-type (<i>Cln3^+/+</i>), heterozygous (<i>Cln3^+/</i>^{<b>Δ</b><i>ex7/8</i>}) and homozygous (<i>Cln3</i><b>^Δ</b>^{<i>ex7/8/</i><b>Δ</b><i>ex7/8</i>}) littermate mice are shown. Rectal body temperatures, which were measured at rest, were slightly elevated in male and female, heterozygous and homozygous <i>Cln3</i><b>^Δ</b>^<i>ex7/8</i> mice, compared to wild-type mice. *, p<0.001 (heterozygous versus wild-type, homozygous versus wild-type). (C) Mean ± SEM values for minimum oxygen consumption (ml/hr) are shown for male (black bars) and female (gray bars) wild-type (<i>Cln3^+/+</i>), heterozygous (<i>Cln3^+/</i>^{<b>Δ</b><i>ex7/8</i>}) and homozygous (<i>Cln3</i><b>^Δ</b>^{<i>ex7/8/</i><b>Δ</b><i>ex7/8</i>}) littermate mice are shown. Minimum oxygen consumption was elevated in male and female heterozygous and homozygous <i>Cln3^Δex7/8</i> mice, compared to wild-type mice. 5–10 mice per group (genotype/sex) were analyzed. *, p<0.001 (heterozygous versus wild-type, homozygous versus wild-type).</p

Electroretinography of 16-month-old <i>Cln3</i>^Δ

<p>^{<b><i>ex7/8</i></b>}<b> mice.</b> (A) Scotopic ERG traces are shown for 5-, 9-, and 16-month old wild-type (<i>Cln3^+/+</i>, black trace, n = 7) and homozygous <i>Cln3</i><b>^Δ</b>^<i>ex7/8</i> (<i>Cln3</i><b>^Δ</b>^{<i>ex7/8/</i><b>Δ</b><i>ex7/8</i>}, red trace, n = 8) mice. The relative amplitudes of the a-wave do not dramatically differ between the wild-type and homozygous <i>Cln3</i><b>^Δ</b>^<i>ex7/8</i> mice. However, the b-wave is drastically reduced in aged homozygous <i>Cln3</i><b>^Δ</b>^<i>ex7/8</i> mice, compared to wild-type littermates. Thus, homozygous <i>Cln3</i><b>^Δ</b>^<i>ex7/8</i> mice exhibit an electronegative ERG at 16-months of age (b/a ratio = 1, versus b/a ratio = 2.4 in wild-type mice). (B) Photopic ERG traces, reflecting cone response, are shown for 5-, 9-, and 16-month-old wild-type (<i>Cln3^+/+</i>, black trace, n = 7) and homozygous <i>Cln3</i><b>^Δ</b>^<i>ex7/8</i> (<i>Cln3</i><b>^Δ</b>^{<i>ex7/8/</i><b>Δ</b><i>ex7/8</i>}, red trace, n = 8) mice. There was a significant genotypic difference in the relative mean amplitudes already at 5 months of age.</p

Subtle genotypic differences in performance of young adult <i>Cln3</i>^Δ

<p>^{<b><i>ex7/8</i></b>}<b> mice in sensory and motor neurological assays.</b> Shown are results of behavioural analyses in a vertical pole-climbing test (A), prepulse inhibition to the acoustic startle response (PPI) (B), acoustic startle response (C), and thermal nociception (D) for female (left) and male (right) littermate control (<i>Cln3^+/+</i>), heterozygous (<i>Cln3^+/</i>^{<b>Δ</b><i>ex7/8</i>}) and homozygous (<i>Cln3</i><b>^Δ</b>^{<i>ex7/8/</i><b>Δ</b><i>ex7/8</i>}) mice (n = 9–10 mice per group). Data are presented as mean ± standard error of the mean (SEM). (A) Homozygous <i>Cln3</i><b>^Δ</b>^<i>ex7/8</i> female mice had an increased latency to descend the pole, compared to female wild-type or heterozygous littermates. In a Kruskal-Wallis test, the genotype effect was p<0.01 (*) for females, with or without heterozygous <i>Cln3</i><b>^Δ</b>^<i>ex7/8</i> mice included in the analysis. (B) Mean %PPI to an acoustic startle, with four prepulse intensities (67, 69, 73, 81 decibels [db]), or with all prepulse intensities averaged (‘global’) are shown. *, ANOVA, p<0.05. (C) The mean ± SEM of the acoustic startle response to 70–120 db sounds is shown for littermate control (<i>Cln3^+/+</i>, circles), heterozygous (<i>Cln3^+/</i>^{<b>Δ</b><i>ex7/8</i>}, squares) and homozygous (<i>Cln3</i><b>^Δ</b>^{<i>ex7/8/</i><b>Δ</b><i>ex7/8</i>}, triangles) <i>Cln3</i><b>^Δ</b>^<i>ex7/8</i> mice. NS = no startle sound. For females, ANOVA, genotype effect was F_(7,11) = 4.63, p<0.05, and post-hoc tests revealed that this was significant at 90 and 100 db (*p<0.05, ***<i>P</i><0.001). No statistically significant differences were detected in the acoustic startle response of males. (D) The mean ± SEM latency to the first sign of pain (seconds = s) in a hot plate assay is shown. *, ANOVA genotype effect p<0.05.</p

Abnormal hematology in peripheral blood from homozygous <i>Cln3</i>^Δ

<p>^{<b><i>ex7/8</i></b>}<b> mice.</b> (A) Mean corpuscular volume (MCV, fL) of peripheral red blood cells from ∼12-week-old mice was measured on an automated analyzer. *, p<0.05, WT and heterozygous mutant mice vs. homozygous mutant mice, unpaired, two-tailed <i>t</i> test. Data shown as mean ± SEM. Percentage of reticulocytes (B) and absolute reticulocyte counts (C) on the specimens analyzed in (A) were determined manually by new methylene blue staining. *, p<0.05, WT and heterozygous mutant mice vs. homozygous mutant mice, unpaired, two-tailed <i>t</i> test. Data shown as mean ± SEM. (D) Linear regression analysis of data from (A) and (C). <i>r²</i> = 0.32, p = 0.02. Datapoints represent individual mice.</p

Bone marrow analysis of <i>Cln3</i>^Δ

<p>^{<b><i>ex7/8</i></b>}<b> mice.</b> Representative images are shown of Wright-Giemsa-stained bone marrow brush cytology, H&E stained sections of formalin-fixed, paraffin embedded tibias, and iron stained brush cytology, from wild-type (<i>Cln3^+/+</i>) and homozygous mutant (<i>Cln3</i><b>^Δ</b>^{<i>ex7/8/</i><b>Δ</b><i>ex7/8</i>}) mice (n = 3 mice per genotype). Stained iron appears blue. Note the reduced amount of stained iron in <i>Cln3</i><b>^Δ</b>^{<i>ex7/8/</i><b>Δ</b><i>ex7/8</i>} marrow, compared to wild-type marrow. Arrow, erythroid element; arrowhead, myeloid element; asterisk, megakaryocyte. Scale bars, top and bottom panels = 25 µm; middle panels = 100 µm.</p

Blood analysis of <i>Cln3</i><b>^Δ</b>^<i>ex7/8</i> mice.

<p>The subset of clinical chemistry and hematological parameters that were found to have genotype-specific differences in the screen are shown, with significantly different values indicated in bold. RBC count, which did not differ, is also shown. Each row of values represents an independent set of measurements. Data represent mean ± SEM. *p<0.05, **p<0.01 (2-way ANOVA for each set of measurements). For the statistical analysis of ferritin levels, one mouse from the group of heterozygous (<i>Cln3^+/</i>^{<b>Δ</b><i>ex7/8</i>}) females was excluded as an outlier. Non-italicized values were determined at the German Mouse Clinic. Italicized values were determined at Massachusetts General Hospital on a separate cohort of mice. The ∼5–6 fL offset in MCV measurements between these sites, as well as the ∼6–7% offset in RDW measurements, are likely due to differences in the automated analyzers used. RBC = red blood cell count, MCV = mean corpuscular volume, Retic. = reticulocyte, RDW = red cell distribution width. Two separate samples from 9–10 mice per group (genotype/sex) were analyzed in the primary screen, and samples from an additional 3–8 mice per group (genotype/sex) were analyzed in follow-up screens.</p

Vacuolation of diverse cell types in homozygous <i>Cln3</i>^Δ

<p>^{<b><i>ex7/8</i></b>}<b> mice.</b> (A) Representative images are shown of Wright-Giemsa stained peripheral blood smears from <i>Cln3^+/+</i> and <i>Cln3</i><b>^Δ</b>^{<i>ex7/8/</i><b>Δ</b><i>ex7/8</i>} littermate mice (scale bar = 10 µm). Note the presence of vacuoles in the cytoplasm of the dark blue stained peripheral blood lymphocyte. (B) Representative images are shown of H&E-stained sections of epididymis from 19-week-old <i>Cln3^+/+</i> and <i>Cln3</i><b>^Δ</b>^{<i>ex7/8/</i><b>Δ</b><i>ex7/8</i>} littermate male mice (scale bar = 50 µm). A representative image of a section of mutant (<i>Cln3</i><b>^Δ</b>^{<i>ex7/8/</i><b>Δ</b><i>ex7/8</i>}) epididymis immunostained for vacuolar ATPase (V-ATPase, green) and aquaporin-9 (AQP9, red), which highlight the apical (luminal) membrane of clear/narrow cells or principal cells, respectively (scale bar = 25 µm). (C) Representative TEM images of <i>Cln3</i><b>^Δ</b>^{<i>ex7/8/</i><b>Δ</b><i>ex7/8</i>} epididymis cross-sections are shown. Note both the giant vacuoles and the multiple smaller vacuoles filling the cytoplasm of the clear cells. Also note the relative absence of electron-dense material inside the vacuoles. Scale bars, left panel = 10 µm; right panel = 2 µm. (D) Representative images of subunit c immunostained <i>Cln3^+/+</i> and <i>Cln3</i><b>^Δ</b>^{<i>ex7/8/</i><b>Δ</b><i>ex7/8</i>} epididymis sections are shown. Asterisks (*) mark some of the large vacuoles. Scale bars = 50 µm. Blood smears and epididymides from at least 10 mice per genotype were analyzed in total, and abnormal vacuolation was observed in all of the <i>Cln3</i><b>^Δ</b>^{<i>ex7/8/</i><b>Δ</b><i>ex7/8</i>} mice and in none of the wild-type or <i>Cln3^+/</i>^{<b>Δ</b><i>ex7/8</i>} mice.</p

Heart analysis of <i>Cln3</i>^Δ

<p>^{<b><i>ex7/8</i></b>}<b> mice.</b> (A) The bar graph depicts normalized heart weights for wild-type (<i>Cln3^+/+</i>), heterozygous (<i>Cln3^+/</i>^{<b>Δ</b><i>ex7/8</i>}), and homozygous (<i>Cln3</i><b>^Δ</b>^{<i>ex7/8/</i><b>Δ</b><i>ex7/8</i>}) littermate 19–20 week old mice. Normalized heart weights represent a ratio of heart weight (mg = milligrams)/body weight (g = grams). Normalized heart weights were slightly increased in heterozygous <i>Cln3</i><b>^Δ</b>^<i>ex7/8</i> mice, and more so in homozygous <i>Cln3</i><b>^Δ</b>^<i>ex7/8</i> mice, compared to wild-type littermates. ANOVA analysis suggested a significant genotype effect (p<0.05). (B) Representative micrographs of hematoxylin and eosin (H&E) stained heart sections from wild-type (<i>Cln3^+/+</i>, n = 8) and homozygous (<i>Cln3</i><b>^Δ</b>^{<i>ex7/8/</i><b>Δ</b><i>ex7/8</i>}, n = 10) littermate 19–20 week old mice are shown, which do not obviously differ from one another in their morphology. Scale bar = 100 µm. (C) Representative micrographs are shown of α-subunit c immunostained heart sections from 19-week old <i>Cln3^+/+</i> and <i>Cln3</i><b>^Δ</b>^{<i>ex7/8/</i><b>Δ</b><i>ex7/8</i>} littermate mice. Note the abundance of subunit c-immunopositive deposits in the <i>Cln3^?ex7/8/</i>^{<b>Δ</b><i>ex7/8</i>} section. Only sparse punctate subunit c immunostaining is present in the <i>Cln3^+/+</i> section. Scale bar = 200 µm. Inset scale bar = 25 µm.</p