Metabolism in HD - Still a relevant mechanism?

Abstract

The polyglutamine expansion within huntingtin is the causative factor in the pathogenesis of Huntington’s disease (HD). Although the underlying mechanisms by which mutant huntingtin causes neuronal dysfunction and degeneration have not been fully elucidated, the compelling evidence suggests that mitochondrial dysfunction and compromised energy metabolism are key players in HD pathogenesis. Longitudinal studies of HD subjects have shown that reductions in glucose utilization before the disease clinical onset. Preferential striatal neurodegeneration, a hallmark of HD pathogenesis, has also been associated with interrupted energy metabolism. Data from genetic HD models indicate that mutant huntingtin disrupts mitochondrial bioenergetics and prevents ATP generation, implying altered energy metabolism as an important component of HD pathogenesis. Here we revisit the evidence of abnormal energy metabolism in the central nervous system of HD patients, overview our current understanding of the molecular mechanisms underlying abnormal metabolism induced by mutant huntingtin, and discuss the promising therapeutic development by halting abnormal metabolism in HD.

Introduction

Huntington disease (HD) is an autosomal dominant neurodegenerative disease which is caused by a CAG repeat expansion in the first exon of the huntingtin gene that encodes huntingtin (Htt) protein^1,2. Individuals who have 36 CAG repeats or more in the huntingtin gene develop the clinical symptoms, including motor, psychiatric, and cognitive abnormalities that cause a progressive loss of functional capacity and shortened life span. HD patients also exhibit profound metabolic abnormalities^3,4. At present, no pharmacological interventions are available to delay the onset or reverse progression of this devastating disease.

Despite remarkable progress in the understanding of the process underlying HD pathogenesis, the molecular mechanisms by which mutant Htt (mHtt) causes disease progression remain uncertain. Compelling data from studies in human HD subjects and experimental HD models suggest that deficits in energy metabolism, due to mitochondrial dysfunction, play a critical role in promoting HD pathogenesis ^5–7. The nature and cause of mitochondria dysfunction in HD is multifactorial, involving direct mHtt-mitochondria interactions and indirect effects via transcriptional dysregulation and trafficking impairment, which compromise mitochondria bioenergetics and dynamics ⁷. Neurons are highly dependent on mitochondria ATP production and Ca²⁺ buffering to maintain excitability and normal synaptic communication ⁸. Moreover, neurons require efficient biogenesis and mitophagy to renew or adapt mitochondria levels throughout their lifespan ^9,10. Therefore, neurons are very sensitive to disturbed energy metabolism.

Here we provide an overview on evidence of metabolism deficits in HD brain and discuss the possible molecular mechanisms underlying the abnormal metabolism in the context of both a toxic gain-of-function from mHtt and the loss of function of normal Htt. The understanding the molecular mechanisms that drive to abnormal metabolism and neurodegeneration should open new avenues for developing promising therapeutic approaches to preserve neuronal function and prevent neurodegeneration in HD.

Deficits in energy metabolism are detected in human HD brain

Studies of cerebral glucose metabolism using F-18 fluorodeoxyglucose positron emission tomography (FDG-PET) provide strong evidence for an impairment of energy metabolism in the caudate putamen and cortex of presymptomatic HD patients ^11,12. Powers and colleagues investigated mitochondrial oxidative metabolism in the striatum of pre-symptomatic HD subjects by direct measurements of the molar ratio of cerebral oxygen metabolism to cerebral glucose metabolism with positron emission tomography and observed selective defect of glycolysis in early HD striatum¹³, these data suggest that metabolic deficit is an early event in HD and metabolic impairment precedes neuropathology and clinical symptoms in HD patients. A further study showed that impaired basal ganglia metabolism is highly correlated with the functional capacity of HD patients and the degree of their motor dysfunction¹⁴. Using magnetic resonance spectroscopy (MRS) imaging, increased lactate levels were observed in the striatum and occipital cortex of HD patients ¹⁵, it may reflect inefficient oxidative phosphorylation which leads to accumulation of lactate from pyruvate via lactate dehydrogenase. In contrast, a MRS study of cerebrospinal fluid from HD patients showed reduced levels of both lactate and citrate may indicate an impairment of both glycolysis and tricarboxylic acid cycle function in HD patients¹⁶.. Various mechanisms that underlie the energy metabolic deficits in HD brain have been proposed, including inhibition of mitochondrial complex II, compromised mitochondrial calcium handling ^17,18, increased oxidative stress ¹⁹, dysfunctional mitochondrial bioenergetics ^20–22, abnormal mitochondria trafficking^22–24, deregulation of key factors controlling mitochondrial biogenesis ²⁵, and deregulated kinases such AMP-activated protein kinase²⁶ and creatine kinase²⁷.

Molecular mechanisms underlying abnormal metabolism in HD (Summarized in Figure 1)

Figure 1.

Summary figure indicating the putative molecular mechanism by which mutant huntingtin (mHtt) induces energy metabolic failure and neurodegeneration in HD.

Inhibition of mitochondrial complex II by mHtt

Postmortem studies show marked deficiency of mitochondrial complex II in the striatum of HD subjects^28–30. Cultured striatal neurons transfected with N-terminus mHtt showed decreased complex II enzymatic activity associated with selective depletion of SDH, and overexpression of complex II/SDH subunits protected cells in this model³¹. Furthermore, expression of full-length mHtt in immortalized striatal progenitor cells (derived from the HdhQ111 knock-in mouse model) decreases complex II activity and increases the sensitivity of cells to calcium-induced decreases in oxygen consumption and mitochondrial membrane potential, whereas overexpression of complex II prevents mitochondrial dysfunction and cell death ^31,32.

mHtt disrupts Ca ²⁺ buffering capacity in mitochondria

Mitochondria play an important role in buffering changing cytoplasmic calcium levels in response to neuronal activity ³³. Mitochondrial calcium transport is powered by the mitochondrial proton gradient, and increased neuronal calcium modifies mitochondrial ATP production by uncoupling oxidative phosphorylation (OXPHOS). Calcium overload may result in discharge of the mitochondrial membrane potential, opening of the mitochondrial permeability transition (MPT) pore, release of cytochrome c, and activation of cell death pathways ³³. Mitochondria isolated from lymphoblast cells of HD patients and HD mouse brain have a reduced membrane potential and depolarize at lower calcium concentrations than control mitochondria²⁰. The reduced mitochondrial ATP levels and decreased ATP/ADP ratio found in mHtt-containing striatal cells is linked to increased calcium influx through NMDA receptors, and cell ATP/ADP ratio is normalized by blocking calcium influx³⁴. It was reported that both wild Htt and mHtt bind to the outer mitochondrial membrane in human neuroblastoma cells and cultured striatal cells¹⁸. Mitochondria incubated with mHtt had increased sensitivity to calcium-induced opening of the MPT and release of cytochrome c, consistent with a direct effect of mHtt on mitochondrial calcium handling¹⁸. It is intriguing to note that striatal mitochondria contain more cyclophilin D than cortical mitochondria and are more sensitive to calcium-induced MPT pore opening³⁵.

mHtt increases oxidative stress

Evidence of enhanced oxidative stress in HD brain includes an increase in accumulation of lipofuscin, a product of unsaturated fatty acid peroxidation that is most prominent in vulnerable striatal neurons³⁰. A significantly increased 8-hydroxydeoxyguanosine (8-OHdG), an oxidized DNA marker, has also been shown in the caudate of HD patients³⁰. In addition, a significant increase in 8-OHdG in mitochondrial DNA of the parietal cortex was found in late stage (Vonsattel grade 3–4) of HD patients. Similarly, 8-OHdG was higher in forebrain tissue and striatum of R6/2 mice at 12 and 14 weeks of age^36,37. Increased oxidative damage to DNA is also detected outside the brain of HD patients^38,39. Oxidative modification of proteins and lipids is increased in the brain of HD subjects and animal models^30,38. Another indicator of increased oxidative stress is the finding that oxidative defense mechanisms including mitochondrial and cytoplasmic superoxide dismutase (MnSOD and Cu-Zn SOD, respectively) are induced in HD brains⁴⁰ and transgenic HD mice⁴¹. Oxidative stress could promote mHtt aggregation and mHtt-induced cell death by impairing proteasome function⁴². These results support the hypothesis on increased oxidative damage in HD.

mHtt impairs mitochondrial bioenergetics

Reduced ATP/ADP ratio is a consequence of mHtt, which has been shown in the striatum of mHtt knock-in mice⁴³, HD postmortem brains⁴⁴, and the lymphoblasts of HD patients³⁴. Increased carbonylation of the mitochondrial enzymes results in decreased mitochondrial enzyme activity and then impaired energy production has been evident in the striatum of HD mice⁴⁵. Further evidence supporting defective mitochondrial bioenergetics in HD is revealed by the direct interaction of mHtt with brain mitochondria^18,20–22. The localization of mHtt to brain mitochondria correlates with age and disease progression in an HD knock-in mouse model²², and results in reduction of mitochondrial ATP generation²².

Mitochondrial spare respiratory capacity is a measure of the ability of mitochondria to generate energy beyond that required for sustaining the basic metabolic needs of the cells, and is important for maintaining homeostasis and survival of neurons. A significant reduction in mitochondrial spare respiratory capacity was reported in human HD fibroblasts and immortalized mHtt expressing mouse striatal cells compared to wild-type cells⁴⁶, indicating that mitochondrial bioenergetics is compromised by mHtt, and supporting a toxic role of mHtt on mitochondrial bioenergetics. Induced pluripotent cells (iPSc) from HD subjects also have reduced mitochondrial spare respiratory capacity and ATP production (unpublished data, personal communication with Dr. Leslie Thompson). Despite these significant observations, it remains unclear how mHtt leads to impaired mitochondrial bioenergetics.

mHtt interrupts mitochondrial trafficking and dynamics

Altered mitochondrial trafficking precedes neuronal dysfunction in both in vitro and in vivo models of HD²³. Full-length mHtt may impair mitochondrial motility in mammalian neurons through both a toxic gain of function from the polyglutamine tract and a loss of function of normal Htt ²³. In addition, it was found that mHtt altered the distribution and reduced the transport rate of mitochondria in the synaptosomes isolated from mHtt knock-in mouse brain ²². However, the mechanisms by which mHtt affects intracellular mitochondrial trafficking are not fully understood ²⁴. A study of HD brains identified a reduced number and altered distribution of mitochondria within vulnerable, calbindin-positive striatal neurons that was more pronounced with disease progression⁴⁷. Reductions of mitochondria were seen preferentially in large- and medium-size mitochondria in conjunction with an increase in levels of dynamin-related protein 1 (Drp1) protein, a mediator of mitochondrial fission, and a decrease in levels of mitofusion 1 (Mfn1), a mediator of mitochondrial fusion ⁴⁷. Kim and colleagues also demonstrated that reductions in peroxisome proliferator-activated receptor (PPAR) gamma coactivator 1 alpha (PGC-1α), a principal regulator of energy metabolism, in HD postmortem brain tissue correlate with reductions in numbers of mitochondria ⁴⁷. These results support the view that altered mitochondrial dynamics represent an important mechanism of mitochondrial dysfunction, and these abnormalities contribute to the mismatch between energy supply and demand that is a recurring event in HD. Interestingly, mHtt has been shown to interact with Drp1, leading to defective mitochondrial axonal transport and synaptic degeneration in postmortem HD brains and primary neurons from transgenic HD mice^48,49.

mHtt reduces PGC-1α and mitochondrial biogenesis

PGC-1α regulates the expression of genes involved in mitochondrial biogenesis and energy homeostasis^25,50. PGC-1α interacts with a number of transcription factors, including NRF-1 and NRF-2 which regulate the expression of mitochondrial respiratory genes ⁵¹. PGC-1α knockout mice exhibit defects in energy metabolism⁵², indicating that PGC-1α plays a central role in modulating energy metabolism. The expression of PGC-1α is repressed by mHtt, due in part to the fact that mHtt interferes with the TAF4/CREB signaling pathway²⁵. In addition, reduced CREB phosphorylation and CRE signaling in mHtt-expressing striatal cells may also contribute to the downregulation of PGC-1α expression ⁴³. PGC-1α target genes are decreased in the postmortem human HD brain ²⁵ and myoblasts from HD patients⁵³. Moreover, PGC-1α mRNA and protein levels are significantly decreased in the brain, liver, brown adipose tissue, muscle of mHtt knock-in mice or HD transgenic mice^53,54, as well in the STHdhQ^111/111 cell line²⁵. Crossing of PGC-1α knockout mice with HD knock-in mice resulted in increased neurodegeneration of striatal neurons and motor abnormalities in the HD mice²⁵. Additionally, expression of PGC-1α partially protects against the toxic effects of mHtt in cultured striatal neurons²⁵. Overall, these data indicate that there is defective PGC-1α functioning and therefore downstream events are likely impaired in HD. mHtt could interfere with transcription of PGC-1α-regulated genes including PGC-1α itself and its target genes which has been shown in the striatum of HD N171-82Q mice and human HD patients⁵⁵. The transcript of PPARγ, a transcription factor that is critical for energy homeostasis, was also downregulated in multiple tissues of an HD mouse model and HD patients⁵⁶. Recently, Soyal and colleagues identified brain- specific isoforms of PGC-1α, including full-length and truncated isoforms, this study indicated differences in regulation of isoform-specific transcripts of PGC-1α as well as potential functional differences between full-length and truncated isoforms, suggesting that newer treatments may be fine-tuned to target the brain-specific isoforms⁵⁷.

mHtt alters AMPK activity

AMPK is a Ser/Thr kinase and major energy sensor that maintains cellular metabolic homeostasis and stimulates pathways that promote energy production or inhibit energy expenditure in response to metabolic stress ^58,59. AMPK comprises three subunits (α, β, and γ) ^60,61, α subunit is the catalytic subunit and has two different isoforms (α1 and α2) ⁶². AMPK activation is known to be associated with a pro-survival role against certain stresses, but the roles and regulation of AMPK in HD pathogenesis are not fully understood. It has been shown that systemic activation of AMPK by anti-diabetic drug metformin extended the shortened lifespan and reduced hindlimb clasping in an HD mouse model⁶³. We recently found that activation of AMPK exhibited a dynamic pattern in an HD cell model, the cells expressing full-length mHtt displayed increased activation of AMPK at early pathogenic phase and then decreased activation of AMPK at late pathogenic phase; moreover, metformin activated AMPK and protected these cells from mHtt toxicity (Jin. et al unpublished data). Additional experiments are needed to evaluate whether the protective effects of metformin are due to AMPK activation in HD models. This is an important issue because AMPK activation may provide distinct functions in different disease stages. It has been reported that overactivation of AMPK-α1 was found in brains of postmortem HD brain and a fragment HD mouse model^26,64, and intrastriatal infusion of AMPK agonist AICAR worsened the motor impairment and neurodegeneration of R6/2 mice ²⁶. However, AMPK activation might be a double-sided sword in HD, early activation may represent a compensatory response to energy deficits induced by mHtt and promote cell survival, but overactivation of AMPK, particularly AMPK-α1, at late stage might be detrimental to cells.

mHtt decreases creatine kinase

The creatine kinase (CK)/phosphocreatine (PCr) system is one of the major machineries controlling proper energy utilization in cells. There are two cytosolic CKs, brain-type CK (CKB) and muscle-type CK (CKM)⁶⁵. Increased PCr concentrations and decreased CKB activities were demonstrated in brains of several HD mouse models (R6/2, N171-82Q, and HdhQ111) and HD patients^44,66. Downregulation of CKB transcripts in the brain of HD mice was also demonstrated by a microarray analysis⁶⁷, suggesting that mHtt might also regulate CKB at the transcriptional level. Moreover, a poor CK/PCr system in HD brains is associated with a reduced ATP/ADP ratio and impaired energy homeostasis, using a microwave fixation method, accumulation of PCr and depletion of ATP were demonstrated in brains of HD mice at an early disease stage⁴⁴.

Besides affecting cellular energy homeostasis, suppression of CKB in HD might also compromise functions of its interacting proteins. One intriguing example is that K-Cl co-transporter 2 (KCC2), which directly binds to CKB and is highly expressed in GABAergic neurons which are selectively vulnerable in HD, was reported to promote spine formation⁶⁸. It would be of great interest to investigate whether inhibition of CKB might account for the loss of spine density ⁶⁹ in HD mice. The detailed mechanism that mediates the suppression of CKB by mHtt is largely uncharacterized. Because CKB is very sensitive to oxidative stress^27,70,71, mHtt enhance ROS production ^30,72, and ROS are likely to mediate suppression of CKB in HD.

Therapeutic approaches by targeting defective metabolism in HD (Summarized in Figure 2)

Figure 2.

Potential therapeutic approaches by targeting mitochondrial dysfunction and improving energy metabolism in HD.

Targeting impaired bioenergetics and metabolism is a beneficial strategy to delay onset given before symptoms begin and prevent/slow disease progression given after onset of disease in HD. A number of promising therapeutics with a particular emphasis on halting mitochondrial dysfunction and improving energy metabolism are developing for treatment of HD, including compounds in preclinical development stage, such as mPTP opening inhibitor, sirtuins modulators, and PPAR activators; and compounds in clinical trials, such as CoQ10 and creatine.

mPTP opening inhibitor

It has been suggested that the neuroprotective properties of cyclosporine A (CsA) are due in part to its ability to prevent mitochondrial permeability transition pore (mPTP) opening in response to high levels of calcium or oxidative stress ^73,74. Exposure to high levels of calcium or oxidative stress results in the mPTP opening of the inner mitochondrial membrane, causing disruption and swelling of mitochondria^74–76. CsA significantly attenuated NMDA-induced calcium peak and loss in the medium-size spiny neurons of YAC128 HD mice⁷⁷. Treatment with CsA protected striatal neurons toxicity induced by 3-NP in vitro and in vivo ⁷⁵. Although CsA is beneficial in HD models, its may not be an idea candidate for chronic treatment in HD due to its powerful immune suppressing effect. Therefore, developing more specific mPTP opening inhibitors is of potential therapeutic benefit by protecting vulnerable neurons populations affected in HD.

Sirtuin modulators

Sirtuins represent a promising new class of conserved deacetylases that play an important role in regulating metabolism⁷⁸. Seven members (SIRT1-7) were identified in mammalian sirtuin family; the role of a few sirtuin members has being explored in HD. SIRT1 deacetylates PGC-1α at specific lysine residues⁷⁹, resulting in increased transcriptional activity of PGC-1α and expression of its target genes. We and others have shown that genetically increasing SIRT1 is neuroprotective in several HD transgenic mouse models, whereas a deficiency of SIRT1 exacerbates the HD phenotype^80,81. An approach to activating SIRT1 is to increase nicotinamide adenine dinucleotide (NAD⁺) levels by administration of nicotinamide precursors ⁸².

Interestingly, nicotinamide improved the motor phenotype in HD mice⁸³. Activation of SIRT3 increases superoxide dismutase 2 activity and enhances antioxidant efficiency. We previously reported that trans-(−)-ε-Viniferin, a compound isolated from natural product, increased SIRT3 levels and protected cells from mHtt toxicity ⁸⁴. Resveratrol (a SIRT1 activator and antioxidant) rescued early neuronal dysfunction induced by mHtt expression in Caenorhabditis elegans⁸⁵ and protected against peripheral deficits in HD mice⁸⁶. In others studies, treatment with resveratrol significantly increased the aerobic capacity in mice⁸⁷. These effects were explained by the fact that in addition to being an antioxidant, resveratrol activates SIRT1 and thus induction of mitochondrial biogenesis which improved mitochondrial function⁸⁷. These and other findings suggest that increase in SIRT1 activity in HD could improve mitochondrial function and maintain metabolism homeostasis in HD. However, the specificity of resveratrol on SIRT1 activity was questioned. Recently, it was reported that SIRT1 can be directly activated through an allosteric mechanism common to chemically diverse sirtuin-activating compounds (STACs), and more specific STACs has been identified by using this system^88,89. We found that a specific STAC identified by this new system prolonged survival and improved motor function in an HD mouse model (Jiang et al., unpublished data). In contrast, Smith and colleagues reported that Selisistat, a SIRT1/Sir2 inhibitor, alleviates pathology in Drosophila and mammalian cell and mouse models of HD⁹⁰. It is intriguing that NF-κB p65 subunit, a reported SIRT2 substrate⁹¹, was deacetylated by Selisistat at the concentrations used in this HD study⁹⁰. It is interesting to know whether Selisistat at the concentrations used in this study also inhibits SIRT2, because SIRT2 inhibition was reported to provide protective effects in an HD model ⁹². The controversial results urge us to further explore the role of sirtuin(s) in different disease stage and develop more specific sirtuin modulators.

PPAR activators

PGC-α is a potent co-activator of the type II nuclear receptor PPAR. Administration of the pan-PPAR agonist, bezafibrate, was efficacious in improving rotarod performance and survival, and atrophy of the striatal medium spiny neurons in HD mice ⁹³. Bezafibrate treatment produced a significant increase in numbers of mitochondria in the striatal spiny neurons in R6/2 HD mice ⁹³. In addition, PPAR γ agonist rosiglitazone penetrates blood-brain barrier and improves mitochondrial biogenesis in the brain⁹⁴. The PPARγ signaling pathway was significantly impaired in the mHtt expressing striatal cells⁹⁵. Pretreatment of mHtt expressing cells with the rosiglitazone prevented the loss of mitochondrial calcium deregulation and oxidative stress overproduction in response to thapsigargin⁹⁵. We demonstrated that rosiglitazone improved motor function in HD mice⁹⁶. These findings suggest that PPAR agonists could be considered in the treatment of HD.

coenzyme Q10 (CoQ10) is critically involved in the electron transport chain⁹⁷ and exerts beneficial effects in mouse models of HD^98–101, though controversial results were also reported in HD models¹⁰² In patients with manifest HD, 600 mg daily of CoQ10 combined with remacemide (CARE-HD) showed a trend toward slowing HD progression with CoQ10 treatment¹⁰³. Subsequently, another study of CoQ10 (Pre2CARE) in manifest HD and healthy controls demonstrated a relative plateau in plasma CoQ10 levels above 2,400 mg daily¹⁰⁴. The shorter duration of trials, compared with typical duration of disease progression, might affect the clinical effects. Moreover, it would be better to identify at-risk subjects and start the treatments earlier. The failure of the CoQ10 treatment may, in part, be due to difficulty reversing the considerable damage needed to cause clinically significant symptoms. As a consequence, a second phase III clinical trial examining the efficacy of CoQ10 at a dose of 2,400 mg daily (2CARE) is presently being carried out by the Huntington Study Group. This trial is enrolling Six hundred eight research participants randomized to CoQ10 or placebo followed for 5 years, with the primary outcome of change in total functional capacity. The 2CARE study will be the largest therapeutic clinical trial to date in HD.

Creatine

Dietary creatine supplementation (1–3%) was shown to delay disease progression by improving aggregate formation, weight loss, impaired motor coordination, brain atrophy, lifespan, and hearing loss in HD mice ^105,106. Nonetheless, results from human trials on dietary creatine supplementation (5–10 g/day) in HD patients have not been very promising to date ¹⁰⁷. Considering the low permeability of the blood-brain barrier to creatine¹⁰⁸, one possible solution is to further increase dosages of dietary creatine in human trials. A phase II prevention and biomarker trials of creatine in at risk HD, neuroimaging demonstrated treatment-related slowing of cortical and striatal atrophy at 6 and 18 months, but the slowing of brain atrophy with creatine treatment was not associated with any clinical benefit¹⁰⁹. A phase III clinical trial of high-dose creatine (40 g/day) for HD patients is currently recruiting participants (CREST-E), the results from the trial will provide further information

Concluding remarks

A deficit in energy metabolism is a prominent feature of HD. The early onset of metabolism-related manifestations in the presymptomatic stage suggests that metabolic deficit occurs in the early cascade of events leading to HD pathogenesis. There is substantial evidence that metabolic alterations associated with mHtt play an important role in HD pathogenesis. Eventually, the ability to monitor disease progression may allow us to treat patients before disease onset and intervene with neuroprotective treatments in order to slow or prevent disease progression. Studies of energy metabolism in HD are therefore having high potential to identify therapeutic targets as well as develop biomarkers. A deeper understanding of mitochondrial biology and correspondingly impaired energy metabolism induced by mHtt will be necessary to develop meaningful therapies to treat HD. As it has been more than 20 years since the discovery of mHtt, an important objective for the next decade of research on HD is to develop interventions that will prevent or slow the progression of these diseases. A full understanding of how mHtt induces compromised metabolism could be an important step in unlocking novel targets and pathways amenable to directed therapy development. Treatments designed to improve energy metabolism are likely to delay onset and slow the pace of disease progression significantly.