Intracranial delivery of CLN2 reduces brain pathology in a mouse model of classical late infantile neuronal ceroid lipofuscinosis

Abstract

Classical late infantile neuronal ceroid lipofuscinosis (cLINCL) is a lysosomal storage disorder caused by mutations in CLN2, which encodes lysosomal tripeptidyl peptidase I (TPP1). Lack of TPP1 results in accumulation of autofluorescent storage material and curvilinear bodies in cells throughout the CNS, leading to progressive neurodegeneration and death typically in childhood. In this study, we injected adeno-associated virus (AAV) vectors containing the human CLN2 cDNA into the brains of CLN2(-/-) mice to determine therapeutic efficacy. AAV2CUhCLN2 or AAV5CUhCLN2 were stereotaxically injected into the motor cortex, thalamus, and cerebellum of both hemispheres at 6 weeks of age, and mice were then killed at 13 weeks after injection. Mice treated with AAV2CUhCLN2 and AAV5CUhCLN2 contained TPP1 activity at each injection tract that was equivalent to 0.5- and 2-fold that of CLN2(+/+) control mice, respectively. Lysosome-associated membrane protein 1 immunostaining and confocal microscopy showed intracellular targeting of TPP1 to the lysosomal compartment. Compared with control animals, there was a marked reduction of autofluorescent storage in the AAV2CUhCLN2 and AAV5CUhCLN2 injected brain regions, as well as adjacent regions, including the striatum and hippocampus. Analysis by electron microscopy confirmed a significant decrease in pathological curvilinear bodies in cells. This study demonstrates that AAV-mediated TPP1 enzyme replacement corrects the hallmark cellular pathologies of cLINCL in the mouse model and raises the possibility of using AAV gene therapy to treat cLINCL patients.

Figure 1.

TPP1 activity in the CLN2^−/− brain at 6 weeks after injection. Brains were cut into 2 mm coronal slabs as shown at the bottom, with corresponding amount of enzyme activity plotted above for the following samples: AAV2_CUhCLN2 ipsilateral hemisphere (black circles), AAV2_CUhCLN2 contralateral hemisphere (gray circles), and control AAV2_CUNULL or saline ipsilateral hemisphere (white circles). TPP1 activity approached wild-type levels in the brain slab (3) that contained the hippocampus and thalamus injection sites. The two adjacent slabs (2 and 4) contained some elevated levels of TPP1 activity. The rostral brain (slab 1), cerebellum (slab 5), and the entire contralateral (uninjected) hemisphere of AAV2_CUhCLN2-treated brains had levels of enzyme activity indistinguishable from AAV2_CUNULL-treated and saline brains. The dashed lines correspond to the relative levels of TPP1 activity in untreated CLN2^+/− and CLN2^+/+ control brains. The total number of animals was n = 6 for AAV2_CUhCLN2 injected and n = 5 for control injected (AAV2_CUNULL and saline). Asterisks in the brain image show location of the injection sites. Data are means ± SEM.

Figure 2.

TPP1 expression in the CLN2^−/− brain at 6 weeks after injection with AAV2_CUhCLN2. TPP1 immunopositive cells were detected in the hippocampus and thalamus of the ipsilateral hemisphere but not in the contralateral hemisphere (A). Close-up of the ipsilateral hippocampus showed that there was no detectable TPP1 immunostaining in untreated brains (B) compared with robust TPP1 immunostaining in AAV2_CUhCLN2-treated brains (C). High-magnification confocal images of cell bodies in the hippocampus demonstrated that TPP1 (D) and LAMP1 (E) were present in an overlapping punctate pattern (F, yellow signal). High-magnification confocal images of CA3 pyramidal dendrites showed positive immunostaining for TPP1 (G). Scale bars: A, 1 mm; B, C, 100 μm; D–G, 20 μm.

Figure 3.

Overlapping regions of TPP1 expression and reduction of autofluorescent storage material at 13 weeks after injection. AAV2_CUhCLN2 (A–D) or AAV2_CUNULL (E, F) was injected into the hippocampus (A, C, E) and thalamus (B, D, F). TPP1 was present in the injected structures (A, B). This resulted in a substantial reduction of autofluorescent storage material in both structures (C, D). In contrast, injection of AAV2_CUNULL did not reduce autofluorescent storage in the hippocampus or thalamus (E, F). Untreated CLN2^+/+ control mice contained undetectable levels of autofluorescence in the hippocampus (G) and thalamus (H). Scale bar, 250 μm.

Figure 4.

Comparison of TPP1 activity in AAV2_CUhCLN2- or AAV5_CUhCLN2-treated brains at 13 weeks after injection. AAV2-injected brains (black circles) contained heterozygote levels of TPP1 activity in brain slabs containing the injection sites (slabs 2, 3, 5). Despite matching titers, AAV5-injected brains (white circles) had substantially higher levels of TPP1 activity in the same corresponding brain slabs. Disparate enzyme activity was also detected in the frontal brain (slab 1) with the two serotype vectors. AAV-treated brains had essentially null levels of TPP1 activity in slab 4. The dashed lines correspond to the relative levels of TPP1 activity in untreated CLN2^+/− and CLN2^+/+ brains. Asterisks in the brain image show location of the injection sites. Data are means ± SEM.

Figure 5.

Comparison of TPP1 expression between AAV2_CUhCLN2 (A, C, F, H, L) and AAV5_CUhCLN2 (B, D, E, G, I–K, M) at 13 weeks after injection. Robust TPP1 immunopositive cells were detected in the thalamus with AAV2 (A) and AAV5 (B). Adjacent sagittal sections contained many cells positive for TPP1 in the motor cortex (C, D). A 100× confocal image of a Purkinje cell with TPP1 (green) in cytoplasmic vesicles consistent with endosomal-lysosomal targeting (E). Double immunohistochemistry showed that the TPP1 (green) surrounded NeuN-positive nuclei (red), confirming that both serotype vectors were neurotrophic (F, G). In contrast, very few cells costained for TPP1 (green) and GFAP (red), demonstrating that astrocytes were inefficiently transduced by AAV2 or AAV5 (H, I). Low (J) and high (K) magnification confocal images of AAV5_CUhCLN2-treated cerebellum showed that many Purkinje cells contained TPP1. There were substantially less Purkinje cells positive for TPP1 in AAV2_CUhCLN2-treated brains (L). Purkinje cell loss in the cerebellum of an AAV5-treated brain (M). Scale bars: A, B, 2 mm; C, D, 50 μm; E, 5 μm; F–I, 20 μm; J, 200 μm; K–M, 40 μm.

Figure 6.

Reduction of brain autofluorescence with AAV2_CUhCLN2 or AAV5_CUhCLN2 at 13 weeks after injection. Shown are the motor cortex (A, D, G), thalamus (B, E, H), and striatum (C, F, I). Untreated CLN2^−/− mice have high levels of autofluorescent storage material at 19 weeks of age (A–C). Injection of AAV2_CUhCLN2 (D–F) or AAV5_CUhCLN2 (G–I) substantially reduced autofluorescent storage. The intensity of autofluorescence was quantified from exposure-matched digital images, and the results were plotted as the percentage of autofluorescent storage to untreated CLN2^−/− control mice (J). The background level of autofluorescence in untreated CLN2^+/+ mice was <5%. A total of four animals were analyzed from each group. Black bars, AAV2_CUhCLN2-treated CLN2^−/− mice; white bars, AAV5_CUhCLN2-treated CLN2^−/− mice. Data are means ± SEM. Scale bar: 250 μm. MC, Motor cortex; ST, striatum; HC, hippocampus; TH, thalamus; CB, cerebellum.

Figure 7.

Electron microscope analysis of curvilinear bodies in AAV-treated and untreated thalamus. Untreated CLN2^−/− brains contain ultrastructural inclusions (arrow) classified as curvilinear bodies (A). In contrast, every cell from CLN2^+/− mice (B) and the majority of cells in CLN2^−/− mice treated with AAV2_CUhCLN2 (C) or AAV5_CUhCLN2 (D) did not contain these inclusions. A random sampling of 120 cells per group were analyzed, and the percentage of cells without or with curvilinear bodies of various sizes were plotted as stacked bar graphs (E). Black bars, The percentage of cells that contain inclusions with a combined area of 9 μm² or greater; gray bars, the percentage of cells that contain inclusions with a combined area of 2–8 μm²; hatched bars, the percentage of cells that contain inclusions with a combined area of 1 μm² or less; white bars, the percentage of cells that contain no inclusions. Scale bar, 1 μm. C, Curvilinear body; N, nucleus.