Cell Metabolism, Volume 4

Supplemental data

Thermoregulatory and metabolic defects in Huntington’s disease transgenic mice implicate PGC-1α in Huntington’s disease neurodegeneration Patrick Weydt, Victor V. Pineda, Anne E. Torrence, Randell T. Libby, Terrence F. Satterfield, Eduardo R. Lazarowski, Merle L. Gilbert, Gregory J. Morton, Theodor K. Bammler, Andrew D. Strand, Libin Cui, Richard P. Beyer, Courtney N. Easley, Annette C. Smith, Dimitri Krainc, Serge Luquet, Ian R. Sweet, Michael W. Schwartz, and Albert R. La Spada Supplemental experimental methods

Histology

Infra-scapular BAT was dissected out and fixed in 4% paraformaldehyde overnight. The tissue

was subsequently dehydrated and sectioned for standard H&E staining (Lin et al., 2004).

Immunohistochemistry

Brains were postfixed overnight in 4% paraformaldehyde in phosphate buffer (PB) and

cryoprotected by overnight incubation in 30% sucrose in PB with 0.1% NaN3. 40 �m frozen

coronal sections were cut on a sliding microtome, and blocked with 1% normal goat serum.

Sections were incubated in 0.1 M PB containing c-Fos anti-serum (1:1000; Calbiochem, San

Diego, CA, USA), 1% normal goat serum, and 0.3% Triton X-100 for 24 h at 4 °C, rinsed three

times in 0.1 M PB and incubated in 0.1 M PB and incubated in fluorescent secondary antibody

(Alexa-Fluor 594; 1:1000; Molecular Probes, Eugene, OR, USA) for 2 hrs at RT. Ventromedial

hypothalamic nucleus images were captured at the same exposure on a Zeiss Axiovert 200M

fluorescent microscope and analyzed using Slidebook software (version 4.01.40). The ImageJ

program was used to count c-Fos positive cells from four adjacent rectangular areas / section.

Table S1. Summary of human caudate microarray data for PGC-1α-dependent

transcription factors and nuclear hormone receptors

The analyzed list of probes for PGC-1α-dependent transcription factors is shown with fold-

change in the human HD caudate sample set given relative to the human caudate control sample

set. Methods for the calculation of individual ‘P’ values are described in the Experimental

Procedures section.

GeneSymbol Probeset ID fold change P.Value Function

CREBBP 202160_at 1.339 <0.001 transcription factor 211808_s_at 1.008 0.381 “ 228177_at 1.030 0.269 “ 235858_at 1.004 0.718 “ ESRRA 1487_at 1.039 0.113 “ 203193_at -1.007 0.656 “ ESRRB 207726_at 1.010 0.233 “ 223858_at 1.002 0.783 “ NCOA1 209105_at -1.130 0.201 “ 209106_at -1.149 0.003 “ 210249_s_at -1.077 0.012 “ 243826_at 1.114 0.028 “ NRF1 204651_at 1.147 0.003 “ 204652_s_at 1.029 0.088 “ 211279_at 1.034 0.577 “ 211280_s_at 1.028 0.387 “ 232700_at 1.092 0.005 “ NRIP1 202599_s_at 1.136 0.047 “ 202600_s_at 1.107 0.069 “ PPARA 226978_at 1.977 <0.001 “ 244689_at 1.327 <0.001 “ 206870_at 1.012 0.560 “ 210771_at 1.097 0.187 “ 223437_at 1.818 <0.001 “ 223438_s_at 1.157 0.001 “ PPARD 208044_s_at 1.035 0.069 “ 210636_at 1.020 0.540 “ 37152_at 1.104 0.032 “ 242218_at -1.001 0.802 “ PPARG 208510_s_at 1.008 0.465 “ 232331_at -1.000 0.941 “ RXRA 202426_s_at 1.100 0.008 “ 202449_s_at 2.050 <0.001 “

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Table S2. Sample subset used for real-time RT-PCR confirmation of human caudate microarray

expression analysis

PM delay = post-mortem delay (in hours); VS Grade = Vonsattel grade; CAGn = CAG repeat allele size

Brain No. genotype Age years Gender PM Delay (h) VS Grade CAGn Cause of deathHC86 HD 46 M 18 1 17/37 Head injuryHC53 HD 56 M 14.5 1 17/43 Bowel obstructionHC51 HD 58 M 4.5 1 16/43 PneumoniaHC103 HD 41 M 11 1 19/39 ndHC82 HD 74 M 16 2 15/42 PneumoniaHC76 HD 71 M 16 2 19/42 PneumoniaHC73 HD 47 M 4 2 19/49 Pneumonia

H114 Control 42 M 14 - - ndH125 Control 58 M 13 - - ndH129 Control 48 M 12 - - ndH86 Control 74 M 15.5 - - ndH113 Control 68 M 19 - - ndH146 Control 61 M 15 - - nd

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Supplemental figures

Figure S1. HD N171-82Q phenotype progression and thermoregulatory defect

A) Correlation of intrascapular chip temperature and rectal probe temperature.

B) To allow comparison of body temperature regulation with other parameters of disease in the

HD N171-82Q mice, we charted body weight in the same cohort of mice whose body

temperatures were charted in Figure 1a. As has been described (Schilling et al., 1999), we

observed significant weight loss in the HD mice with disease progression (90 days of age; p <

.05 by two-way ANOVA).

C-E) Using a standard accelerating rotarod paradigm (Garden et al., 2002), we noted progressive

impairment in rotarod performance in the HD mice in comparison to controls with age. (12 wks:

p = n.s.; 16 wks: p < 0.05; 20 wks: p < 0.05; all analyses were by two-way ANOVA).

F) Individual temperature charts for the HD N171-82Q cohort. All HD mice show body

temperature decreases with progression of their disease, with certain mice suffering dramatic

drops in body temperature prior to their death. While hypothermia is a reliable indicator of

impending death, hypothermic HD mice usually survive for days with temperatures < 35°C.

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Figure S2. The htt transgene is expressed in brown fat

RNAs were isolated from infrascapular brown fat samples from non-transgenic mice (WT) and

HD N171-82Q mice (HD). RT-PCR amplification of the htt transgene with human-specific

primers yielded a positive signal for only HD brown fat in the presence of RT enzyme (upper

panel). Successful RT-PCR amplification of GAPDH served as a positive control (lower panel).

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Figure S3. Cold-challenge activates the ventromedial nucleus of the hypothalamus in WT

and HD-N171-82Q mice

c-fos protein expression in the ventromedial nucleus of the hypothalamus in WT (A) and HD-

N171-82Q (B) mice that were each subjected to a 3 hr cold challenge. As shown here, WT and

HD-N171-82Q mice displayed a similar degree of neuronal activation in response to cold-

challenge. The ImageJ program was used to count c-fos positive cells from four adjacent

rectangular areas spanning the ventral lateral preoptic nucleus. There was no significant

difference in the number of c-fos positive cells between WT and HD-N171-82Q mice (p = 0.645

by one-way ANOVA). 3V = third ventricle; VLPO = ventral lateral preoptic nucleus; MPOM

= medial preoptic nucleus, medial subdivision.

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Figure S4. UCP-1 expression alterations and PGC-1α target gene alterations in HD BAT

A) HD transgenic mice fail to up-regulate UCP-1 protein expression during cold challenge.

Western blot analysis of brown fat UCP-1 protein levels in cold challenged HD N171-82Q mice

(HD) and non-transgenic littermate controls (WT) indicates up-regulation of UCP-1 in WT mice

at the end of the 3 hour cold challenge in comparison to HD mice. Staining of SDS PAGE gels

prior to transfer confirmed equivalent loading of brown fat protein samples (not shown).

B) Impaired UCP-1 protein up-regulation in norepinephrine-treated HD primary brown

adipocytes. Protein lysates obtained from primary brown adipocyte cultures from individual

non-transgenic (WT) and HD N171-82Q (HD) mice were immunoblotted with an anti-UCP-1

antibody. Much lower levels of UCP-1 protein are apparent in primary brown adipocyte samples

from HD mice in comparison to WT mice. Blots were reprobed with an anti-βactin antibody to

assure equivalent loading (data not shown).

C) Expression analysis of PGC-1α mitochondrial gene targets in HD BAT. We performed real-

time RT-PCR analysis of BAT RNAs, obtained from sets of 20 week-old HD 82Q (red) and WT

mice (black), and observed a significant reduction or trend toward reduction in PGC-1α gene

targets involved in mitochondrial energy production (p = 0.035 for COX4; p = 0.072 for

COX7a1; p = 0.036 for LDH-B, p = .092 for TFAM, all by t-test). These reductions were

consistent with a decrease in functional mitochondria and depressed ATP/ADP ratios (Figure 2).

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Figure S5. Timeline for feeding protocol

After 2 – 3 days of acclimation to the metabolic chamber, mice were studied at ‘Baseline’ with

ad libitum feeding, followed by an 18 hr ‘Fasting’ period, and then permitted to eat ad libitum

again for 1 day. The time points for the respective body composition measurements are so

indicated. Please see the Experimental Procedures section for a detailed description of the entire

protocol.

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Figure S6. Microarray expression analysis of PGC-1α-dependent transcription factors

Here we see a heat map comparing caudate nucleus expression of 10 PGC-1α-dependent

transcription factors for 32 Grade 0 – 2 HD patients (adjacent to gold bar) and 32 matched

controls (adjacent to blue bar). Note that intensity of red signal corresponds to the degree of

relative up-regulation, while the intensity of green signal corresponds to the degree of relative

down-regulation. Unlike the heat map for PGC-1α target genes, many PGC-1α-dependent

transcription factors showed a trend toward up-regulation in HD patients in comparison to

controls. Among the interrogated transcription factors, PPARα, RXRα, and NRF-1 displayed

the most significant expression increases. Please see Supplementary Table 2 for statistical values

for the probe data displayed on this heat map.

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Supplemental references

Garden, G. A., Libby, R. T., Fu, Y. H., Kinoshita, Y., Huang, J., Possin, D. E., Smith, A. C., Martinez, R. A., Fine, G. C., Grote, S. K., et al. (2002). Polyglutamine-expanded ataxin-7 promotes non-cell-autonomous purkinje cell degeneration and displays proteolytic cleavage in ataxic transgenic mice. J. Neurosci. 22, 4897-4905. Schilling, G., Becher, M. W., Sharp, A. H., Jinnah, H. A., Duan, K., Kotzuk, J. A., Slunt, H. H., Ratovitski, T., Cooper, J. K., Jenkins, N. A., et al. (1999). Intranuclear inclusions and neuritic aggregates in transgenic mice expressing a mutant N-terminal fragment of huntingtin. Hum. Mol. Genet. 8, 397-407.