CNS-expressed cathepsin D prevents lymphopenia in a murine model of congenital neuronal ceroid lipofuscinosis

Abstract

Deficiency in Cathepsin D (CtsD), the major cellular lysosomal aspartic proteinase, causes the congenital form of neuronal ceroid lipofuscinoses (NCLs). CtsD-deficient mice show severe visceral lesions like lymphopenia in addition to their central nervous system (CNS) phenotype of ceroid accumulation, microglia activation, and seizures. Here we demonstrate that re-expression of CtsD within the CNS but not re-expression of CtsD in visceral organs prevented both central and visceral pathologies of CtsD(-/-) mice. Our results suggest that CtsD was substantially secreted from CNS neurons and drained from CNS to periphery via lymphatic routes. Through this drainage, CNS-expressed CtsD acts as an important modulator of immune system maintenance and peripheral tissue homeostasis. These effects depended on enzymatic activity and not on proposed functions of CtsD as an extracellular ligand. Our results furthermore demonstrate that the prominent accumulation of ceroid/lipofuscin and activation of microglia in brains of CtsD(-/-) are not lethal factors but can be tolerated by the rodent CNS.

Figure 1

CNS application of AAV-CtsD prolonged lifetime of CtsD^−/− mice. Survival times in days are shown for CtsD^−/− mice injected with control virus (n = 6) or PBS (n = 8) (A), injected into visceral organs with AAV-CtsD (n = 10) (B), injected into brain either unilaterally (n = 4, black circles) or bilaterally (n = 3, white circles) with AAV-CtsD (total n = 7) (C), injected into brain plus periphery with AAV-CtsD (n = 6) (D), and injected with the enzymatically inactive CtsD_Asn231 mutant into the brain (n = 7) (E). Survival times were significantly different between controls (PBS/AAV-EGFP) and AAV-CtsD brain-injected animals, and between animals survived for about 60, 130, or 190 days after AAV-CtsD brain + visceral injection, as assessed by Student’s t test at P < 0.01.

Figure 2

AAV-mediated CtsD expression prevents ceroid accumulation and microglia activation in CtsD^−/− mice. Immunohistochemical staining for activated microglia (MCA711, red, as overlay with nuclear DAPI stain, blue) at day p27 in the thalamus of CtsD^+/− mice (A), CtsD^−/− mice (B), and CtsD^−/− mice injected into CNS with AAV-CtsD (C). High-power micrographs show deposition of autofluorescent ceroid (green) in CtsD^−/− mice (D) and fulminate accumulation of microglia in vicinity to these deposits (E), whereas both ceroid and activated microglia are absent in AAV-CtsD injected CtsD^−/− mice (F). D and E represents the rectangle in panel B. Scale bar = 500 μm (A–C), 50 μm (D–F). Representative pictures taken from n = 4 or 5 per condition.

Figure 3

Integrity of visceral organs after re-expression of CtsD in CNS and recurrence of disease after adolescence. Overview of brain, gastro-intestinal system, spleen, and liver morphology and FACS analysis of CD4⁺/CD8+ double positive thymocytes, at day p 26 of CtsD +/+ mice (A), CtsD^−/− mice AAV-EGFP injected (B), and CtsD^−/− mice AAV-CtsD injected into CNS (C). Tissue morphology around day p60 is shown for CtsD^+/− mice (D), for AAV-CtsD brain injected^−/− mice of healthy phenotype (E), and for AAV-CtsD brain injected mice of moribund phenotype (F). Scale bar = 12 mm. Weight loss over time of AAV-CtsD brain injected animals (solid line) and untreated CtsD^−/− animals (dotted line) as compared with sex and age matched littermates is shown (G).

Figure 4

Re-expression of CtsD within CNS prevents lymphopenia in CtsD^−/− mice. The percentage of CD4⁺/CD8⁺ double positive thymocytes was determined from thymi isolated at days p21 (A), p24 (B), and p26 (C) from +/+, +/−, untreated −/−, and AAV-CtsD brain injected −/− mice by FACS analysis. n = 3 to 5 animals for each diagram in A, n = 1 for each diagram in B, demonstrating the spectrum of CD4⁺/CD8+ thymocytes depletion at this state, and n = 4 to 6 animals for each diagram in C.

Figure 5

Quantitative analysis of viral vector spread and CtsD delivery to CNS and visceral tissues. Representative Western blot for detection of EGFP in visceral tissues after CNS injection of AAV-EGFP into CtsD^−/− mice is shown (A). Western blot analysis of CtsD levels at p22 is shown for control mice (B), for untreated −/− mice (C), for −/− mice injected into CNS with AAV-CtsD (D), and for −/− mice injected into periphery with AAV-CtsD (E). Each blot shown is representative of tissue analysis of n = 3 animals per condition. Blot A was probed with an anti-EGFP antibody, blots B–E were probed with an anti-CtsD antibody. In B–E a size standard for CtsD from control brain was used to distinguish CtsD immunoreactivity from background signals. Please note that in E a batch of standard containing higher protein concentration than those used in B–D was used. Otherwise, each lane of the Western blots represents 40 μg of loaded protein.

Figure 6

CNS ceroid accumulation is not responsible for death of CtsD^−/− mice. Representative brain sections from CtsD^−/− mouse injected with AAV-CtsD bilaterally into CNS and presenting with a completely moribund phenotype at day p62 are shown in A, C, E, G, and I, whereas contralateral brain sections from a CtsD^−/− mouse injected with AAV-CtsD unilaterally into CNS and additionally into periphery presenting with a healthy phenotype at p133 is shown in B, D, F, H, and J. Note that J was taken from a more rostral part of the cerebellum as compared with I. Pictures are overlays of wide field micrographs showing ceroid autofluorescence in green, activated microglia stained by MCA711 in red, and nuclear counter stain by DAPI in blue.