Citalopram Reduces Aggregation of ATXN3 in a YAC Transgenic Mouse Model of Machado-Joseph Disease

Abstract

Machado-Joseph disease, also known as spinocerebellar ataxia type 3, is a fatal polyglutamine disease with no disease-modifying treatment. The selective serotonin reuptake inhibitor citalopram was shown in nematode and mouse models to be a compelling repurposing candidate for Machado-Joseph disease therapeutics. We sought to confirm the efficacy of citalopram to decrease ATXN3 aggregation in an unrelated mouse model of Machado-Joseph disease. Four-week-old YACMJD84.2 mice and non-transgenic littermates were given citalopram 8 mg/kg in drinking water or water for 10 weeks. At the end of treatment, brains were collected for biochemical and pathological analyses. Brains of citalopram-treated YACMJD84.2 mice showed an approximate 50% decrease in the percentage of cells containing ATXN3-positive inclusions in the substantia nigra and three examined brainstem nuclei compared to controls. No differences in ATXN3 inclusion load were observed in deep cerebellar nuclei of mice. Citalopram effect on ATXN3 aggregate burden was corroborated by immunoblotting analysis. While lysates from the brainstem and cervical spinal cord of citalopram-treated mice showed a decrease in all soluble forms of ATXN3 and a trend toward reduction of insoluble ATXN3, no differences in ATXN3 levels were found between cerebella of citalopram-treated and vehicle-treated mice. Citalopram treatment altered levels of select components of the cellular protein homeostatic machinery that may be expected to enhance the capacity to refold and/or degrade mutant ATXN3. The results here obtained in a second independent mouse model of Machado-Joseph disease further support citalopram as a potential drug to be repurposed for this fatal disorder.

Keywords: Neurodegeneration; Polyglutamine; Proteinopathy; Spinocerebellar ataxia; Therapy.

Figure 1:. Citalopram decreases ATXN3-positive intranuclear inclusions in the midbrain and hindbrain of Q84 mice.

(A) Four-week-old Q84 mice were orally treated with citalopram ~8 mg/kg or drinking water (vehicle) for 10 weeks, when they were euthanized and brains were collected for pathological assessment and protein analysis. (B) Brain slices were immunostained with anti-ATXN3 antibody (1H9) and cells containing ATXN3-positive intranuclear (nuc.) inclusions (black arrowheads) were counted in pontine nuclei, facial nuclei, vestibular nuclei, substantia nigra, and deep cerebellar nuclei (DCN) (n = 6–8 mice per condition, 1–4 slices per mouse). Graphs representing the quantification of cell count for each region show a robust decrease in number of cells containing ATXN3 intranuclear inclusions in all regions of citalopram-treated Q84 mice compared to vehicle-treated mice, except for the DCN. Scale bar= 50 μm. Bars represent the average percentage of cells with ATXN3 inclusions relative to vehicle-treated Q84 mice (± SEM), *P < 0.05 and **P < 0.01, normalized for total area using Visiopharm software.

Figure 2:. ATXN3 levels are reduced in the brainstem and spinal cord but not the cerebellum of Q84 mice treated with citalopram.

(A, B) Anti-ATXN3 immunoblotting of soluble protein extracts shows decreased HMW ATXN3, mutant human ATXN3, and endogenous mouse ATXN3 abundance in the brainstem of citalopram-treated mice (numbers 11–20) compared to vehicle-treated mice (numbers 1–10) (males 1–6 and 11–16; females 7–10 and 17–20). (B) Soluble protein extracts from spinal cord also show decrease levels of all forms of ATXN3. (C) Soluble proteins from cerebella of the same mice do not reveal any difference between treatment groups. Graphs show quantification of protein bands by densitometry. Bars represent the average percentage of protein species relative to vehicle-treated mice, corrected for Gapdh (± SEM). (D, E) Filter trap assay shows a trend toward reduction of insoluble ATXN3 in brainstem (P = 0.232) and in the spinal cord (P = 0.095) of citalopram-treated mice. (F) No differences in insoluble ATXN3 were found for cerebellar lysates (P = 0.421). Bars in the graph represent the average level of insoluble ATXN3 relative to vehicle-treated mice (± SEM). Statistical significance is indicated as *P < 0.05, **P < 0.01 and ***P < 0.001. Black circles indicate mice, whose results were excluded from statistical analysis because they harbored significantly lower CAG repeat numbers compared with the average (CAG)_n of the mouse group. Grey circle represents a mouse whose band density was excluded from statistical analysis for being positioned in the last lane of the gel and consequently of unreliable value due to technical reasons.

Figure 3:. 12 week-old Q84 mice show decreased levels of key molecular chaperones in the spinal cord but not cerebellum compared to wt littermates.

Western blot analysis of total soluble protein lysates from spinal cords of Q84 mice show decreased levels of Hsp90α, Hsp90β, and Hsp40 relative to controls (A). No significant changes were observed for any assessed chaperones in cerebellar extracts of Q84 mice (B). (A & B) Left panels show immunoblots of indicated proteins in soluble fractions of spinal cord or cerebellum from Q84 and control littermates (Supplementary Table 2). Right panels show graphs representing the quantification of protein bands by densitometry. Bars represent the average percentage of protein species relative to wt mice, corrected for Gapdh (± SEM). Statistical significance is indicated as *P < 0.05 and **P < 0.01.

Figure 4:. Citalopram reduces levels of Hsp90β in the brainstem and spinal cord and abundance of Hsp40 in the cerebellum of Q84 mice.

Citalopram-treated mice show significantly decreased levels of Hsp90β in brainstem (A) and spinal cord (B) extracts, and reduced amount of Hsp40 in cerebellar lysates (C). (A, B, and C) Left panels show immunoblots of indicated proteins in soluble fractions of brainstem, spinal cord and cerebellum. Right panels display quantification of band intensity, with values normalized to Gapdh. Bars represent the average percentage of protein relative to vehicle-treated mice (± SEM). Statistical significance is indicated as **P < 0.01. Mice 1, 2, and 20 were excluded from statistical analysis.

Figure 5:. Q84 mice treated with citalopram reveal slight alterations of macroautophagy components in spinal cord and cerebellum.

Assessment of macroautophagy markers shows increased levels of p62 in the spinal cord (B), and of p62 and beclin-1 in the cerebellum (C) of citalopram-treated mice compared to controls. Citalopram treatment did not affect levels of LC3 in any of the three regions (A-C). (A, B, and C) Left panels show immunoblots of indicated proteins in soluble fractions of brainstem, spinal cord and cerebellum. Right panels display quantification of band intensity, with values normalized to Gapdh. Bars represent the average percentage of protein relative to vehicle-treated mice (± SEM). Statistical significance is indicated as *P < 0.05 and **P < 0.01. Mice 1, 2, and 20 were excluded from statistical analysis. Diamond (◆) represents p62 bands.