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Large-scale phenotyping of an accurate genetic mouse model of JNCL identifies novel early pathology outside the central nervous system.

Staropoli JF, Haliw L, Biswas S, Garrett L, Hölter SM, Becker L, Skosyrski S, Da Silva-Buttkus P, Calzada-Wack J, Neff F, Rathkolb B, Rozman J, Schrewe A, Adler T, Puk O, Sun M, Favor J, Racz I, Bekeredjian R, Busch DH, Graw J, Klingenspor M, Klopstock T, Wolf E, Wurst W, Zimmer A, Lopez E, Harati H, Hill E, Krause DS, Guide J, Dragileva E, Gale E, Wheeler VC, Boustany RM, Brown DE, Breton S, Ruether K, Gailus-Durner V, Fuchs H, de Angelis MH, Cotman SL - PLoS ONE (2012)

Bottom Line: 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 Cln3(Δ) (ex7/8) 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 Cln3(Δ) (ex7/8) neonates, and to a greater extent in older animals.

View Article: PubMed Central - PubMed

Affiliation: Molecular Neurogenetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, Massachusetts, United States of America.

ABSTRACT
Cln3(Δex7/8) 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 Cln3(Δex7/8) mice. Homozygous Cln3(Δex7/8) mice, congenic on a C57BL/6N background, displayed subtle deficits in sensory and motor tasks at 10-14 weeks of age. Homozygous Cln3(Δex7/8) 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 Cln3(Δex7/8) mice, which were also seen to a lesser extent in heterozygous Cln3(Δex7/8) 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 Cln3(Δ) (ex7/8) 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 Cln3(Δ) (ex7/8) neonates, and to a greater extent in older animals. Early onset, severe vacuolation in clear cells of the epididymis of male homozygous Cln3(Δ) (ex7/8) mice was also observed. These data highlight additional organ systems in which to study CLN3 function, and early phenotypes have been established in homozygous Cln3(Δ) (ex7/8) mice that merit further study for JNCL biomarker development.

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Heart analysis of Cln3Δex7/8 mice. (A) The bar graph depicts normalized heart weights for wild-type (Cln3+/+), heterozygous (Cln3+/Δex7/8), and homozygous (Cln3Δex7/8/Δex7/8) 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 Cln3Δex7/8 mice, and more so in homozygous Cln3Δex7/8 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 (Cln3+/+, n = 8) and homozygous (Cln3Δex7/8/Δex7/8, 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 Cln3+/+ and Cln3Δex7/8/Δex7/8 littermate mice. Note the abundance of subunit c-immunopositive deposits in the Cln3?ex7/8/Δex7/8 section. Only sparse punctate subunit c immunostaining is present in the Cln3+/+ section. Scale bar = 200 µm. Inset scale bar = 25 µm.
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pone-0038310-g004: Heart analysis of Cln3Δex7/8 mice. (A) The bar graph depicts normalized heart weights for wild-type (Cln3+/+), heterozygous (Cln3+/Δex7/8), and homozygous (Cln3Δex7/8/Δex7/8) 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 Cln3Δex7/8 mice, and more so in homozygous Cln3Δex7/8 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 (Cln3+/+, n = 8) and homozygous (Cln3Δex7/8/Δex7/8, 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 Cln3+/+ and Cln3Δex7/8/Δex7/8 littermate mice. Note the abundance of subunit c-immunopositive deposits in the Cln3?ex7/8/Δex7/8 section. Only sparse punctate subunit c immunostaining is present in the Cln3+/+ section. Scale bar = 200 µm. Inset scale bar = 25 µm.

Mentions: Increasing evidence indicates abnormal cardiovascular health in JNCL patients [3], [4]. To assess the cardiovascular status of Cln3Δex7/8 mice, blood pressure (12–13 weeks of age), pulse rate (16–17 weeks of age), echocardiography parameters (16–17 weeks of age), and serum N-terminal pro atrial natriuretic peptide (Nt-proANP) levels (at 18–19 weeks of age) were measured and analyzed. None of these parameters were significantly altered in heterozygous or homozygous Cln3Δex7/8 mutant mice at these ages, compared to wild-type littermate mice (see Figs. S7, S8, Table S3). However, normalized heart weight, measured at 20 weeks of age, was slightly increased in heterozygous and homozygous Cln3Δex7/8 mice, compared to wild-type littermate mice (ANOVA, p<0.05; Fig. 4A). Despite this difference, in further histological assessment of heart from 19-week-old mice, we did not detect any obvious signs of pathological cardiac hypertrophy (Fig. 4B). Not surprisingly, subunit c-positive lysosomal storage material was evident in homozygous Cln3Δex7/8 mice (Fig. 4C), while no storage material was observed in Cln3Δex7/8 heterozygotes (data not shown). We also immunostained heart sections with an antibody recognizing nuclear factor of activated T-cells (NFAT), which is a central regulator of the signaling pathways mediating cardiac hypertrophy and enters the nucleus upon activation of hypertrophic signaling pathways [34]. No significant differences in nuclear NFAT levels were detected in heart sections from homozygous Cln3Δex7/8 mice, compared to those from wild-type littermate mice (data not shown). Therefore, these results suggest that the common JNCL mutation in the mouse leads to an increased resting metabolism, without significant differences in overall cardiovascular function in young adults.


Large-scale phenotyping of an accurate genetic mouse model of JNCL identifies novel early pathology outside the central nervous system.

Staropoli JF, Haliw L, Biswas S, Garrett L, Hölter SM, Becker L, Skosyrski S, Da Silva-Buttkus P, Calzada-Wack J, Neff F, Rathkolb B, Rozman J, Schrewe A, Adler T, Puk O, Sun M, Favor J, Racz I, Bekeredjian R, Busch DH, Graw J, Klingenspor M, Klopstock T, Wolf E, Wurst W, Zimmer A, Lopez E, Harati H, Hill E, Krause DS, Guide J, Dragileva E, Gale E, Wheeler VC, Boustany RM, Brown DE, Breton S, Ruether K, Gailus-Durner V, Fuchs H, de Angelis MH, Cotman SL - PLoS ONE (2012)

Heart analysis of Cln3Δex7/8 mice. (A) The bar graph depicts normalized heart weights for wild-type (Cln3+/+), heterozygous (Cln3+/Δex7/8), and homozygous (Cln3Δex7/8/Δex7/8) 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 Cln3Δex7/8 mice, and more so in homozygous Cln3Δex7/8 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 (Cln3+/+, n = 8) and homozygous (Cln3Δex7/8/Δex7/8, 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 Cln3+/+ and Cln3Δex7/8/Δex7/8 littermate mice. Note the abundance of subunit c-immunopositive deposits in the Cln3?ex7/8/Δex7/8 section. Only sparse punctate subunit c immunostaining is present in the Cln3+/+ section. Scale bar = 200 µm. Inset scale bar = 25 µm.
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Related In: Results  -  Collection

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pone-0038310-g004: Heart analysis of Cln3Δex7/8 mice. (A) The bar graph depicts normalized heart weights for wild-type (Cln3+/+), heterozygous (Cln3+/Δex7/8), and homozygous (Cln3Δex7/8/Δex7/8) 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 Cln3Δex7/8 mice, and more so in homozygous Cln3Δex7/8 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 (Cln3+/+, n = 8) and homozygous (Cln3Δex7/8/Δex7/8, 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 Cln3+/+ and Cln3Δex7/8/Δex7/8 littermate mice. Note the abundance of subunit c-immunopositive deposits in the Cln3?ex7/8/Δex7/8 section. Only sparse punctate subunit c immunostaining is present in the Cln3+/+ section. Scale bar = 200 µm. Inset scale bar = 25 µm.
Mentions: Increasing evidence indicates abnormal cardiovascular health in JNCL patients [3], [4]. To assess the cardiovascular status of Cln3Δex7/8 mice, blood pressure (12–13 weeks of age), pulse rate (16–17 weeks of age), echocardiography parameters (16–17 weeks of age), and serum N-terminal pro atrial natriuretic peptide (Nt-proANP) levels (at 18–19 weeks of age) were measured and analyzed. None of these parameters were significantly altered in heterozygous or homozygous Cln3Δex7/8 mutant mice at these ages, compared to wild-type littermate mice (see Figs. S7, S8, Table S3). However, normalized heart weight, measured at 20 weeks of age, was slightly increased in heterozygous and homozygous Cln3Δex7/8 mice, compared to wild-type littermate mice (ANOVA, p<0.05; Fig. 4A). Despite this difference, in further histological assessment of heart from 19-week-old mice, we did not detect any obvious signs of pathological cardiac hypertrophy (Fig. 4B). Not surprisingly, subunit c-positive lysosomal storage material was evident in homozygous Cln3Δex7/8 mice (Fig. 4C), while no storage material was observed in Cln3Δex7/8 heterozygotes (data not shown). We also immunostained heart sections with an antibody recognizing nuclear factor of activated T-cells (NFAT), which is a central regulator of the signaling pathways mediating cardiac hypertrophy and enters the nucleus upon activation of hypertrophic signaling pathways [34]. No significant differences in nuclear NFAT levels were detected in heart sections from homozygous Cln3Δex7/8 mice, compared to those from wild-type littermate mice (data not shown). Therefore, these results suggest that the common JNCL mutation in the mouse leads to an increased resting metabolism, without significant differences in overall cardiovascular function in young adults.

Bottom Line: 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 Cln3(Δ) (ex7/8) 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 Cln3(Δ) (ex7/8) neonates, and to a greater extent in older animals.

View Article: PubMed Central - PubMed

Affiliation: Molecular Neurogenetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, Massachusetts, United States of America.

ABSTRACT
Cln3(Δex7/8) 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 Cln3(Δex7/8) mice. Homozygous Cln3(Δex7/8) mice, congenic on a C57BL/6N background, displayed subtle deficits in sensory and motor tasks at 10-14 weeks of age. Homozygous Cln3(Δex7/8) 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 Cln3(Δex7/8) mice, which were also seen to a lesser extent in heterozygous Cln3(Δex7/8) 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 Cln3(Δ) (ex7/8) 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 Cln3(Δ) (ex7/8) neonates, and to a greater extent in older animals. Early onset, severe vacuolation in clear cells of the epididymis of male homozygous Cln3(Δ) (ex7/8) mice was also observed. These data highlight additional organ systems in which to study CLN3 function, and early phenotypes have been established in homozygous Cln3(Δ) (ex7/8) mice that merit further study for JNCL biomarker development.

Show MeSH
Related in: MedlinePlus