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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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Cellular vacuolation is specific to CLN3 dysfunction.Representative images are shown of Wright-Giemsa stained peripheral blood smears and H&E-stained sections of male epididymis from mice representing three different forms of NCL. Ages of the mice at the time of sample collection are indicated. Note the obvious vacuolation of peripheral blood lymphocytes and clear cells of the epididymis in young homozygous Cln3Δex7/8 mice (Cln3Δex7/8/Δex7/8), shown at postnatal day 7 (P7) and 4 weeks of age (4 wk), respectively. Note the lack of abnormal vacuoles in Cln6nclf/nclf and Ctsd−/− mice, which are models of variant late-infantile and congenital NCL, respectively. N = 3−4 mice per genotype. Scale bars, top panels = 10 µm; bottom panels = 25 µm.
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pone-0038310-g009: Cellular vacuolation is specific to CLN3 dysfunction.Representative images are shown of Wright-Giemsa stained peripheral blood smears and H&E-stained sections of male epididymis from mice representing three different forms of NCL. Ages of the mice at the time of sample collection are indicated. Note the obvious vacuolation of peripheral blood lymphocytes and clear cells of the epididymis in young homozygous Cln3Δex7/8 mice (Cln3Δex7/8/Δex7/8), shown at postnatal day 7 (P7) and 4 weeks of age (4 wk), respectively. Note the lack of abnormal vacuoles in Cln6nclf/nclf and Ctsd−/− mice, which are models of variant late-infantile and congenital NCL, respectively. N = 3−4 mice per genotype. Scale bars, top panels = 10 µm; bottom panels = 25 µm.

Mentions: Lymphocyte vacuolation, among the NCLs, is thought to be diagnostic for CLN3 mutation [40]. Therefore, we sought to determine whether the vacuolation phenotypes uncovered in this study were also unique to Cln3Δex7/8 mice, versus mouse models for two other forms of NCL. Peripheral blood smears were prepared from 12-week old Cln6nclf mice, which model a variant late-infantile form of NCL (CLN6, MIM#601780) [43], [44], [45], and from postnatal day 7 (P7) Ctsd knock-out mice, which model the most severe form of NCL (congenital NCL; CLN10, MIM#610127) [46], [47]. Unlike the vacuolated appearance of the lymphocytes from 12-week-old and P7 homozygous Cln3Δex7/8 mice (Figs. 8A, 9), there were no morphological differences in homozygous Cln6nclf/nclf or in Ctsd−/− mouse peripheral blood lymphocytes (Fig. 9). Notably, however, we did observe a significant reduction in numbers of peripheral blood lymphocytes in Ctsd−/− mice (∼50% reduced from age-matched control mice, as assessed by visual inspection of peripheral blood smears). This is consistent with a previously reported finding of progressive lymphopenia in the thymus and spleen of Ctsd−/− mice [46]. Similar to our lymphocyte analysis, we also found no evidence for morphological differences in the epididymal clear cells from homozygous Cln6nclf or Ctsd knock-out male mice, as compared to wild-type littermate mice (Fig. 9).


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)

Cellular vacuolation is specific to CLN3 dysfunction.Representative images are shown of Wright-Giemsa stained peripheral blood smears and H&E-stained sections of male epididymis from mice representing three different forms of NCL. Ages of the mice at the time of sample collection are indicated. Note the obvious vacuolation of peripheral blood lymphocytes and clear cells of the epididymis in young homozygous Cln3Δex7/8 mice (Cln3Δex7/8/Δex7/8), shown at postnatal day 7 (P7) and 4 weeks of age (4 wk), respectively. Note the lack of abnormal vacuoles in Cln6nclf/nclf and Ctsd−/− mice, which are models of variant late-infantile and congenital NCL, respectively. N = 3−4 mice per genotype. Scale bars, top panels = 10 µm; bottom panels = 25 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368842&req=5

pone-0038310-g009: Cellular vacuolation is specific to CLN3 dysfunction.Representative images are shown of Wright-Giemsa stained peripheral blood smears and H&E-stained sections of male epididymis from mice representing three different forms of NCL. Ages of the mice at the time of sample collection are indicated. Note the obvious vacuolation of peripheral blood lymphocytes and clear cells of the epididymis in young homozygous Cln3Δex7/8 mice (Cln3Δex7/8/Δex7/8), shown at postnatal day 7 (P7) and 4 weeks of age (4 wk), respectively. Note the lack of abnormal vacuoles in Cln6nclf/nclf and Ctsd−/− mice, which are models of variant late-infantile and congenital NCL, respectively. N = 3−4 mice per genotype. Scale bars, top panels = 10 µm; bottom panels = 25 µm.
Mentions: Lymphocyte vacuolation, among the NCLs, is thought to be diagnostic for CLN3 mutation [40]. Therefore, we sought to determine whether the vacuolation phenotypes uncovered in this study were also unique to Cln3Δex7/8 mice, versus mouse models for two other forms of NCL. Peripheral blood smears were prepared from 12-week old Cln6nclf mice, which model a variant late-infantile form of NCL (CLN6, MIM#601780) [43], [44], [45], and from postnatal day 7 (P7) Ctsd knock-out mice, which model the most severe form of NCL (congenital NCL; CLN10, MIM#610127) [46], [47]. Unlike the vacuolated appearance of the lymphocytes from 12-week-old and P7 homozygous Cln3Δex7/8 mice (Figs. 8A, 9), there were no morphological differences in homozygous Cln6nclf/nclf or in Ctsd−/− mouse peripheral blood lymphocytes (Fig. 9). Notably, however, we did observe a significant reduction in numbers of peripheral blood lymphocytes in Ctsd−/− mice (∼50% reduced from age-matched control mice, as assessed by visual inspection of peripheral blood smears). This is consistent with a previously reported finding of progressive lymphopenia in the thymus and spleen of Ctsd−/− mice [46]. Similar to our lymphocyte analysis, we also found no evidence for morphological differences in the epididymal clear cells from homozygous Cln6nclf or Ctsd knock-out male mice, as compared to wild-type littermate mice (Fig. 9).

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