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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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Vacuolation of diverse cell types in homozygous Cln3Δex7/8 mice. (A) Representative images are shown of Wright-Giemsa stained peripheral blood smears from Cln3+/+ and Cln3Δex7/8/Δex7/8 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 Cln3+/+ and Cln3Δex7/8/Δex7/8 littermate male mice (scale bar = 50 µm). A representative image of a section of mutant (Cln3Δex7/8/Δex7/8) 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 Cln3Δex7/8/Δex7/8 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 Cln3+/+ and Cln3Δex7/8/Δex7/8 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 Cln3Δex7/8/Δex7/8 mice and in none of the wild-type or Cln3+/Δex7/8 mice.
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pone-0038310-g008: Vacuolation of diverse cell types in homozygous Cln3Δex7/8 mice. (A) Representative images are shown of Wright-Giemsa stained peripheral blood smears from Cln3+/+ and Cln3Δex7/8/Δex7/8 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 Cln3+/+ and Cln3Δex7/8/Δex7/8 littermate male mice (scale bar = 50 µm). A representative image of a section of mutant (Cln3Δex7/8/Δex7/8) 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 Cln3Δex7/8/Δex7/8 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 Cln3+/+ and Cln3Δex7/8/Δex7/8 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 Cln3Δex7/8/Δex7/8 mice and in none of the wild-type or Cln3+/Δex7/8 mice.

Mentions: Evaluation of peripheral blood smears for vacuolated lymphocytes is a useful diagnostic tool in the workup of JNCL patients [40], [41]. In this study, we similarly detected abnormal vacuolation in ∼5–15% of the peripheral blood lymphocytes from homozygous Cln3Δex7/8 mice, where the proportion of lymphocytes with a vacuolated appearance tended to increase with age (8.3±1 at postnatal day 7, versus 14.8±2 at 16 weeks of age). The numbers of vacuolated lymphocytes in wild-type and heterozygous littermate mice were typically <5% of the total counted lymphocytes (Fig. 8A).


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)

Vacuolation of diverse cell types in homozygous Cln3Δex7/8 mice. (A) Representative images are shown of Wright-Giemsa stained peripheral blood smears from Cln3+/+ and Cln3Δex7/8/Δex7/8 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 Cln3+/+ and Cln3Δex7/8/Δex7/8 littermate male mice (scale bar = 50 µm). A representative image of a section of mutant (Cln3Δex7/8/Δex7/8) 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 Cln3Δex7/8/Δex7/8 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 Cln3+/+ and Cln3Δex7/8/Δex7/8 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 Cln3Δex7/8/Δex7/8 mice and in none of the wild-type or Cln3+/Δex7/8 mice.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368842&req=5

pone-0038310-g008: Vacuolation of diverse cell types in homozygous Cln3Δex7/8 mice. (A) Representative images are shown of Wright-Giemsa stained peripheral blood smears from Cln3+/+ and Cln3Δex7/8/Δex7/8 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 Cln3+/+ and Cln3Δex7/8/Δex7/8 littermate male mice (scale bar = 50 µm). A representative image of a section of mutant (Cln3Δex7/8/Δex7/8) 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 Cln3Δex7/8/Δex7/8 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 Cln3+/+ and Cln3Δex7/8/Δex7/8 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 Cln3Δex7/8/Δex7/8 mice and in none of the wild-type or Cln3+/Δex7/8 mice.
Mentions: Evaluation of peripheral blood smears for vacuolated lymphocytes is a useful diagnostic tool in the workup of JNCL patients [40], [41]. In this study, we similarly detected abnormal vacuolation in ∼5–15% of the peripheral blood lymphocytes from homozygous Cln3Δex7/8 mice, where the proportion of lymphocytes with a vacuolated appearance tended to increase with age (8.3±1 at postnatal day 7, versus 14.8±2 at 16 weeks of age). The numbers of vacuolated lymphocytes in wild-type and heterozygous littermate mice were typically <5% of the total counted lymphocytes (Fig. 8A).

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