Modulation of Brain Cholesterol Metabolism through CYP46A1 Overexpression for Rett Syndrome

Abstract

Rett syndrome (RTT) is a rare neurodevelopmental disorder caused by mutation in the X-linked gene methyl-CpG-binding protein 2 (Mecp2), a ubiquitously expressed transcriptional regulator. RTT results in mental retardation and developmental regression that affects approximately 1 in 10,000 females. Currently, there is no curative treatment for RTT. Thus, it is crucial to develop new therapeutic approaches for children suffering from RTT. Several studies suggested that RTT is linked with defects in cholesterol homeostasis, but for the first time, therapeutic evaluation is carried out by modulating this pathway. Moreover, AAV-based CYP46A1 overexpression, the enzyme involved in cholesterol pathway, has been demonstrated to be efficient in several neurodegenerative diseases. Based on these data, we strongly believe that CYP46A1 could be a relevant therapeutic target for RTT. Herein, we evaluated the effects of intravenous AAVPHP.eB-hCYP46A1-HA delivery in male and female Mecp2-deficient mice. The applied AAVPHP.eB-hCYP46A1 transduced essential neurons of the central nervous system (CNS). CYP46A1 overexpression alleviates behavioral alterations in both male and female Mecp2 knockout mice and extends the lifespan in Mecp2-deficient males. Several parameters related to cholesterol pathway are improved and correction of mitochondrial activity is demonstrated in treated mice, which highlighted the clear therapeutic benefit of CYP46A1 through the neuroprotection effect. IV delivery of AAVPHP.eB-CYP46A1 is perfectly well tolerated with no inflammation observed in the CNS of the treated mice. Altogether, our results strongly suggest that CYP46A1 is a relevant target and overexpression could alleviate the phenotype of Rett patients.

Keywords: AAVPHP.eB; Rett syndrome; cholesterol pathway; gene therapy.

Figure 1

Experimental design for the assessment of the therapeutic efficacy of intravenous AAVPHP.eB-hCYP46A1-HA administration in Mecp2 knockout mouse model (Mecp2 KO) for Rett syndrome. (A) Study of the therapeutic efficacy of AAVPHP.eB-hCYP46A1-HA treatment in male Mecp2−/y mice. Mecp2 KO males aged at 3 weeks received an injection of AAVPHP.eB-hCYP46A1-HA at 5.1011vg total intravenously by retro-orbital injection. Animals necropsied either 3 weeks (named cohort 1 with WT (n = 8); untreated (NT; n = 8); treated (n = 7) Mecp2 KO mice) or 7 weeks (named cohort 2; n = 8 for each groups) after AAV administration. Another cohort (named survival cohort) maintained to study the survival of Mecp2−/y mice (WT (n = 13); NT (n = 45); treated (n = 13) Mecp2 KO). Behavioral tests (weight, clasping tests) performed before injection of the treatment and each week after the treatment administration. Different organs (CNS and peripheral organs) collected to perform histological and molecular analysis. (B) Study of the therapeutic efficacy of AAVPHP.eB-hCYP46A1-HA treatment in Mecp2+/− female mice. Mecp2 KO females aged at 12 weeks received an injection of AAVPHP.eB-hCYP46A1-HA at 5.1011vg total intravenously by retro-orbital injection. Animals sacrificed 23 weeks (named female cohort; WT (n = 10); NT (n = 9); treated (n = 9) Mecp2 KO mice) after AAV administration. Behavioral tests (weight, clasping tests) performed before injection of the treatment and every two weeks after the treatment administration. Different organs (CNS and peripheral organs) collected to perform histological and molecular analysis.

Figure 2

AAVPHP.eB-hCYP46A1-HA efficiently transduces central nervous system of 6-week-old and 10-week-old Mecp2-/y knockout mice. (A,B) Biodistribution of the AAVPHP.eB-hCYP46A1-HA in central nervous system (A) and peripheral organs (B) in 6-week-old Mecp2 KO mice, thus 3 weeks after intravenous injection (n = 7). A symbol was used for each mouse. VGC for vector genome copy number per 2n genome. (C,D) Biodistribution of the AAVPHP.eB-hCYP46A1-HA in central nervous system (C) and peripheral organs (D) in 10-week-old Mecp2 KO males, thus 7 weeks after intravenous injection (n = 8). A symbol was used for each mouse. (E–K) Immunofluorescence detection of hCYP46A1-HA (red) on sagittal sections in the brain (E) and high magnification of different brain areas (F–K), i.e., cerebral cortex (F), striatum (G), thalamus (H), hippocampus (I), hypoglossal nucleus (J), and cerebellum (K), in 6-week-old treated Mecp2 KO mice with AAVPHP.eB-hCYP46A1-HA. (L–Q) Immunofluorescence detection of hCYP46A1-HA (red) and of NeuN or CB (green, CB only for cerebellum) on sagittal sections in the cortex (L), striatum (M), thalamus (N), hippocampus (O), medulla (P), and cerebellum (Q) in 6-week-old treated Mecp2 KO male mice with AAVPHP.eB-hCYP46A1-HA. Insert in (L–Q) shows a co-localization of hCYP46A1-HA in neurons or Purkinje cells (NeuN, CB, green). (R) Quantification of HA positive cells in NeuN or CB in different brain areas. (S,T) Immunofluorescence detection of hCYP46A1-HA (red) and of GFAP (S) or of Iba1 (T) on sagittal sections in the medulla. Insert in (S,T) shows a co-localization of hCYP46A1-HA in astrocytes or microglial cells (GFAP or Iba1, green). (U,V) Quantification of HA positive cells in GFAP (U) or Iba1 (V) in different brain areas. Data are represented as mean ± SEM. CB for calbindin. Scale Bars: 1000 µm (E); 100 µm (F–Q,S,T).

Figure 3

AAVPHP.eB-CYP46A1-HA efficiently transduces central nervous system of Mecp2+/− female mice. (A,B) Biodistribution of the AAVPHP.eB-hCYP46A1- HA in central nervous system (A) and peripheral organs (B) in 35-week-old Mecp2+/− female mice, 23 weeks after intravenous injection of AAVPHP.eB-CYP46A1- HA (n = 9). A symbol was used for each mouse. VGC for vector genome copy number per 2n genome. (C–I) Immunofluorescence detection of CYP46A1-HA (red) on sagittal sections in the brain (C) and high magnification of different brain areas (×40) (D–I), i.e., cerebral cortex (D), striatum (E), thalamus (F), hippocampus (G), hypoglossal nucleus (H), and cerebellum (I) in 35-week-treated Mecp2 KO female mice with AAVPHP.eB-hCYP46A1-HA. (J–O) Immunofluorescence detection of hCYP46A1-HA (red) and of NeuN or CB (green, CB only for cerebellum) on sagittal sections in the cortex (J), striatum (K), thalamus (L), hippocampus (M), medulla (N), and cerebellum (O) in 35-week-old treated Mecp2 KO females with AAVPHP.eB-hCYP46A1-HA. Insert in (J–O) shows a co-localization of hCYP46A1-HA in neurons or Purkinje cells (NeuN, CB, green). (P) Quantification of HA positive cells in NeuN or CB in different brain areas. (Q,S) Immunofluorescence detection of hCYP46A1-HA (red) and of GFAP (Q) or of Iba1 (S) on sagittal sections in the cortex. Insert in (Q,S) shows a co-localization of hCYP46A1-HA in astrocytes or microglial cells (GFAP or Iba1, green). (R,T) Quantification of HA positive cells in GFAP (R) or Iba1 (T) in different brain areas. CB for calbindin. Data are represented as mean ± SEM. Scale bars: 1000 µm (C); 500 µm (N); 100 µm (J–M,O,Q,S).

Figure 4

Injection of AAVPHP.eB-hCYP46A1-HA in Mecp2 KO mice leads to an overexpression of hCYP46A1 in the brain of treated Mecp2 KO mice. Quantitative expression of human CYP46A1 and murine CYP46A1 in brain of treated Mecp2 KO male aged at 6 (A) or 10 weeks (B,C) and female mice aged at 35 weeks (D,E). Expression values are normalized to those for WT mice. Data are represented as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.0001. Black stars for p value compared to WT and grey stars to compare to Mecp2 KO. CYP46A1 for cholesterol 24S-hydroxylase.

Figure 5

Overexpression of hCYP46A1-HA in Mecp2 KO mice improves motor impairment and the survival of 10 days for male mice. (A) Weight follow-up of WT, untreated, AAVPhP.eB-null, or treated Mecp2 KO male mice. Red arrow shows the day of AAVPHP.eB-hCYP46A1-HA administration. (B) Clasping score follow-up of WT, untreated, AAVPhP.eB-null, or treated Mecp2 KO male mice. (C) Weight follow-up of WT, untreated, or treated Mecp2 KO female mice. Red arrow shows the day of AAVPHP.eB-hCYP46A1-HA administration. (D) Clasping score follow-up in WT, untreated, or treated Mecp2 KO female mice. (E) Survival curve for WT, untreated, AAVPhP.eB-null, or AAVPHP.eB-hCYP46A1-HA Mecp2 KO male mice. Data are represented as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.0001. Black star for p value compared to WT and grey stars to compare to Mecp2 KO and Blue stars to compare to AAVPHP.eB-null Mecp2 mice.

Figure 6

Overexpression of hCYP46A1-HA in Mecp2 KO males increases the level of some proteins or genes involved in cholesterol pathways and mitochondrial activity. (A) Levels of cholesterol, 7-lathostherol, 8-Dehydrocholesterol (8-DHC), 7-Dehydrocholesterol (7-DHC), desmosterol, lanostherol, 24-hydroxycholesterol (24-OH), 25-hydroxycholesterol (25-OH), and 27-hydroxycholesterol (27-OH) on brain from WT, untreated, or treated Mecp2 KO male mice aged 6 weeks. (B) Quantitative expression of ApoE, SREBP1, SREBP2, HMGCR, DHCR7, and DGAT1 on brain from WT, untreated, or treated MECP2 KO male mice aged 6 weeks. Expression values are normalized to those for WT mice. (C–H) Quantitative expression of ApoE (C), SREBP1 (D), SREBP2 (E), HMGCR (F), DHCR7 (G), and DGAT1 (H) on brain from in WT, untreated, or treated Mecp2 KO males aged 10 weeks. Expression values are normalized to those of WT mice. (I–N) Quantitative expression of ApoE (I), SREBP1 (J), SREBP2 (K), HMGCR (L), DHCR7 (M), and DGAT1 (N) on brain from in WT, untreated, or treated Mecp2 KO females aged 35 weeks. (O) Mitochondrial aconitase activity on brain from in WT, untreated, or treated Mecp KO male mice aged 10 weeks. (P) Mitochondrial aconitase activity on brain from in WT, untreated, or treated Mecp2 KO female mice aged 35 weeks. Expression values are normalized to those for WT mice. ApoE: Apolipoprotein E; SREBP1: Sterol Regulatory Element-Binding Protein 1; SREBP2: Sterol Regulatory Element-Binding Protein 2; HMGCR: 3-Hydroxy-3-Methylglutaryl-CoA Reductase; DHCR7: 7-Dehydrocholesterol Reductase; DGAT1: Diacylglycerol O-Acyltransferase 1. Data are represented as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.0001. Black stars for p value compared to WT and grey stars to compare to Mecp2 KO.

Figure 7

An injection of AAVPHP.eB-hCYP46A1-HA in 6-week-old Mecp2 KO males does not lead to neuroinflammation. (A–C) Immunohistochemistry detection of IBA1 on sagittal sections in the brain of 6-week-old WT, untreated, or treated MECP2-/y male mice with AAVPHP.eB-hCYP46A1-HA. Insets are high magnification of tissue section. (D) Quantification of IBA1-positive cells per mm² in cerebral cortex, striatum, hippocampus, and cerebellum of WT, untreated, and treated Mecp2 KO males. (E–G) Immunohistochemistry detection of GFAP on sagittal sections in the brain of 6-week-old WT, untreated, or treated Mecp2 KO males with AAVPHP.eB-hCYP46A1-HA. Insets are high magnification of tissue section. (H) Quantification of GFAP-positive cells per mm² in cerebral cortex, striatum, hippocampus, and cerebellum of WT, untreated, and treated Mecp2 KO males. (I–K) Hematoxylin–eosin staining on sagittal sections in the brain of 6-week-old WT, untreated, or treated Mecp2 KO males with AAVPHP.eB-hCYP46A1-HA. Insets are high magnification of tissue section. (L) Quantification of numbers of Purkinje cells per mm in cerebellum of WT, untreated, and treated Mecp2 KO male mice. Scale bar = 250 µm (A–G) or 100 µm (I–K). Data are represented as mean ± SEM. * p < 0.05; ** p < 0.01. Black stars for p value compared to WT and grey stars to compare to Mecp2 KO.

Figure 8

An injection of AAVPHP.eB-hCYP46A1-HA in 35-week-old Mecp2 KO females does not lead to a severe neuroinflammation. (A–C) Immunofluorescence detection of Iba1 on sagittal sections in the brain of 35-week-old WT, untreated, or treated Mecp2 KO females with AAVPHP.eB-hCYP46A1-HA. (D) Quantification of Iba1-positive cells per mm² in cerebral cortex, striatum, hippocampus, and cerebellum of WT, untreated, and treated Mecp2 KO females. Insets are high magnification of tissue section. (E–G) Immunofluorescence detection of GFAP on sagittal sections in the brain of 35-week-old WT, untreated, or treated Mecp2 female mice with AAVPHP.eB-hCYP46A1-HA. (H) Quantification of GFAP-positive cells per mm² in cerebral cortex, striatum, hippocampus, and cerebellum of WT, untreated, and treated Mecp2 KO female mice. Insets are high magnification of tissue section. (I–K) Nissl staining on sagittal sections in the brain of 35-week-old WT, untreated, or treated Mecp2 KO females with AAVPHP.eB-hCYP46A1-HA. (L) Quantification of numbers of Purkinje cells per mm in cerebellum of WT, untreated, and treated Mecp2 KO females. Insets are high magnification of tissue section. Scale bar = 200 µm (A–G) or 100 µm (I–K). Data are represented as mean ± SEM. * p < 0.05.