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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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Subunit c immunohistochemistry of major hematopoietic tissues from 12-week-old Cln3Δex7/8 mice.Representative images from bone marrow brush cytology, tibia cross-sections (‘Bone marrow histology’), and liver and spleen sections immunostained for subunit c are shown for wild-type (Cln3+/+) and homozygous mutant (Cln3Δex7/8/Δex7/8) mice (n = 2−5 mice per tissue/genotype). Inset in Cln3Δex7/8/Δex7/8 bone marrow cytology panel (top right panel) shows a sea-blue histiocyte (arrows) from an H&E stained preparation. Sea-blue histiocytes were not found in wild-type bone marrow cytology preparations. Brown stain reflects subunit c-positive storage material, which is most prominent in cells that appear morphologically consistent with macrophages. Arrowheads mark examples of subunit c filled Kupffer cells in liver, also a macrophage lineage cell. Insets in histology panels show lower power magnification of subunit c immunostain. Scale bars = 25 µm.
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pone-0038310-g007: Subunit c immunohistochemistry of major hematopoietic tissues from 12-week-old Cln3Δex7/8 mice.Representative images from bone marrow brush cytology, tibia cross-sections (‘Bone marrow histology’), and liver and spleen sections immunostained for subunit c are shown for wild-type (Cln3+/+) and homozygous mutant (Cln3Δex7/8/Δex7/8) mice (n = 2−5 mice per tissue/genotype). Inset in Cln3Δex7/8/Δex7/8 bone marrow cytology panel (top right panel) shows a sea-blue histiocyte (arrows) from an H&E stained preparation. Sea-blue histiocytes were not found in wild-type bone marrow cytology preparations. Brown stain reflects subunit c-positive storage material, which is most prominent in cells that appear morphologically consistent with macrophages. Arrowheads mark examples of subunit c filled Kupffer cells in liver, also a macrophage lineage cell. Insets in histology panels show lower power magnification of subunit c immunostain. Scale bars = 25 µm.

Mentions: During our histological analysis of the bone marrow, we also noted the presence of sea-blue histiocytes, macrophages filled with ceroid lipofuscin storage material, in the marrow from homozygous Cln3Δex7/8 mice (shown in inset of Fig. 7). A similar finding has been reported in bone marrow from JNCL patients [39]. Thus, to more thoroughly determine the extent to which hematopoietic tissues in homozygous Cln3Δex7/8 mice exhibit the pathologic hallmark of JNCL, storage of mitochondrial subunit c, we analyzed bone marrow, spleen, and liver by subunit c immunohistochemistry. Only faint, punctate staining, likely corresponding to normal endogenous mitochondrial subunit c, was observed in tissues from normal mice. By contrast, consistent with the presence of relatively frequent sea-blue histiocytes in the Wright-Giemsa stained samples, bone marrow from homozygous Cln3Δex7/8 mice showed a striking accumulation of subunit c in cells that were morphologically consistent with macrophages (Fig. 7). Accumulation of subunit c was also noted in the macrophage-rich red pulp of spleen from homozygous Cln3Δex7/8 mice, and, as previously described [8], subunit c accumulation was also abundant in liver hepatocytes, particularly those surrounding the central venules of hepatic lobules, as well as in cells likely corresponding to Kupffer cells, liver-resident macrophages (Fig. 7). Thus, subunit c storage is prominent in multiple hematopoietic tissues from homozygous Cln3Δex7/8 mice, and is particularly abundant in macrophage-lineage cells.


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)

Subunit c immunohistochemistry of major hematopoietic tissues from 12-week-old Cln3Δex7/8 mice.Representative images from bone marrow brush cytology, tibia cross-sections (‘Bone marrow histology’), and liver and spleen sections immunostained for subunit c are shown for wild-type (Cln3+/+) and homozygous mutant (Cln3Δex7/8/Δex7/8) mice (n = 2−5 mice per tissue/genotype). Inset in Cln3Δex7/8/Δex7/8 bone marrow cytology panel (top right panel) shows a sea-blue histiocyte (arrows) from an H&E stained preparation. Sea-blue histiocytes were not found in wild-type bone marrow cytology preparations. Brown stain reflects subunit c-positive storage material, which is most prominent in cells that appear morphologically consistent with macrophages. Arrowheads mark examples of subunit c filled Kupffer cells in liver, also a macrophage lineage cell. Insets in histology panels show lower power magnification of subunit c immunostain. Scale bars = 25 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368842&req=5

pone-0038310-g007: Subunit c immunohistochemistry of major hematopoietic tissues from 12-week-old Cln3Δex7/8 mice.Representative images from bone marrow brush cytology, tibia cross-sections (‘Bone marrow histology’), and liver and spleen sections immunostained for subunit c are shown for wild-type (Cln3+/+) and homozygous mutant (Cln3Δex7/8/Δex7/8) mice (n = 2−5 mice per tissue/genotype). Inset in Cln3Δex7/8/Δex7/8 bone marrow cytology panel (top right panel) shows a sea-blue histiocyte (arrows) from an H&E stained preparation. Sea-blue histiocytes were not found in wild-type bone marrow cytology preparations. Brown stain reflects subunit c-positive storage material, which is most prominent in cells that appear morphologically consistent with macrophages. Arrowheads mark examples of subunit c filled Kupffer cells in liver, also a macrophage lineage cell. Insets in histology panels show lower power magnification of subunit c immunostain. Scale bars = 25 µm.
Mentions: During our histological analysis of the bone marrow, we also noted the presence of sea-blue histiocytes, macrophages filled with ceroid lipofuscin storage material, in the marrow from homozygous Cln3Δex7/8 mice (shown in inset of Fig. 7). A similar finding has been reported in bone marrow from JNCL patients [39]. Thus, to more thoroughly determine the extent to which hematopoietic tissues in homozygous Cln3Δex7/8 mice exhibit the pathologic hallmark of JNCL, storage of mitochondrial subunit c, we analyzed bone marrow, spleen, and liver by subunit c immunohistochemistry. Only faint, punctate staining, likely corresponding to normal endogenous mitochondrial subunit c, was observed in tissues from normal mice. By contrast, consistent with the presence of relatively frequent sea-blue histiocytes in the Wright-Giemsa stained samples, bone marrow from homozygous Cln3Δex7/8 mice showed a striking accumulation of subunit c in cells that were morphologically consistent with macrophages (Fig. 7). Accumulation of subunit c was also noted in the macrophage-rich red pulp of spleen from homozygous Cln3Δex7/8 mice, and, as previously described [8], subunit c accumulation was also abundant in liver hepatocytes, particularly those surrounding the central venules of hepatic lobules, as well as in cells likely corresponding to Kupffer cells, liver-resident macrophages (Fig. 7). Thus, subunit c storage is prominent in multiple hematopoietic tissues from homozygous Cln3Δex7/8 mice, and is particularly abundant in macrophage-lineage cells.

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