Sortilin inhibition treats multiple neurodegenerative lysosomal storage disorders

Abstract

Lysosomal storage disorders (LSDs) are a genetically and clinically diverse group of diseases characterized by lysosomal dysfunction. Batten disease is a family of severe LSDs primarily impacting the central nervous system. Here we show that AF38469, a small molecule inhibitor of sortilin, improves lysosomal and glial pathology across multiple LSD models. Live-cell imaging and comparative transcriptomics demonstrates that the transcription factor EB (TFEB), an upstream regulator of lysosomal biogenesis, is activated upon treatment with AF38469. Utilizing CLN2 and CLN3 Batten disease mouse models, we performed a short-term efficacy study and show that treatment with AF38469 prevents the accumulation of lysosomal storage material and the development of neuroinflammation, key disease associated pathologies. Tremor phenotypes, an early behavioral phenotype in the CLN2 disease model, were also completely rescued. These findings reveal sortilin inhibition as a novel and highly efficacious therapeutic modality for the treatment of multiple forms of Batten disease.

Figure 1.. Inhibition of sortilin through AF38469 (40 nM, 400 nM, 4μM) impacts the accumulation of ASM in cell models of Batten disease.

Mouse embryonic fibroblasts (MEFs) and primary cortical neurons (PNCs) were isolated from E15.5 embryos from Cln1^R151X, Cln2^R207X, Cln3^Δex7/8, Cln6^nclf, and Cln8^mnd mouse lines. Cln11^−/− fibroblasts were isolated from adult Cln11^−/− mice. All cell lines were dosed with drug-containing media on day in vitro (DIV) 3 and DIV5 and analyzed on DIV7 using the CellInsight CX7 High-Content Screening Platform (CX7) (A) Representative ASM accumulation in Cln3^Δex7/8 MEFs and PNCs. Upon treatment with AF38469 (40 nM, 400 nM, 4μM), ASM accumulation was (B-F) reduced in Cln1^R151X Cln2^R207X, Cln3^Δex7/8, Cln6^nclf, and Cln8^mnd MEFs; (B-D, F) reduced in Cln1^R151X Cln2^R207X, Cln3^Δex7/8, and Cln8^mnd PNCs; and (E, G) increased in Cln6^nclf PNCs and Cln11^−/− MEFs. Mean ± S.E.M.. One-way ANOVA with a Šidák post-hoc test, *^/#p<0.05, **^/##p<0.01, ***^/###p<0.001, ****^/####p<0.0001. Hashsigns indicate comparison to WT, asterisks indicate comparison to mutant vehicle. MEFs and PNCs n=1000-4000 and 4000-20000 cells/treatment group, respectively. Scale bar, 100 μm, Inset scale bar, 50 μm.

Figure 2. Inhibition of sortilin through acute treatment with AF38469 stimulates TFEB and TFE3 nuclear translocation and upregulation of TFEB gene targets.

Wild type Neuro 2A cells were transfected with pEGFP-N1-TFEB or pEGFP-N1-TFE3 and acutely treated with vehicle or AF38469 (40 nM). (A) Treatment with AF38469 significantly stimulated TFEB and TFE3 nuclear translocation after 90 minutes of treatment as measured by live cell imaging. Expression analysis of lysosomal differentially expressed genes (DEGs) in wild type AF38469 (40 nM) treated MEFs compared to vehicle treated (B) showing up and downregulation of lysosomal DEGs not regulated by TFEB compared to (C) showing distinct pattern of upregulation in TFEB targeted lysosomal DEGs organized by adjusted p-value and fold change. (D) Expression analysis of all DEGs. (E) Table of select enriched upregulated and downregulated DAVID GO terms from DEG analysis. Mean ± S.E.M. One-way ANOVA, Dunnett’s multiple comparisons. *p <0.05, **p<0.01, ****p<0.0001. A: n=5000-6500 cells/treatment. For B-F: RNA collected from 3 wells/treatment. Adjusted p-value and fold change.

Figure 3. Short-term treatment with AF38469 improves histopathological and behavioral outcomes in Cln2^R207X mice.

(A) Homozygous Cln2^R207X and littermatewild type mice received continuous treatment with AF38469 or vehicle via drinking water starting at wean until 11 weeks of age. (B) Treatment with AF38469 (0.03 μg/ml) significantly increased PPT1 enzyme activity levels in Cln2^R207X treated mice. (C) Treatment with AF38469 had no impact on TPP1 enzyme activity levels Cln2^R207X treated mice. (D, E) AF38469 treatment (0.3 μg/ml) in Cln2^R207X mice significantly prevented SubC accumulation and had no impact on microglial reactivity (CD68⁺) or astroglial activation (GFAP⁺) in the S1BF of the somatosensory cortex and CA3 of the hippocampus. (F) Treated Cln2^R207X mice (0.3 μg/ml) saw significant prevention of accumulation of mitochondrial ATP synthase subunit C (SubC⁺) and reduction of microgliosis (CD68⁺) in the VPM/VPL of the thalamus with no impact on astrocytosis (GFAP⁺). (G) Treatment with AF38469 had no impact on CLN2 Batten disease pathology in the dentate gyrus and hilus regions of the hippocampus. (H-K) Tremor index scores were reduced and restored to wild type levels in AF38469 treated Cln2^R207X mice when compared to vehicle treated mice. Mean ± S.E.M. (B-G) Nested one-way ANOVA with a Šidák post-hoc test (H-K) One-way ANOVA with a Šidák post-hoc test *^/#p<0.05, **^/##p<0.01, ***^/###p<0.001, ****^/####p<0.0001. Hashsigns indicate comparison to WT, asterisks indicate comparison to mutant vehicle. Scale bar, 100 μm. n = 6-8 animals/treatment.

Figure 4. Short-term treatment with AF38469 improves histopathological outcomes in Cln3^Δex7/8 mice.

(A) Homozygous Cln3^Δex7/8 and wild type mice received continuous treatment with AF38469 or vehicle via drinking water starting at wean until 16 weeks of age. B) Treatment with AF38469 had no impact on PPT1 enzyme activity levels in Cln3^Δex7/8 treated mice (C) Treatment with AF38469 significantly increased TPP1 enzyme activity levels in Cln3^Δex7/8 treated mice. (D, E) In the S1BF of the somatosensory cortex and CA3 of the hippocampus, AF38469 treatment in Cln3^Δex7/8 mice significantly prevented SubC accumulation and microgliosis (CD68⁺). Treatment with the low and high dose significantly prevented astrocyte activation in the S1BF and CA3, respectively. (F, G) Treated Cln3^Δex7/8 mice saw significant prevention of accumulation of mitochondrial ATP synthase subunit C (SubC⁺) and reduction of microgliosis and astrocytosis (CD68⁺, GFAP⁺) in the VPM/VPL of the thalamus and dentate gyrus and hilus regions of the hippocampus. Mean ± S.E.M. Nested one-way ANOVA with a Šidák post-hoc test *^/#p<0.05, **^/##p<0.01, ***^/###p<0.001, ****^/####p<0.0001. Hashsigns indicate comparison to WT, asterisks indicate comparison to mutant vehicle. Scale bar, 100 μm. n =6-8 animals/treatment.