Early Deficits in Glycolysis Are Specific to Striatal Neurons from a Rat Model of Huntington Disease
et al. (2013) Early Deficits in Glycolysis Are Specific to Striatal Neurons from a Rat
Model of Huntington Disease. PLoS ONE 8(11): e81528. doi:10.1371/journal.pone.0081528
Early Deficits in Glycolysis Are Specific to Striatal Neurons from a Rat Model of Huntington Disease
Caroline Gouarn 0
Gwenalle Tardif 0
Jennifer Tracz 0
Virginie Latyszenok 0
Magali Michaud 0
Emily Clemens 0
Libo Yu-Taeger 0
Huu Phuc Nguyen 0
Thierry Bordet 0
Rebecca M. Pruss 0
Hemachandra Reddy, Oregon Health & Science University, United States of America
0 1 Trophos, Parc Scientifique de Luminy, Luminy Biotech Entreprises , Marseille , France , 2 Institute of Medical Genetics and Applied Genomics, University of Tuebingen , Tuebingen, Germany, 3 Center for Rare Diseases , University of Tuebingen , Tuebingen , Germany
In Huntington disease (HD), there is increasing evidence for a link between mutant huntingtin expression, mitochondrial dysfunction, energetic deficits and neurodegeneration but the precise nature, causes and order of these events remain to be determined. In this work, our objective was to evaluate mitochondrial respiratory function in intact, non-permeabilized, neurons derived from a transgenic rat model for HD compared to their wild type littermates by measuring oxygen consumption rates and extracellular acidification rates. Although HD striatal neurons had similar respiratory capacity as those from their wild-type littermates when they were incubated in rich medium containing a supra-physiological glucose concentration (25 mM), pyruvate and amino acids, respiratory defects emerged when cells were incubated in media containing only a physiological cerebral level of glucose (2.5 mM). According to the concept that glucose is not the sole substrate used by the brain for neuronal energy production, we provide evidence that primary neurons can use lactate as well as pyruvate to fuel the mitochondrial respiratory chain. In contrast to glucose, we found no major deficits in HD striatal neurons' capacity to use pyruvate as a respiratory substrate compared to wild type littermates. Additionally, we used extracellular acidification rates to confirm a reduction in anaerobic glycolysis in the same cells. Interestingly, the metabolic disturbances observed in striatal neurons were not seen in primary cortical neurons, a brain region affected in later stages of HD. In conclusion, our results argue for a dysfunction in glycolysis, which might precede any defects in the respiratory chain itself, and these are early events in the onset of disease.
Funding: Funding was provided by Trophos and by the European Union under the 7th Framework Program for RTD - Project MitoTarget - Grant
Agreement HEALTH-F2-2008-223388. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing interests: The authors have the following interests. This study was partly funded by Trophos, the employer of CG, GT, JT, VL, MM, TB and
RMP and who hold Trophos shares or stock options. There are no patents, products in development or marketed products to declare. This does not alter
the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.
Huntington disease (HD) is a hereditary neurodegenerative
disorder caused by a CAG repeat extension in the coding
region of the huntingtin gene, leading to striatal atrophy which
later expands to the cerebral cortex and other subcortical brain
regions [1,2]. Clinically, the disease is characterized by
psychiatric symptoms, movement disorders, progressive
dementia and also by pronounced weight loss despite
sustained caloric intake  supporting to the hypothesis of
impaired ATP synthesis in HD [4,5]. This was further confirmed
by the detection of significant alterations in the glucose
concentration by brain imaging [6-8] and in the concentration of
energetic metabolites (mainly N-acetylaspartate, glutamine/
glutamate, and lactate) in brain or in the cerebrospinal fluid of
HD patients [9-15]. Whether this results from reduced
mitochondrial ATP synthesis and/or reduced glycolytic ATP
levels is not known. The observation of a severe reduction in
the activity of the mitochondrial respiratory chain complexes
II/III and a milder reduction in the activity of complex IV in the
caudate/putamen from post-mortem brain samples suggested
that mitochondrial abnormalities may underlie HD pathogenesis
[16-18]. However, whether respiratory chain impairment is the
cause or the consequence of neuronal loss in HD remains
unclear, since such defects were not observed in
presymptomatic patients [19,20].
To further address the precise nature and the role of
metabolic and mitochondrial dysfunction in HD, studies were
performed in genetic models of HD, particularly in mice
expressing full-length mutant huntingtin (fl-mHtt). As observed
in pre-symptomatic and early HD patients, no major impairment
in the enzymatic activity of the mitochondrial respiratory chain
complexes IIV was evidenced in either the striatum or the
sensorimotor cortex of these mice . By contrast, deficits in
respiration rate and ATP production reported in STHdh Q111
striatal cell lines derived from knock-in mice with 111 CAG
repeats introduced into the mouse HTT homologue Hdh
[21-23]. However, the impairment could not be assigned to
defects in individual respiratory complexes in these cells.
Moreover, differences in mitochondrial respiratory rates were
no longer present when using isolated mitochondria from the
same cell lines , suggesting that detection of some
mitochondria deficits may only be detected in intact cells. In
that sense, Oliveira and colleagues measured mitochondrial
respiratory rates in intact, non-permeabilized, primary striatal
neurons from Hdh150 knock-in mice and did not find any deficit
under resting condition when compared to wild type controls
. However, when challenged with an energy-demanding
stimulus (NMDA-receptor activation) and incubated in
pyruvate-based media to accentuate mitochondrial metabolism,
Hdh150 neurons were more vulnerable to calcium overload
than neurons from their wild-type littermates . These results
highlight the importance of assessing mitochondrial function in
the cellular environment including both neuronal cell bodies
and neurites. It is also important to survey the use of various
glycolytic and oxidative substrates to better understand how
cells cope with metabolic demands in situ.
To pursue these questions, we investigated mitochondrial
respiration in specific primary neuronal subpopulations cultured
from a new HD transgenic rat model expressing fl-mHtt and all
regulatory elements integrated from a bacterial artificial
chromosome (BACHD rats). These rats display a robust, earlier
onset and faster progressive HD-like phenotype even in
heterozygous transgenic rats compared to previously described
HD rats expressing a mHtt fragment . Oxygen consumption
rates and anaerobic glycolysis were measured using a variety
of substrates in situ, directly in the culture well, preserving the
neuronal network integrity.
Materials and Methods
All experiments were approved and performed in accordance
with internal institutional guidelines; Trophos is an accredited
institution for animal experimentation in France (French
ministry of Agriculture, Agreements No. B-13-055-15) and in
strict accordance with national and European regulations
(Directive 86/609/EEC of the European Economic Community
regarding the protection of animals used for experimental and
other scientific purposes). All efforts were made to avoid or
minimize animal suffering and to reduce and refine the
Male heterozygous transgenic BACHD rats (HD) expressing
high levels of fl-mHtt protein containing 97 CAG/CAA repeats
(line LY.005)  were bred with female wild type (WT) rats
obtained from Elevage Janvier (Le Genest Saint Isle, France).
The rats were housed in a controlled environment (room
temperature 22C 2; reverse 12h light dark cycle, 50% 5
humidity) with food and water available ad libitum. Pregnant
female rats were euthanized at embryonic day 17 (E17) by
gradual fill CO2 overdose. Rats were placed in a hermetic box
then exposed to a mixture of O2/CO2 (40-60% respectively)
until sleep was induced. The % CO2 was then progressively
increased up to 100% while O2 was decreased (0.5L/min every
30 sec. for 3 min up to 5L/min) and then maintained for 4-5
Rat embryos from a single pregnant female rat were used for
each independent experiment. All embryos were genotyped to
identify WT and HD animals. During genotyping, brains were
maintained in Hibernate conservation medium (BrainBits) at
4C. The embryos were genotyped with a PCR-based assay
using DNA from tail tissue. The mHTT gene product was
detected using the primers
5AGGTCGGTGCAGAGGCTCCTC-3 (Eurofins). PCR
conditions were: 30 cycles at 94C (30 s), 60C (30 s), 72C (1
Primary neuronal cultures
E17 primary striatal neurons were prepared by an adapted
method previously described . Briefly, striata were
thoroughly minced into 1 mm sections, washed in HBSS
medium (Invitrogen) complemented with 7 mM HEPES
(Invitrogen) and 0.45% glucose (w/v) (Sigma) and incubated for
15 min at 37C with 0.25% of trypsin (Invitrogen). The tissues
were mechanically dissociated by several pipetting and
centrifugation steps (5 min at 250 x g). Primary striatal neurons
were seeded onto poly-ornithine (Invitrogen) and laminin (BD)
coated Seahorse 24-well plates at a density of 150,000 cells/
well in Neurobasal media (Invitrogen), complemented with
2% B-27 supplement (Gibco) and 1 mM pyruvate (Invitrogen).
The two cell populations (WT and HD) were seeded in different
wells of the same plate with 4 to 8 replicates per plate
depending on the protocol. The cells were incubated for 7 days
at 37C in a humidified incubator in an atmosphere of 95% O2,
5% CO2. E17 primary cortical neurons were prepared using a
similar protocol and seeded at a density of 110,000 cells/well.
Striatal and cortical neuronal culture purity was controlled by
immunofluorescence using NeuN antibody (Millipore) to identify
neurons, GFAP (Millipore) for astrocytes and F4/80 (Santa
Cruz) for microglial cells. Both striatal and cortical neuronal
cultures were 98% pure. The presence of mHtt, already in the
form of aggregates, was confirmed in primary striatal and
cortical neuronal cultures derived from HD rats (see in Protocol
S1 and Figure S1).
Oxygen consumption rate and extracellular
Cellular oxygen consumption and extracellular acidification
rate (ECAR) reflecting lactate release were concomitantly
measured using a XF24 extracellular flux analyzer (Seahorse
Bioscience) as previously described .
Respiration experiments in substrate-rich medium
Experiments in substrate-rich media were performed using
Dulbeccos Modified Eagles Medium (DMEM) containing 25
mM glucose without bicarbonate (Invitrogen) and
complemented with 1 mM fresh pyruvate (assay medium). One
hour before the start of the respiration measurements, the
original culture medium was replaced by assay medium and
plates were placed into the XF analyzer calibrated at 37C.
After 10 min of equilibration, OCR was measured for 1.5 min to
establish a baseline rate. The medium was then gently mixed
for 2 min followed by a 3 min pause to restore normal oxygen
tension in the microenvironment surrounding the cells. After
baseline measurements, oligomycin (final concentration 0.5
g/ml) (Sigma) was injected into each well in order to inhibit
mitochondrial ATP synthase to determine proton
leakdependent OCR (State 4o). After this, the uncoupling agent
carbonilcyanide p-triflouromethoxyphenylhydrazone (FCCP;
final concentration 2 M) (Sigma) was injected to determine
maximal OCR (state 3u). Finally, rotenone (final concentration
75 nM) (Sigma) and antimycin A (final concentration 150 ng/ml)
(Sigma) were injected to inhibit complex 1 and complex 3
respectively and to completely abolish mitochondrial
respiration. After each injection step, two measurement cycles
(measurement + mix + pause) were performed (Figure 1) and
after rotenone and antimycin A injection, 4 measurement
cycles were performed. Preliminary experiments determined
the optimal concentration of these agents to optimally inhibit or
stimulate respiration without inducing toxicity.
Substrate-dependent respiration experiments
To assay the ability of different substrates to support
respiration, experiments were designed using a simple
substrate-free modified Krebs Henseleit Buffer (KHB) (NaCl
111 mM, KCl 4.7 mM, MgSO4 2 mM, Na2HPO4 1.2 mM, pH
7.4), complemented with 2 mM GlutaMAX (Invitrogen). One
hour before the start of the assay, the original culture medium
was replaced by KHB and the plates were placed in an
incubator at 37C without CO2 and then in the XF analyzer.
After 10 min of equilibration in the instrument, OCR was
measured for 1.5 min to establish a substrate free basal rate.
Medium was then gently mixed for 2 min followed by a 3 min
pause to restore normal oxygen tension in the
microenvironment surrounding the cells. After baseline
measurements, the substrates D-glucose (Sigma), pyruvate
(Sigma) or lactate (Sigma) were injected and OCR was
measured for 1.5 min followed by injections of oligomycin,
FCCP and rotenone plus antimycin A as described above. After
each injection, two or three measurement cycles
(measurement + mix + pause) were performed.
Respiration parameters calculation
When oxygen consumption rate measurements ended, cells
were incubated for 20 minutes with 2 g/ml calcein-AM to
measure the relative neuronal cell density and viability in each
well. Only experiments with a similar cell density based on
calcein fluorescence between WT and HD (Students t test p >
0.05) were validated and analyzed. Respiration parameters
were calculated from data obtained during the steps of OCR
measurement. For basal respiration and oligomycin respiration,
the last OCR measurement of the cycle was selected for
calculation. For FCCP the first OCR measurement of the cycle
was selected to avoid the effect of substrates depletion on
maximal OCR. For further details please refer to the legend of
the respective figures. Because non-mitochondrial oxygen
consumption rate (measured after antimycin A injection) was
overestimated due to the fact that FCCP injection activated
non-mitochondrial oxidase and because the rate was negligible
(according to preliminary assays), we decided to not remove
non-mitochondrial OCR from all measurements. Cell
respiratory control ratio (RCR) was calculated by dividing the
maximal rate (uncoupled) by the oligomycin rate (proton leak).
Spare respiratory capacity was calculated by subtracting the
basal rate from the maximal (uncoupled) rate. Coupling
efficiency was calculated by dividing the fraction of basal
mitochondrial oxygen consumption used for ATP synthesis
(basal rate minus oligomycin rate) by the basal rate. The
substrate response was determined by dividing the OCR
obtained after substrate addition (glucose, pyruvate, lactate, or
combinations) by the substrate-free basal rate in KHB.
Data are expressed as means SEM. Comparisons
between two groups were performed using an unpaired
Students t test. Comparisons of several groups or differences
between treatments and experimental groups were conducted
using a one way or two way analysis of variance (ANOVA)
followed by Dunnetts post test. A p-value of less than 0.05 was
considered statistically significant.
Characterization of WT and HD striatal neuron
respiration in substrate-rich medium
E17 primary striatal neurons from HD and WT rat embryos
were cultured for 7 days in Neurobasal medium and then
placed in DMEM with similar concentrations of amino acids,
vitamins and glucose (25 mM) to measure OCR. Their
response to the respiration analysis paradigm in substrate rich
medium is shown in Figure 1A. Basal OCR is strongly
controlled by ATP turnover and to a lesser extent by substrate
oxidation and proton leak . Addition of oligomycin induced a
decrease in OCR and under this condition (state 4o),
respiration is strongly controlled by proton leak kinetics and
partially by substrate oxidation . In contrast, OCR sensitive
to oligomycin is strongly driven by the proton flux
accompanying ATP synthesis. Addition of the uncoupler
(FCCP) caused an increase in OCR until the maximum
respiration rate was reached (state3u). This respiratory rate
reflects the maximal respiratory chain activity as well as the
maximal substrate oxidation rate that is achievable . Finally,
addition of rotenone (complex 1 inhibitor) and antimycin A
(complex 3 inhibitor) totally blocked the respiratory chain and
the residual OCR represented non-mitochondrial respiration,
probably driven by NADPH oxidase activity . In these
experiments performed in substrate rich DMEM, no differences
in OCR or respiratory parameters were detectable between WT
Figure 1. No differences in respiratory parameters are detected between HD and WT striatal neurons incubated in
substrate-rich medium. OCR measurements were performed in bicarbonate- and HEPES-free DMEM containing 25 mM glucose
complemented with pyruvate (1 mM) and began 10 min after cell plate installation in the Seahorse analyser. A) OCR was measured
sequentially in basal conditions (time points 1-3), after injections of oligomycin (oligo; time points 4 and 5), FCCP (time points 6 and
7), rotenone (time points 8 and 9) and then antimycin A (AA; time points 10 and 11) at the indicated times. Data shown are mean
SEM from a representative experiment (eight replicates). Respiratory parameters were derived from OCR measured during each
phase of the experiment as shown in A: B) respiratory control ratio = [FCCP rate (time point 6) / oligo rate (time point 5)]; C)
coupling efficiency = [basal rate (time point 3) oligo rate (time point 5)] / basal rate (time point 3); D) spare respiratory capacity =
[FCCP rate (time point 6) basal rate (time point 3)]. Respiratory parameters shown in panels B-D are mean SEM calculated from
all replicates (eight replicates in each of three independent experiments).
and HD striatal neurons (Figure 1). Similarly, HD striatal
neurons showed no deficits in RCR, which detects substrate
oxidation efficiency and respiration due to proton leak, but not
driven by ATP synthesis (Figure 1B), coupling efficiency, which
is strongly controlled by ATP turnover and proton leak (Figure
1C) or spare respiratory capacity, which reflects the capacity of
the respiratory chain to respond to an increase in energy
demand and the ability of substrates to provide fuel (Figure
Glucose supported respiration in striatal neurons
In first experiments, the medium used to measure respiration
contained 25 mM glucose to mimic their original culture
conditions. This is a high, supra-physiological concentration
compared to the glucose concentrations found in brain that are
estimated to be around 2.5 mM in rats . To check whether
there was any difference in the ability of HD striatal neurons to
use more physiological levels of glucose as well as various
other substrates to support oxidative phosphorylation, we
performed a series of experiments adding specific substrates to
cells incubated in KHB. Different concentrations of glucose
were injected (final concentrations of 2.5 - 25 mM) followed by
oligomycin, FCCP, and rotenone + antimycin A injections
(Figure 2A). In the absence of glucose, neurons were able to
maintain basal respiration but they were not able to respond to
FCCP stimulation (Figure 2A). All concentrations of glucose
between 2.5 and 25 mM induced a similar increase in basal
OCR (Figure 2B) but even though basal respiration was similar
in the presence of physiological and supra-physiological
glucose concentrations (Figure 2B), FCCP-stimulated
respiration and RCR were glucose-concentration dependent
(Figure 2C). These results suggest that the physiological
glucose concentration found in brain, although sufficient to
support respiration under basal conditions (low energy
demand) may be limiting under conditions of high respiratory
Neurons can use lactate and pyruvate to support
Increasing evidence supports the idea that glucose is not the
sole energy substrate for neurons in the brain. Oxidative
substrates such as pyruvate or even lactate provided by glial
cells could be supplementary fuel for neurons . Results
presented in Figure 3 reinforced this hypothesis. WT neurons
were incubated for 1 hour in KHB medium lacking metabolic
substrates then provided with various concentrations of
pyruvate (Figure 3A) or lactate (Figure 3B). Then respiratory
parameters were derived following injection of oligomycin,
FCCP, and rotenone + antimycin A. As observed in the
previous study assaying the response to glucose, in the
absence of substrates, neurons were able to maintain basal
respiration but were not able to respond to FCCP stimulation
(Figure 3A; 3B). Addition of either pyruvate or lactate increased
basal respiration as previously seen with glucose and restored
the neurons capacity to respond to FCCP stimulation (Figure
3A, 3B). In the case of pyruvate, a similar basal respiration rate
was detected over the concentration range tested (1-30 mM)
but maximal respiration and RCR reached a plateau between 1
and 10 mM (Figure 3A, 3C, 3E). Addition of 1 or 10 mM lactate
resulted in a similar basal respiration rate, which was
significantly increased in the presence of 30 mM lactate (Figure
3B-F). FCCP stimulated respiration was also supported by
lactate but to a lesser extent than pyruvate. This can be
explained by the observation that 30 mM lactate induced a
significant increase in proton leak respiration in addition to ATP
synthesis-driven respiration (Figure 3G). This was not the case
for 30 mM pyruvate (Figure 3D). Because 30 mM lactate
increased mitochondrial proton leak, potentially disturbing
mitochondrial function, this concentration was not considered in
further analysis of RCR as a function of pyruvate and lactate
concentrations (Figure 3I). We observed a
concentrationdependent increase in RCR for both lactate and pyruvate,
plateauing at 10 mM for pyruvate. Comparing the RCR
between 10 mM pyruvate and lactate shows that pyruvate is
clearly a better substrate, most likely due to the fact that lactate
must first be converted to pyruvate.
Respiration in WT and HD striatal neurons supported
by pyruvate or lactate
We also evaluated HD and WT striatal neurons capacity to
use alternative substrates, pyruvate and lactate according the
protocol set up in Figure 3. The concentration chosen for this
exploration was 10 mM for each substrate, which was the
concentration inducing the maximal OCR response under
stimulated conditions without causing proton leak. As
previously shown in Figure 4, after more than one hour of
substrate deprivation, OCR was similar between WT and HD
Figure 2. Respiration parameters measured in WT striatal neurons as a function of glucose concentration. On the day of
the respiration experiments, medium was replaced with substrate-free KHB and cells were incubated for 1 h at 37C. OCR
measurements began 10 min after cell plate installation in the Seahorse analyser. A) After measuring the initial OCR in the absence
of substrates, various concentrations of glucose in KHB (vehicle; Veh) were injected (final concentrations: 0, 2.5, 5 or 25 mM). OCR
was then measured in the presence of substrate and following successive injections of oligomycin (oligo), FCCP, then rotenone and
antimycin A (rot + AA) at the indicated times. Data are expressed as the percentage of the basal value measured at the time point 3
(four replicates). p < 0.05, p < 0.01, p < 0.001 (0 mM vs. 2.5 mM glucose); # p < 0.05, ## p < 0.01, ### p < 0.001 (0 mM
vs. 5 mM glucose); * p < 0.05, ** p < 0.01, ***p < 0.001 (0 mM vs. 25 mM glucose). Respiratory parameters were derived from OCR
measured during each of these phases; B) glucose response = [basal glucose rate (time point 6) / basal free substrate rate (time
point 3)]; C) respiratory control ratio = [FCCP rate (time point 9) / oligomycin rate (time point 8)]. * p < 0.05 compared to 2.5 mM
Figure 3. Striatal neurons can use alternative substrates to glucose to support respiration. On the day of the respiration
experiments, medium was replaced with substrate-free KHB and cells were incubated as described in Figure 2. After measuring the
initial OCR in the absence of substrates, various concentrations of pyruvate (A) or lactate (B) in KHB (vehicle; Veh) were injected
(final concentrations: 0, 1, 10 or 30 mM). OCR was then measured in the presence of substrate and following successive injections
of oligomycin (oligo), FCCP, then rotenone and antimycin A (rot + AA) at the indicated times. Data are expressed as the percentage
of the basal value measured at time point 3 (four replicates). p < 0.05, p < 0.01, p < 0.001 (0 mM vs. 1 mM pyruvate or
lactate); # p < 0.05, ## p < 0.01, ### p < 0.001 (0 mM vs. 10 mM pyruvate or lactate); * p < 0.05, ** p < 0.01, ***p < 0.001 (0 mM vs.
30 mM pyruvate or lactate). Respiratory parameters were derived from the various phases following pyruvate (C-E) or lactate (F-H)
injection: substrate response = [basal pyruvate or lactate rate (time point 6) / basal substrate free rate (time point 3)]; proton leak
rate (time point 7); respiratory control ratio = [FCCP rate (time point 9) / oligomycin rate (time point 8)]. * p < 0.05, ** p < 0.01
compared to 1 mM pyruvate (C-E) or lactate (F-H). I). Respiratory control ratio expressed as a function of pyruvate (1, 3, 10 or 30
mM) or lactate (1, 3 or 10 mM) concentration.
neurons (Figure 5A, 5D). Pyruvate addition induced a
significant increase in basal OCR and restored neurons
capacity to respond to FCCP (Figure 5A). No significant
differences were detected in HD striatal neurons compared to
WT (Figure 5A-C). Lactate addition induced a similar increase
in basal OCR as pyruvate, which was again not different
between WT and HD neurons (Figure 5D). Even though
respiration in the presence of 10 mM lactate was similar or only
slightly lower in HD neurons, the resulting RCR and spare
respiratory capacity appeared to be significantly lower in
FCCP-stimulated HD neurons compare to WT (Figure 5E-F).
Figure 4. OCR measurements performed using a physiological glucose concentration revealed respiratory deficits in HD
striatal neurons. OCR measurements were performed as described in Figures 2. A) OCR measurements were taken following
injection of glucose (final concentration, 2.5 mM), oligomycin (oligo) then FCCP at the indicated times. Data shown are mean SEM
from a representative experiment. Respiratory parameters were derived from the various phases following glucose injection (B-C):
respiratory control ratio = [FCCP rate (time point 10) / oligo rate (time point 9)]; glucose spare respiratory capacity = [FCCP rate
(time point 10) glucose rate (time point 7)]. Data shown are mean SEM from all replicates (six replicates in each of three
independent experiments). * p < 0.05, ** p < 0.01 comparing HD vs. WT.
To see how neurons respond to a mixture of metabolic
substrates, OCR was measured in WT and HD striatal neurons
in the presence of 2.5 mM glucose followed by addition of 10
mM lactate or pyruvate (Figure 6). Neither pyruvate nor lactate
Figure 5. Comparison of HD and WT striatal neurons ability to use pyruvate and lactate in the absence of glucose. OCR
measurements were performed as described in Figure 3. OCR measurements were taken following injection of 10 mM pyruvate (A)
or lactate (D), oligomycin (oligo) and FCCP (2 M) at the indicated times. Data shown are mean SEM from a representative
experiment (six or ten replicates for pyruvate or lactate, respectively). Respiratory parameters were derived from the various phases
following pyruvate (B-C) or lactate (E-F) injection: respiratory control ratio = [FCCP rate (time point 9) / oligo rate (time point 8)];
spare respiratory capacity = [FCCP rate (time point 9) pyruvate or lactate rate (time point 6)]. Data for respiratory parameters are
mean SEM calculated from all replicates (three independent experiments). * p < 0.05 comparing HD vs. WT.
increased basal OCR further when neurons were already
supplied with a physiological glucose concentration (Figure
6AD), suggesting that glucose alone was sufficient to support
basic neuron energy requirements. Moreover, this result
confirmed that under basal conditions, respiration is probably
strongly controlled by the rate of mitochondrial ATP turnover or
proton leak and is not limited by the rate of substrate oxidation.
FCCP injection induced a large increase in OCR in the two
conditions tested. In the presence of glucose and pyruvate,
RCR was higher compared to glucose or pyruvate alone (9.2
0.8, 5.4 0.4, 3.5 0.4; glucose + pyruvate RCR, pyruvate
RCR, glucose RCR, respectively; mean SEM). Similar results
were obtained with lactate, with higher RCR in the presence of
glucose and lactate compared to glucose or lactate alone (6.2
0.5, 3.3 0.2, 3.5 0.4; glucose + lactate RCR, lactate RCR,
glucose RCR, respectively; mean SEM). These results
suggest that in the presence of a physiological glucose
concentration, maximal respiration can be improved when
alternative substrates such as pyruvate or lactate are available.
However, it can be seen that FCCP-stimulated OCR was
significantly lower in HD compared to WT striatal neurons when
supplied with either pyruvate or lactate in addition to a
physiological glucose concentration (Figure 6A-D). This shows
that the respiratory defects observed under stress conditions in
Figure 6. HD striatal neurons have a lower capacity to use glucose under high respiratory demands, even when
supplemented with pyruvate or lactate. Striatal neurons were cultivated for 7 days as described in Figure 1. One hour before the
start of the respiration experiments, medium was replaced with substrate-free KHB complemented with 2.5 mM glucose. OCR
measurements were performed as described in Figure 3. OCR measurements following injection of 10 mM pyruvate (A) or lactate
(D), oligomycin (oligo) and FCCP (2 M) at the indicated times. Data shown are mean SEM from a representative experiment (five
or four replicates for pyruvate or lactate, respectively). Respiratory parameters were derived from the various phases following
pyruvate (B-C) or lactate (E-F) injection: respiratory control ratio = [FCCP rate (time point 9) / oligo rate (time point 8)]; spare
respiratory capacity = [FCCP rate (time point 9) - pyruvate or lactate rate (time point 6)]. Data for respiratory parameters are mean
SEM calculated from all replicates (three independent experiments). * p < 0.05, ** p < 0.01 comparing HD vs. WT.
the presence of physiological concentration of glucose (Figure
4) were not completely compensated by the presence of
alternative substrates, such as pyruvate or lactate.
Comparison of ECAR in WT and HD primary striatal
To investigate if OCR deficits observed in the presence of
2.5 mM glucose in HD primary striatal neurons were associated
with a defect in anaerobic glycolysis, we measured ECAR in
HD and WT striatal neurons. First, we observed under basal
conditions that ECAR in WT striatal neurons remained stable
regardless of the glucose concentration used (Figure 7A). After
oligomycin injection, the inhibition of ATP synthesis by the
respiratory chain resulted in ECAR activation as previously
described . Interestingly, this activation was glucose
concentration dependent (Figure 7A). Because FCCP
increases H+ extrusion, ECAR could not be used as a measure
of glycolysis under uncoupling conditions; therefore, data
obtained after FCCP injection was not analyzed. Then, ECAR
was measured in WT and HD primary striatal neurons
incubated either in rich substrate DMEM or in KHB containing
2.5 mM glucose (Figure 7B). Under basal conditions, no
significant difference was observed between WT and HD
neurons. In contrast, oligomycin-stimulated ECAR as an
indicator of glycolysis was significantly lower in HD neurons
both in rich and low glucose medium, suggesting a specific
defect in their maximal anaerobic glycolysis capacity. At the
same time, intracellular pH measured using
Invitrogen, 0.3M) was similar in WT and HD neurons
suggesting no difference in lactate release between the two
genotypes (data not shown).
Comparison of respiration parameters in WT and HD
To investigate if the deficits seen in HD striatal neurons were
common to all types of neurons, we performed similar
experiments using primary cortical neurons, a brain region
affected later in Huntington disease . In contrast to striatal
neurons, no respiratory deficits were detected in the presence
of 2.5 mM glucose alone under basal or uncoupling conditions
(Figure 8A) and RCR was similar between HD and WT cortical
neurons. Additionally, in the presence of glucose (2.5 mM) and
pyruvate (10 mM) or lactate (10 mM), no defect was detected
in HD cortical neurons in any of the respiratory parameters
(Figure 8B-C). ECAR measurements were also performed in
presence of 2.5 mM glucose in primary cortical neurons and
results did not show any defects in HD compared to WT
In this study, we measured the ability of various substrates to
support mitochondrial respiration in intact primary striatal and
cortical neurons obtained from WT and HD rat embryos. These
studies were designed to detect potential metabolic differences
in HD neurons that could contribute to the pattern of
neurodegeneration found in HD. We detected no specific
mitochondrial respiratory chain defects in either cortical or
striatal neurons from HD rats. By contrast, we found defects in
glycolysis that are present even in cells prepared from
embryos, but only in primary striatal and not cortical neurons
derived from this rat HD model even though mHtt aggregates
are present in both types of neurons (see Figure S1).
As previously mentioned, most of the studies exploring
mitochondrial defects in HD models were done using brain
extracts containing heterogeneous mixtures of neuronal and
glial mitochondria, which may have precluded clear
conclusions. In this study, we monitored metabolic rates in
specific primary neuronal subpopulations (cortical or striatal)
attached to the culture plates after 7 days in culture using
extracellular flux measurement technology . This allowed
us to measure OCR in intact cells including all mitochondria
populations located in neuronal cell bodies as well as neurites.
Indeed, mitochondria localized in dendrites, axon shafts and
presynaptic terminals play a key role in neuronal metabolism
and in neuroplasticity  and these structures are largely left
behind when neurons are detached from their culture surface
for respiration measurements; this mitochondrial fraction is
important especially when considering that axonal transport is
disturbed in HD . In addition, these studies highlighted the
importance of alternate substrates to support neuronal
respiration along with physiological glucose concentrations
found in brain, particularly when respiration demand is high.
The normal plasma glucose concentration is around 4.5 mM;
however, the glucose concentration in the CNS is only about
2.5 mM [31,36]. Neurobasal medium, commonly used to
culture primary neurons contains 25 mM glucose, a
supraphysiological concentration. We found that glucose
dosedependently increased neuronal spare respiratory capacity and
RCR and that 2.5 mM glucose was clearly suboptimal to
support neurons under high respiratory demand. We then
found that both lactate and pyruvate were individually able to
support mitochondrial respiration in intact neurons and that
these alternative substrates could provide additional respiratory
capacity beyond that provided by CNS levels of glucose when
neurons were challenged with an uncoupling agent. These
results highlight the importance of neuronal glial coupling
whereby glucose utilization by astrocytes results in the release
of lactate that can then be taken up and used by neurons to
support respiration [37-39]. Lactate conversion to pyruvate may
take place preferentially in nerve terminals depending on
differential distribution of LDH isoforms found in neuron cell
bodies and terminals . Furthermore, MCT1 transporters
have recently been implicated in lactate exchange between
oligodendrocytes and neuronal axons . Interestingly, we
observed an additive effect of glucose and pyruvate on
maximal, FCCP-stimulated OCR (refer to maximal OCR in
Figures 4, 5, 6). These results support the existence of a
glucose dependent but pyruvate independent system
transferring electrons to the respiratory chain in neurons.
Indeed, the glycerol 3-phosphate shuttle directly transfers
electrons to coenzyme Q in the mitochondrial inner membrane.
This shuttle, active in neurons, is regulated by NADH/NAD+
and calcium and depends on the first step of glycolysis [42-44].
When HD striatal neurons were incubated in substrate-rich
media containing a supra-physiological glucose concentration,
no respiratory deficits were detected. These results are
consistent with previous results obtained in HD knock-in mouse
primary striatal neurons  where mitochondrial respiration
was monitored in medium containing 15 mM glucose. In
addition to these results, we now show that when the
respiration demand is high, HD striatal neurons exhibit
respiratory deficits when supplied with 2.5 mM glucose either
alone or when supplemented with lactate or pyruvate. When
pyruvate was the sole substrate present in the assay media, no
significant respiratory deficits were observed in HD striatal
neurons. These results argue for an alteration in glucose
uptake and/or in glycolysis in HD rather than any defects in the
respiratory chain itself. This hypothesis is further supported by
the fact that deficits were only detected in the presence of
physiological glucose concentrations but not when glucose was
abundant. Moreover, because glucose defects in OCR were
not compensated by pyruvate addition, it appears that a
Figure 7. HD striatal neurons have a lower capacity to use anaerobic glycolysis in the presence of high and low glucose
concentrations. Striatal neurons were cultivated for 7 days in Neurobasal medium containing 25 mM glucose and
complemented with B27 and pyruvate (1 mM). A) On the day of the respiration experiments, medium was replaced with
substratefree KHB and WT cells were incubated for 1 h at 37C. ECAR was measured after injection of different concentrations of glucose
then oligomycin. Data are mean SEM and derived from the same experiment presented in Figure 2. * p < 0.05, ** p < 0.01 vs. 2.5
mM glucose. B) On the day of the respiration experiments, cells were incubated in 2.5 mM glucose in KHB medium or
bicarbonateand HEPES-free DMEM containing 25 mM glucose complemented with pyruvate (1 mM) for 1 h at 37C. ECAR was measured
under basal conditions and after oligomycin stimulation. Data are mean SEM from all independent experiments (2.5 mM glucose:
five replicates in each of five experiments, n=25; 25 mM glucose: eleven replicates in each of seven experiments, n=77). . * p<0.05,
** p < 0.01 comparing WT vs. HD.
Figure 8. HD cortical neurons had no metabolic deficits regardless of the substrate supplied. HD and WT cortical neurons
were cultivated for 7 days in Neurobasal medium complemented with B27 and pyruvate (1 mM). One hour before the start of
respiration experiments, medium was replaced with substrate-free KHB and incubated at 37C before being placed in the Seahorse
analyser. OCR measurements were performed as described in Figures 2 and 3. OCR measurements following injection of (A) 2.5
mM glucose, (B) 10 mM pyruvate or (C) 10 mM lactate followed by oligomycin (oligo) and FCCP (2 M) at the indicated times. D)
ECAR was measured in the presence of 2.5 mM glucose under basal conditions and after oligomycin stimulation. Data shown in
panels A-C are from representative experiments while data in panel D is the mean SEM calculated from all replicates (eight
replicates for each substrate in each of two independent experiments)..
glucose-dependent but pyruvate independent electron transfer
pathway is down regulated in HD primary striatal neurons.
Extracellular acidification is a valid indicator of the anaerobic
glycolysis rate (metabolic pathway converting glucose into
lactate) and a valuable tool for analysing cellular bioenergetics
[29,45]. Here, we observed that ECAR deficits were detected in
HD striatal neurons both when incubated in culture medium
containing a supra-physiological glucose concentration but also
in KHB containing 2.5 mM glucose. Taken together, the OCR
and ECAR data strongly support the hypothesis that striatal
glycolysis deficits occur early in HD pathogenesis.
Dysregulation in glycolysis has been reported in several
studies in animal and cellular HD models or in patients
[20,46-48]. Additionally, Powers and colleagues reported a
preserved mitochondrial oxidative metabolism in early HD
patients with striatal atrophy, indicating that defects in
respiratory chain enzymes observed in post mortem brain are
either not sufficient to explain oxidative phosphorylation
impairments or are not present early in the time course of the
disease . They also reported in the same early HD patients
a decrease in cerebral glucose metabolism indicating a
selective impairment of striatal glycolytic metabolism . Very
recently, Zala et al. showed that the glycolytic enzyme,
GAPDH, is located on neuron vesicles and that local glycolysis
powers vesicular fast axonal transport. Additionally, the authors
demonstrated that huntingtin is a scaffold that joins GAPDH to
these vesicles, suggesting that mutations in huntingtin could
perturb glycolysis-generated ATP necessary for vesicle motility
Interestingly, in our study, even though mHtt aggregates are
present in both cell types, metabolic defects were only detected
in embryonic striatal neurons and not in cortical neurons, a
brain region affected later in Huntington disease progression.
Indeed, cortical neurons showed neither OCR deficits nor
ECAR alterations in the presence of a physiological
concentration of glucose. These results provide additional
insight into the mechanisms of selective striatal
neurodegeneration and into the relative metabolic vulnerability
of different cellular populations to mHtt toxicity. In the light of
the present study, it might be argued that striatal neurons seem
to have different metabolic requirements compared to cortex
and may have a reduced capacity to manage substrate
deprivation. Indeed, we can observe in Figures 4A and 8A that
glucose injection did not have the same impact on basal OCR
in striatal and cortical neurons. In striatal neurons a significant
increase of 31.5 5.8% was observed in response to glucose
injection; for cortical neurons, which had a higher basal OCR in
the absence of glucose, the response was only 9 2.9%.
In summary, early glycolysis defects are found specifically in
HD striatal neurons. These subtle defects, observed only with
levels of glucose found in brain, may only have adverse
consequences after prolonged stress or in combination with
other age-related declines in metabolism, explaining why
neurodegeneration only becomes evident in HD gene carriers
in middle age. These results can be related to new insights in
pre-symptomatic carriers of apolipoprotein E4, who show
reduced cerebral glucose metabolism even before A
aggregation and decades before the onset of AD pathology
[50,51]. These brain functional abnormalities in
neurodegenerative disease gene carriers argue for early
prevention therapies, decades before the onset of cognitive or
Figure S1. Confirmation of mhtt expression in primary
neuronal cultures. Primary striatal and cortical neurons from
HD rats express mhtt. SDS-insoluble and thus aggregated
proteins were trapped on a nitrocellulose membrane and
probed with a polyQ-specific antibody. The presence of
aggregated polyQ-containing protein in primary striatal and
cortical cultures from HD rat embryos but not their WT
littermates indicates the expression of aggregated forms of
mhtt in these neurons. Each dot is the extract from a separate
neuronal culture, prepared from an individual WT or HD
We are grateful to all the members of the Mitotarget consortium
for their helpful comments throughout the project. We also
thank colleagues at Trophos for their assistance and comments
during the course of this work.
Conceived and designed the experiments: CG LEC HPN TB
RMP. Performed the experiments: CG GT JT VL LEC MM.
Analyzed the data: CG LEC HPN TB RMP. Contributed
reagents/materials/analysis tools: LEC LY-T HPN. Wrote the
manuscript: CG LEC HPN TB RMP.
1. Bruyn GW ( 1979 ) Huntington's chorea . Tijdschr Ziekenverpl 32 : 101 - 105 . PubMed: 154197 .
2. Vonsattel JP , Myers RH , Stevens TJ , Ferrante RJ , Bird ED et al. ( 1985 ) Neuropathological classification of Huntington's disease . J Neuropathol Exp Neurol 44 : 559 - 577 . doi:10.1097/ 00005072 - 198511000 -00003. PubMed: 2932539 .
3. Djouss L , Knowlton B , Cupples LA , Marder K , Shoulson I et al. ( 2002 ) Weight loss in early stage of Huntington's disease . Neurology 59 : 1325 - 1330 . Available online at: doi:10.1212/01.WNL. 0000031791.10922.CF. PubMed: 12427878.
4. Browne SE ( 2008 ) Mitochondria and Huntington's disease pathogenesis: insight from genetic and chemical models . Ann N Y Acad Sci 1147 : 358 - 382 . doi:10.1196/annals.1427.018. PubMed: 19076457 .
5. van der Burg JM , Bacos K , Wood NI , Lindqvist A , Wierup N et al. ( 2008 ) Increased metabolism in the R6/2 mouse model of Huntington's disease . Neurobiol Dis 29 : 41 - 51 . doi:10.1016/j.nbd. 2007 .07.029. PubMed: 17920283 .
6. Hayden MR , Martin WR , Stoessl AJ , Clark C , Hollenberg S et al. ( 1986 ) Positron emission tomography in the early diagnosis of Huntington's disease . Neurology 36 : 888 - 894 . doi:10.1212/WNL.36.7.888. PubMed: 2940474 .
7. Kuhl DE , Phelps ME , Markham CH , Metter EJ , Riege WH et al. ( 1982 ) Cerebral metabolism and atrophy in Huntington's disease determined by 18FDG and computed tomographic scan . Ann Neurol 12 : 425 - 434 . doi:10.1002/ana.410120504. PubMed: 6217782 .
8. Kuwert T , Lange HW , Langen KJ , Herzog H , Aulich A et al. ( 1990 ) Cortical and subcortical glucose consumption measured by PET in patients with Huntington's disease . Brain 113 ( 5 ): 1405 - 1423 . doi: 10.1093/brain/113.5.1405. PubMed: 2147116 .
9. Jenkins BG , Koroshetz WJ , Beal MF , Rosen BR ( 1993 ) Evidence for impairment of energy metabolism in vivo in Huntington's disease using localized 1H NMR spectroscopy . Neurology 43 : 2689 - 2695 . doi: 10.1212/WNL.43.12.2689. PubMed: 8255479 .
10. Koroshetz WJ , Jenkins BG , Rosen BR , Beal MF ( 1997 ) Energy metabolism defects in Huntington's disease and effects of coenzyme Q10 . Ann Neurol 41 : 160 - 165 . doi:10.1002/ana.410410206. PubMed: 9029064 .
11. Martin WR , Wieler M , Hanstock CC ( 2007 ) Is brain lactate increased in Huntington's disease ? J Neurol Sci 263 : 70 - 74 . doi:10.1016/j.jns. 2007 .05.035. PubMed: 17655868 .
12. Reynolds NC Jr., Prost RW , Mark LP ( 2005 ) Heterogeneity in 1H-MRS profiles of presymptomatic and early manifest Huntington's disease . Brain Res 1031 : 82 - 89 . doi:10.1016/j.brainres. 2004 .10.030. PubMed: 15621015 .
13. Snchez-Pernaute R , Garca-Segura JM , del Barrio Alba A, Viao J , de Ybenes JG ( 1999 ) Clinical correlation of striatal 1H MRS changes in Huntington's disease . Neurology 53 : 806 - 812 . doi:10.1212/WNL. 53.4.806. PubMed: 10489045 .
14. Unschuld PG , Edden RA , Carass A , Liu X , Shanahan M et al. ( 2012 ) Brain metabolite alterations and cognitive dysfunction in early Huntington's disease . Mov Disord 27 : 895 - 902 . doi:10.1002/mds. 25010. PubMed: 22649062 .
15. van den Bogaard SJ , Dumas EM , Teeuwisse WM , Kan HE , Webb A et al. ( 2011 ) Exploratory 7-Tesla magnetic resonance spectroscopy in Huntington's disease provides in vivo evidence for impaired energy metabolism . J Neurol 258 : 2230 - 2239 . doi:10.1007/ s00415- 011 - 6099 -5. PubMed: 21614431 .
16. Gu M , Gash MT , Mann VM , Javoy-Agid F , Cooper JM et al. ( 1996 ) Mitochondrial defect in Huntington's disease caudate nucleus . Ann Neurol 39 : 385 - 389 . doi:10.1002/ana.410390317. PubMed: 8602759 .
17. Browne SE , Bowling AC , MacGarvey U , Baik MJ , Berger SC et al. ( 1997 ) Oxidative damage and metabolic dysfunction in Huntington's disease: selective vulnerability of the basal ganglia . Ann Neurol 41 : 646 - 653 . doi:10.1002/ana.410410514. PubMed: 9153527 .
18. Damiano M , Galvan L , Dglon N , Brouillet E ( 2010 ) Mitochondria in Huntington's disease . Biochim Biophys Acta 1802 : 52 - 61 . doi:10.1016/ j.bbadis. 2009 .07.012. PubMed: 19682570 .
19. Guidetti P , Charles V Chen EY , Reddy PH , Kordower JH et al. ( 2001 ) Early degenerative changes in transgenic mice expressing mutant huntingtin involve dendritic abnormalities but no impairment of mitochondrial energy production . Exp Neurol 169 : 340 - 350 . doi: 10.1006/exnr.2000.7626. PubMed: 11358447 .
20. Powers WJ , Videen TO , Markham J , McGee-Minnich L , AntenorDorsey JV et al. ( 2007 ) Selective defect of in vivo glycolysis in early Huntington's disease striatum . Proc Natl Acad Sci U S A 104 : 2945 - 2949 . doi:10.1073/pnas.0609833104. PubMed: 17299049 .
21. Milakovic T , Johnson GV ( 2005 ) Mitochondrial respiration and ATP production are significantly impaired in striatal cells expressing mutant huntingtin . J Biol Chem 280 : 30773 - 30782 . doi:10.1074/ jbc.M504749200. PubMed: 15983033.
22. Seong IS , Ivanova E , Lee JM , Choo YS , Fossale E et al. ( 2005 ) HD CAG repeat implicates a dominant property of huntingtin in mitochondrial energy metabolism . Hum Mol Genet 14 : 2871 - 2880 . doi: 10.1093/hmg/ddi319. PubMed: 16115812 .
23. Siddiqui A , Rivera-Snchez S , Castro MD , Acevedo-Torres K , Rane A et al. ( 2012 ) Mitochondrial DNA damage Is associated with reduced mitochondrial bioenergetics in Huntington's disease . Free Radic Biol Med , 53 : 1478 - 88 . PubMed: 22709585 .
24. Milakovic T , Quintanilla RA , Johnson GV ( 2006 ) Mutant huntingtin expression induces mitochondrial calcium handling defects in clonal striatal cells: functional consequences . J Biol Chem 281 : 34785 - 34795 . doi:10.1074/jbc.M603845200. PubMed: 16973623.
25. Oliveira JM , Jekabsons MB , Chen S , Lin A , Rego AC et al. ( 2007 ) Mitochondrial dysfunction in Huntington's disease: the bioenergetics of isolated and in situ mitochondria from transgenic mice . J Neurochem 101 : 241 - 249 . doi:10.1111/j.1471- 4159 . 2006 .04361.x. PubMed: 17394466.
26. Oliveira JM , Chen S , Almeida S , Riley R , Gonalves J et al. ( 2006 ) Mitochondrial-dependent Ca2+ handling in Huntington's disease striatal cells: effect of histone deacetylase inhibitors . J Neurosci 26 : 11174 - 11186 . doi:10.1523/JNEUROSCI.3004- 06 . 2006 . PubMed: 17065457.
27. Yu-Taeger L , Petrasch-Parwez E , Osmand AP , Redensek A , Metzger S et al. ( 2012 ) A Novel BACHD Transgenic Rat Exhibits Characteristic Neuropathological Features of Huntington Disease . J Neurosci 32 : 15426 - 15438 . doi:10.1523/JNEUROSCI.1148- 12 . 2012 . PubMed: 23115180.
28. Friedman WJ , Ibez CF , Hallbk F , Persson H , Cain LD et al. ( 1993 ) Differential actions of neurotrophins in the locus coeruleus and basal forebrain . Exp Neurol 119 : 72 - 78 . doi:10.1006/exnr.1993.1007. PubMed: 8432352 .
29. Wu M , Neilson A , Swift AL , Moran R , Tamagnine J et al. ( 2007 ) Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells . Am J Physiol Cell Physiol 292 : C125 - C136 . PubMed: 16971499.
30. Brand MD , Nicholls DG ( 2011 ) Assessing mitochondrial dysfunction in cells . Biochem J 435 : 297 - 312 . doi:10.1042/BJ20110162. PubMed: 21726199 .
31. Silver IA , Ereciska M ( 1994 ) Extracellular glucose concentration in mammalian brain: continuous monitoring of changes during increased neuronal activity and upon limitation in oxygen supply in normo-, hypo-, and hyperglycemic animals . J Neurosci 14 : 5068 - 5076 . PubMed: 8046468 .
32. Magistretti PJ , Pellerin L ( 1999 ) Cellular mechanisms of brain energy metabolism and their relevance to functional brain imaging . Philos Trans R Soc Lond B Biol Sci 354 : 1155 - 1163 . doi:10.1098/rstb. 1999.0471. PubMed: 10466143 .
33. Han I , You Y , Kordower JH , Brady ST , Morfini GA ( 2010 ) Differential vulnerability of neurons in Huntington's disease: the role of cell typespecific features . J Neurochem 113 : 1073 - 1091 . PubMed: 20236390 .
34. Cheng A , Hou Y , Mattson MP ( 2010 ) Mitochondria and neuroplasticity . ASN. New_Eur 2: e00045. PubMed: 20957078.
35. Li XJ , Orr AL , Li S ( 2010 ) Impaired mitochondrial trafficking in Huntington's disease . Biochim Biophys Acta 1802 : 62 - 65 . doi:10.1016/ j.bbadis. 2009 .06.008. PubMed: 19591925 .
36. Routh VH ( 2002 ) Glucose-sensing neurons: are they physiologically relevant? Physiol Behav 76 : 403 - 413 . doi:10.1016/ S0031-9384(02)00761-8 . PubMed: 12117577 .
37. Magistretti PJ , Sorg O , Naichen Y , Pellerin L , de Rham S et al. ( 1994 ) Regulation of astrocyte energy metabolism by neurotransmitters . Ren Physiol Biochem 17 : 168 - 171 . PubMed: 7518950 .
38. Pellerin L , Bouzier-Sore AK , Aubert A , Serres S , Merle M et al. ( 2007 ) Activity-dependent regulation of energy metabolism by astrocytes: an update . Glia 55 : 1251 - 1262 . doi:10.1002/glia.20528. PubMed: 17659524 .
39. Pellerin L , Magistretti PJ ( 1994 ) Glutamate uptake into astrocytes stimulates aerobic glycolysis: a mechanism coupling neuronal activity to glucose utilization . Proc Natl Acad Sci U S A 91 : 10625 - 10629 . doi: 10.1073/pnas.91.22.10625. PubMed: 7938003 .
40. O'Brien J , Kla KM , Hopkins IB , Malecki EA , McKenna MC ( 2007 ) Kinetic parameters and lactate dehydrogenase isozyme activities support possible lactate utilization by neurons . Neurochem Res 32 : 597 - 607 . doi:10.1007/s11064- 006 - 9132 -9. PubMed: 17006762 .
41. Fnfschilling U , Supplie LM , Mahad D , Boretius S , Saab AS et al. ( 2012 ) Glycolytic oligodendrocytes maintain myelin and long-term axonal integrity . Nature 485 : 517 - 521 . PubMed: 22622581 .
42. McKenna MC , Waagepetersen HS , Schousboe A , Sonnewald U ( 2006 ) Neuronal and astrocytic shuttle mechanisms for cytosolic-mitochondrial transfer of reducing equivalents: current evidence and pharmacological tools . Biochem Pharmacol 71 : 399 - 407 . doi:10.1016/j.bcp. 2005 .10.011. PubMed: 16368075 .
43. Nguyen NH , Brthe A , Hassel B ( 2003 ) Neuronal uptake and metabolism of glycerol and the neuronal expression of mitochondrial glycerol-3-phosphate dehydrogenase . J Neurochem 85 : 831 - 842 . doi: 10.1046/j.1471- 4159 . 2003 .01762.x. PubMed: 12716415.
44. Ramos M , del Arco A , Pardo B , Martinez-Serrano A , Martinez-Morales JR et al. ( 2003 ) Developmental changes in the Ca2+-regulated mitochondrial aspartate-glutamate carrier aralar1 in brain and prominent expression in the spinal cord . Brain Res. Dev Brain Res 143 : 33 - 46 . doi:10.1016/S0165-3806(03) 00097 - X .
45. Gohil VM , Sheth SA , Nilsson R , Wojtovich AP , Lee JH et al. ( 2010 ) Nutrient-sensitized screening for drugs that shift energy metabolism from mitochondrial respiration to glycolysis . Nat Biotechnol 28 : 249 - 255 . PubMed: 20160716 .
46. Ferreira IL , Cunha-Oliveira T , Nascimento MV , Ribeiro M , Proena MT et al. ( 2011 ) Bioenergetic dysfunction in Huntington's disease human cybrids . Exp Neurol 231 : 127 - 134 . doi:10.1016/j.expneurol. 2011 .05.024. PubMed: 21684277 .
47. Olh J , Klivnyi P , Gardin G , Vcsei L , Orosz F et al. ( 2008 ) Increased glucose metabolism and ATP level in brain tissue of Huntington's disease transgenic mice . FEBS J 275 : 4740 - 4755 . doi: 10.1111/j.1742- 4658 . 2008 .06612.x. PubMed: 18721135.
48. Jin YN , Hwang WY , Jo C , Johnson GV ( 2012 ) Metabolic state determines sensitivity to cellular stress in Huntington disease: normalization by activation of PPARgamma. PLOS ONE 7: e30406 . doi:10.1371/journal.pone.0030406. PubMed: 22276192 .
49. Zala D , Hinckelmann MV , Yu H , Lyra da Cunha MM , Liot G et al. ( 2013 ) Vesicular glycolysis provides on-board energy for fast axonal transport . Cell 152 : 479 - 491 . doi:10.1016/j.cell. 2012 .12.029. PubMed: 23374344 .
50. Jagust WJ , Landau SM ( 2012 ) Apolipoprotein E , Not Fibrillar betaAmyloid, Reduces Cerebral Glucose Metabolism in Normal. Aging - J Neuroscience 32 : 18227 - 18233 . doi:10.1523/JNEUROSCI. 3266- 12 . 2012 .
51. Reiman EM , Chen K , Alexander GE , Caselli RJ , Bandy D et al. ( 2004 ) Functional brain abnormalities in young adults at genetic risk for lateonset . Alzheimer'S Dementia - Proc Natl Acad Sci U S A 101 : 284 - 289 . doi:10.1073/pnas.2635903100.