Evaluation of Rett Syndrome Symptom Improvement by Metabolic Modulators in Mecp2‐Mutant Mice

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

Mouse models recapitulate many symptoms of Rett Syndrome, an X?linked disorder caused by mutations in methyl?CpG?binding protein 2 (MECP2 ). The study of Mecp2 ?null male mice has provided insight into pathogenesis of the disorder?most recently, dysregulation of cholesterol and lipid metabolism. Perisymptomatic treatment with statin drugs successfully mitigates the effects of this metabolic syndrome, increases longevity, and improves motor function. Described here is a metabolic drug screening protocol and timeline for symptom evaluation in Mecp2 ?mutant mice. Specifically, mice are treated twice weekly with a compound of interest alongside subjective health assessments, bi?weekly body composition measurements, and blood chemistries. Throughout treatment, behavioral phenotyping tests are carried out at specific time points. This protocol is highly adaptable to other neurological diseases; however, the time for completion depends on the specific mutant model under study. The protocol highlights the use of techniques described in several different Current Protocols in Mouse Biology articles to carry out testing in a preclinical model. Curr. Protoc. Mouse Biol . 3:187?204 © 2013 by John Wiley & Sons, Inc.

Keywords: drug treatment; cholesterol; lipid; behavioral assessment; Rett Syndrome; neurodevelopmental disorders; metabolism

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Introduction
Basic Protocol 1: Treatment of Mecp2‐Mutant Mouse Models with Metabolic Modulators
Support Protocol 1: Accelerating Rotarod‐Repeated Measures Assessment
Support Protocol 2: Open‐Field Activity Assessment
Support Protocol 3: Prepulse Inhibition of Acoustic Startle Reflex
Support Protocol 4: Unrestrained Whole‐Body Plethysmography
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Treatment of Mecp2‐Mutant Mouse Models with Metabolic Modulators

Materials

Age‐ and sex‐matched Mecp2‐ mutant mice and wild‐type littermates (e.g., B6.Mecp2^tm1.1Bird ; The Jackson Laboratory, JaxMice cat. no. 003890; or other mutants or strain backgrounds; Katz et al., )
Compound of interest
Appropriate vehicle for compound (e.g., sterile saline, ethanol, and/or DMSO)
Liquid N₂

Scale for weighing compounds
Glass vials for storing organic compounds
Appropriate protective equipment for handling compounds (follow manufacturer's guidelines)
Appropriate equipment for compound administration (e.g., 10‐ml syringes with ∼26‐G needles for subcutaneous injection)
Waste container for sharp items
Mouse whole‐body composition analyzer (e.g., Bruker MiniSpec NMR; optional)
Serum separator tubes (e.g., BD Vacutainer Blood Collection tubes)
∼25‐G needles and syringes for cardiac punctures
Cryo‐safe tubes for tissue collection

Additional reagents and equipment for mouse blood collection (Donovan and Brown, ; Rathkolb et al., ), subjective health assessments according to guidelines proposed for preclinical evaluation in mice (Katz et al., ), euthanasia (method to be determined by experimenter and institution, performed in accordance with relevant animal care and use guidelines and regulations; Donovan and Brown, ), post‐mortem dissection and examination (Antal et al., ), clinical chemistry procedures (Rathkolb et al., ), measurement of lipids and lipoproteins in mice (Tailleux and Staels, ), and characterization of whole‐body cholesterol fluxes in mice (Brufau and Groen, )

Support Protocol 1: Accelerating Rotarod‐Repeated Measures Assessment

Materials

Treated and control, age‐ and sex‐matched Mecp2‐ mutant mice and wild‐type littermates (e.g., B6.Mecp2^tm1.1Bird ; The Jackson Laboratory, JaxMice cat. no. 003890; or other mutants or strain backgrounds; Katz et al., )
Ethanol

Rotarod apparatus for mice and rats (Stoelting, cat. no. 52790) or equivalent (also see Brooks et al., )
Isolated room without noise or traffic
Adjustable light source
Light monitor
White noise source
Noise meter

Additional reagents and equipment for rotarod testing (Brooks et al., )

Support Protocol 2: Open‐Field Activity Assessment

Materials

Treated and control, age‐ and sex‐matched Mecp2‐ mutant mice and wild‐type littermates (e.g., B6.Mecp2^tm1.1Bird ; The Jackson Laboratory, JaxMice cat. no. 003890; or other mutants or strain backgrounds; Katz et al., )
Ethanol

Animal activity monitors: open‐field mouse configuration (AccuScan Fusion monitor, http://www.accuscan‐usa.com, or equivalent; also see Thomas et al., )
Isolated room without noise or traffic
Adjustable light source
Light monitor
White noise source
Noise meter

Additional reagents and equipment for open‐field testing (Thomas et al., )

Support Protocol 3: Prepulse Inhibition of Acoustic Startle Reflex

Materials

Treated and control, age‐ and sex‐matched Mecp2‐ mutant mice and wild‐type littermates (e.g., B6.Mecp2^tm1.1Bird ; The Jackson Laboratory, JaxMice cat. no. 003890; or other mutants or strain backgrounds; Katz et al., )
Ethanol

Startle Response Lab system (San Diego Instruments, or equivalent) and associated software
Isolated room without noise or traffic

Additional reagents and equipment for prepulse inhibition testing (Ouagazzal and Meziane, )

Support Protocol 4: Unrestrained Whole‐Body Plethysmography

Materials

Treated and control, age‐ and sex‐matched Mecp2‐ mutant mice and wild‐type littermates (e.g., B6.Mecp2^tm1.1Bird ; The Jackson Laboratory, JaxMice cat. no. 003890; or other mutants or strain backgrounds; Katz et al., )
Unrestrained whole‐body plethysmography system for mouse (Buxco, EMMS PLY 310, http://www.electromedsys.com/, or equivalent), with associated software
Isolated room without noise or traffic
Chart for recording activity

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Figures

Figure 1. A simplified schematic of cholesterol and lipid synthesis including major regulatory elements and feedback points. Pathway members are in black, enzymes in red, transcription factors in blue, and hormones in green. Both cholesterol and lipid synthesis are regulated by AMPK and downstream factors including the ACCs and SREBPs. Cholesterol itself is part of a negative feedback mechanism that adjusts levels of the rate‐limiting enzymes HMGCR and SQLE. Lipid synthesis is regulated in part by the accumulation of lipids in adipocytes, which produce adipokines, such as leptin and adiponectin. In turn, these adipokines regulate AMPK levels.

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Figure 2. An example timeline used by the authors for cohorts of Mecp2^tm1.1Bird /Y going through treatment with metabolic modulators and phenotypic evaluation. Abbreviations: OFA, open‐field assessment; PPI, prepulse inhibition of acoustic startle response; Pleth, plethysmography.

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Figure 3. Summary of recommended phenotyping schedule for Mecp2 ‐null mice.

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Figure 4. Comparison of (A ) serum cholesterol levels and (B ) liver lipid levels in 8‐month‐old female mice (striped bars) and 10‐week‐old male mice (solid bars). (A ) Serum cholesterol levels do not differ between male and female wild‐type mice, or between male and female statin‐treated 129. Mecp2^tm1.1Bird mice. When not treated with statin drugs, however, serum cholesterol levels in aged 129. Mecp2^tm1.1Bird /+ (female) mice are significantly lower than in the younger 129. Mecp2^tm1.1Bird /Y (male) mice. (B ) Liver lipid levels are trend higher in 8‐month‐old female mice than 10‐week‐old male mice across all genotypes and treatment groups, likely due to age. However, this difference is only significant in 129. Mecp2 ‐mutant mice and most striking in those that have not been treated with statin drugs. Derived from data in prior publication in Buchovecky et al. ().

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Literature Cited

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