AAV-Mediated GALC Gene Therapy Rescues Alpha-Synucleinopathy in the Spinal Cord of a Leukodystrophic Lysosomal Storage Disease Mouse Model

Abstract

Krabbe's disease (KD) is primarily a demyelinating disorder, but recent studies have identified the presence of neuronal protein aggregates in the brain, at least partially composed by alpha-synuclein (α-syn). The role of this protein aggregation in the pathogenesis of KD is largely unknown, but it has added KD to a growing list of lysosomal storage diseases that can be also be considered as proteinopathies. While the presence of these protein aggregates within the KD brain is now appreciated, the remainder of the central nervous system (CNS) remains uncharacterized. This study is the first to report the presence of thioflavin-S reactive inclusions throughout the spinal cord of both murine and human spinal tissue. Stereological analysis revealed the temporal and spatial accumulation of these inclusions within the neurons of the ventral spinal cord vs. those located in the dorsal cord. This study also confirmed that these thio-S positive accumulations are present within neuronal populations and are made up at least in part by α-syn in both the twitcher mouse and cord autopsied material from affected human patients. Significantly, neonatal gene therapy for galactosylceramidase, a treatment that strongly improves the survival and health of KD mice, but not bone marrow transplantation prevents the formation of these inclusions in spinal neurons. These results expand the understanding of α-syn protein aggregation within the CNS of individuals afflicted with KD and underlines the tractability of this problem via early gene therapy, with potential impact to other synucleinopathies such as PD.

Keywords: Parkinson's disease; alpha-synuclein; globoid cell leukodystrophy; proteinopathies; spinal cord.

Figure 1

Thioflavin-S accumulation in spinal cord across life span. Spinal sections from the cervical cord of post-natal day 7 (P7) (C), P15 (E), P30 (G), and P45 (I) twitcher mice were stained with thioflavin-S (thio-S) to reveal presence of protein inclusions. There is a progressive accumulation across lifespan with the earliest detection at P7 primarily present only in the ventral horn (C, red arrows). A similar pattern of accumulation was noted within the lumbar horns at the same time-points P7 (D, red arrows), P15 (F), P30 (H), and P45 (J). Cervical (A) and lumbar (B) spinal sections from a P45 wild type (WT) mouse reveal the background staining and confirm a lack of thio-S reactive material. Images depicted are representative of the thio-S inclusion burden within the cervical and lumbar regions of the spine. They do not represent a precise spinal level. Scale bars represent 0.5 mm.

Figure 2

Thioflavin-S quantification and comparison between ventral and dorsal spinal cords across life-span. Stereological analysis was performed to quantify thioflavin-S (thio-S) accumulation and compare thio-S density (inclusions/mm³) between ventral and dorsal spinal cord (A–D). Significantly more thio-S inclusions were observed in the ventral cord as compared to the dorsal at all timepoints (A–C) expect post-natal day 45 (P45) (D). Significance between means analyzed by ANOVA and Tukey's post-hoc analysis with (*) indicating p < 0.05, (**) p < 0.01. n = 3–4 animals. Results are presented as mean ± error of the mean.

Figure 3

Quantification and comparison of thioflavin-S accumulations across life-span. Stereological analysis of thioflavin-S accumulation density (inclusions/mm³) was performed. The ventral cervical and lumbar cords revealed large early accumulations at P7 and P15 with relatively slight additional accumulation over the final two time points (A,C). The dorsal spinal cord (B,D) showed a more protracted accumulation across life span. Psychopsine levels were measured in the cervical ventral (CV), cervical dorsal (CD), lumbar ventral (LV), and lumbar dorsal (LD) cord regions. This was compared across post-natal development (E) and in correlation to thio-s accumulation (F). (A–D) Significance between means analyzed by ANOVA and Tukey's post-hoc analysis with (*) indicating p < 0.05, (**) p < 0.01, and (***) p < 0.001, n = 3–4 animals. (E,F) Significance between means analyzed by ANOVA and Tukey's post-hoc analysis with (*) indicating p < 0.05 and (****) p < 0.0001 in comparison between CV and CD; (####) p < 0.0001 in comparison between LV and LD, n = 2–3 animals. Results are presented as mean ± error of the mean.

Figure 4

Thioflavin-S accumulations present in neuronal cell populations of spinal cord. Spinal tissue from TWI (A) and WT (B) animals was stained with thioflavin (thio-S) and the neuronal marker NeuN, which found thio-S inclusions within neurons of TWI but not within neurons of WT animals. Dual-staining in TWI with thio-S (C) and alpha-synuclein (α-syn) (D) revealed colocalization (F) of a-syn with the thio-S inclusions. Cell nuclei are highlighted by DAPI (E). Scale bars represents (A–F): 20 μm.

Figure 5

Alpha-synuclein staining in human Krabbe disease patients. Human spinal tissue from a patient with Krabbe (A–F) was stained with thioflavin-S (A,D) and for α-synuclein (α-syn) (B,E). Overlay of the two channels demonstrated colocalization of thioflavin-S reactive material with α-syn in cells of both the dorsal horn (C) and ventral horn (F). (G–I) [corresponding to the boxes in (D–F), respectively] show a cell with thioflavin-S and α-syn positive material as highlighted by the blue arrows compared to an unaffected cell highlighted by the white arrow. Cell comparison between panels is highlighted by white dashed line. Affected patients were both 2 years of age and in terminal stage of disease. Last, spinal tissue stained concurrently from an unaffected control case is shown in (J). Scale bar represents (A–F): 40 μm; (G–J): 20 μm.

Figure 6

Thioflavin-S positive inclusions are prevented with AAV-GALC gene therapy but not bone marrow transplantation. The extent of thio-S positive inclusions in the TWI (A) spinal cord, was not significantly improved with bone marrow transplantation (BMT) at either a P40 (D) or an aged (85 days of age) (E) timepoint. Treatment with AAV gene therapy either alone (F,G) or in combination with BMT (H,I) prevented the development of thio-S inclusions at both an early (P40) and late (aged, 350–550 days of age) timepoint. The staining present in the AAV and AAV+BMT treated TWI was comparable to WT mice at the same timepoints (B,C). Observations present in n = 3 animals for all groups except aged TWI-AAV which n = 2. Scale bars represent 0.5 mm.