The Use of the Mitochondrial Toxin 3-NP to Uncover Cellular Dysfunction in Huntington’s Disease

Degenerative diseases that affect the nervous system are characterized by a progressive alteration of specific neuronal populations and normally end in cell death. Some neurodegenerative disorders exhibit a clear genetic origin, such as in the case of Huntington’s Disease (HD); however, most neurodegenerative diseases do not have a genetic cause, suggesting that other mechanisms cause these alterations.


Huntington Disease
Huntington's Disease, also called Huntington's chorea because of the presence of rapid and incessant choreic movements accompanied by cognitive and psychiatric alterations, is an inherited neurodegenerative disease that affects cell projections in a specific region of the brain known as the nucleus striatum. This neurodegenerative illness was described in 1872 by George Huntington, although some features that resemble HD had already been described earlier (Walker, 2007).
The symptoms manifest in the third or fourth decade of life in most cases, and progression of the illness is slow (15 to 20 years). In comparison to other neurodegenerative diseases, the development of HD is associated with the mutation of a single gene called Interesting Transcrip 15 (IT15), or the Hd gene. The mutation originates the expansion of the CAG nucleotide repeats in a single protein called huntingtin; wich is called mutant huntingtin (mhtt), when the CAG expansion is present (Taylor et al., 2002). This single mutation produces diverse cellular, physiological and anatomical changes; however, the cellular mechanisms underlying HD neurodegeneration are not yet fully understood.
A number of studies have shown that mitochondria from HD patients and animal models are damaged (Jenkings et al., 1993), suggesting that disturbances in the cellular metabolism of HD patients originate via mitochondrial dysfunction (Beal, 2005). Deficits in energy metabolism become manifest in the pre-symptomatic and symptomatic HD brain and peripheral tissues (Kuhl et al., 1982;Mazziotta et al., 1987;Grafton et al., 1990;Koroshetz et al., 1997;Lodi et al., 2000). In particular, factors related to mitochondrial functioning seem to underlie the selective vulnerability of striatal cells (Saft et al., 2005;Seong et al., 2005); for example, the reduction of the chaperone, protease and intramembrane mitochondrial molecule Omi/HTr2 (Inagaki et al., 2008), and alterations in oxidative phosphorylation functioning in general (Pickrell et al., 2011). Post-mortem studies on HD brain tissue showed decreased activity in complexes II, III and IV of the mitochondrial respiratory chain (Gu et al., 1996). Also, animal models of HD showed deficits in mitochondrial respiration ; for example; the systemic administration of mitochondrial toxins or inhibitors generated striatal pathology and movement disorders such as chorea and dystonia, which resemble HD (Ludolph et al., 1991;Browne, 2008). In fact, accidental ingestion of the irreversible mitochondrial inhibitor 3-nitropropionic acid (3-NP) was found to cause striatal degeneration and the HD phenotype. Moreover, the systemic administration of 3-NP caused striatal cell loss and movement alterations in rats  and primates (Palfi et al., 1996;Dautry et al., 2000). However, an analysis of unbiased gene expression showed that changes in energy metabolism in mhtt of transgenic mice compared to 3-NP treated animals were different: while 3-NP affected mitochondrial pathway gene expression, the effects of mhtt on metabolism were extramitochondrial (Lee et al., 2007), which suggested that mitochondrial toxins such as 3-NP do not quite cause HD pathology (Olivera, 2010). However, mhtt generates mitochondrial dysfunction in HD (Grunewald & Beal 1999) and reductions in ATP generation (Seong et al., 2005), and it also alters Ca 2+ buffering (Reddy et al., 2009) and mitochondrial trafficking (Li et al., 2010). Therefore, irrespective of whether mhtt impacts mitochondria directly or secondarily, the repercussions of mitochondrial dysfunction are devastating to cells and may underlie the disruptions in numerous cellular processes, resulting in HD pathogenesis. As a result, the identification of respiratory chain changes in complex II of respiratory chain in HD post-mortem brains led to the use of mitochondrial complex II inhibitors to generate toxicity models that replicate aspects of HD striatal pathology in vivo. Thus, studies on mitochondrial toxins are relevant and important for understanding defects in cellular metabolism and the energetic pathogenesis of Huntington's Disease.

3-Nitropropionic acid (3-NP)
3-NP is a highly specific, time dependent and irreversible inhibitor of succinate dehydrogenase (SDH) and the Krebs cycle (Alston et al., 1977). The levels of inhibition of this enzyme by 3-NP correlate well with the levels of inhibition of the tricarboxylic acid cycle (Henry et al., 2002).
It is known that 3-NP imitates the symptoms of dystonia, glutaric aciduria, Leber's disease and HD (Novotny et al., 1986;Janavs and Aminoff 1998;Strauss and Morton, 2003), but after discovering that the accidental ingestion of 3-NP (He et al., 1995;Ming, 1995) caused damage that was concentrated in the striatum, 3-NP was used in experimental models to study the cellular mechanisms underlying striatal neural degeneration (Alexi et al., 1998).
The initial studies suggested that excitotoxicity plays a central role in the physiological and cellular effects of 3-NP on striatal degeneration (Hamilton and Gould, 1987;Novelli et al., 1988;Zeevalk and Nicklas, 1990) because of the presence of massive glutamatergic afferents in the nuclei (Di Figlia et al., 1990); also, metabolic insults were suggested as playing a part in the mechanistic damage Brouillet et al., 1999). Chronic treatment with 3-NP in rats produced astrogliosis and selective degeneration of medium-sized spiny neurons, similar to the neurochemical and histological pathology observed in post-mortem HD tissue. Interestingly, 3-NP treatment was found to retain terminals from large cholinergic interneurons and NADPH-diaphorase-positive aspiny interneurons Brouillet et al., 1999).
The type of death triggered by 3-NP treatment depends on how the toxin was administered: intraparenchymal applications induce ischaemic injury features while intraperitoneal applications induce striatal degeneration, which shows more of an HD phenotype (Borlongan et al., 1997a). Another concern about the 3-NP pharmacological model of HD is related to the variations in cellular damage, which depend on whether a study is carried out in vitro or in vivo, whether rats or mice are used, whether 3-NP is administered intrastriatally or intraperitoneally, whether the treatment is acute or chronic, and whether low, sub-toxic or toxic concentrations are provided (Brouillet et al., 1999(Brouillet et al., , 2005. 3-NP administered by subcutaneous (s.c.) or intraperitoneal (i.p.) injections is more toxic in rats than in mice. In rodents, the toxicity of 3-NP depends on the strain (Brouillet et al., 2005). Fisher rats, for example, are more susceptible to 3-NP toxicity than Sprague-Dawley, Wistar and Lewis strains (Ouary et al., 2000), and C57BL/6 and Balb/c mice are more resistant to 3-NP toxicity than 129SVEMS and FVB/n mice (Gabrielson et al., 2001). The strain-dependent differences observed following 3-NP intoxication are probably related to differences in elimination/detoxification of the compound (Ouary et al., 2000). Vulnerability to different agents is not restricted to animal strains; it occurs in all human groups and is due to genetic variations that give rise to different responses to drugs (Weinshilboum et al., 2003;Weinshilboum and Wang, 2006), so we need to be cautious when generalizing about the results obtained among different strains.
Other important factors are the age and gender of the animals used in experimental protocols . Interestingly, female rats are less sensitive to 3-NP than males, which suggests that oestrogen protection can affect the degree of sensitivity (Nishino et al., 1998;Mogami et al., 2002).
The method used for 3-NP delivery also influences the physiological effect; acute treatments of a single i.p. dose of 3-NP were found to lead to striatal degeneration within 6-12 h after injection (Alexi et al., 1998;Brouillet et al. 1999). Sub-chronic treatments consisting of daily repeated i.p. injections led to striatal degeneration over a few days Schulz et al., 1996;Guyot et al., 1997). Chronic treatments (of more than 5 days up to 4 weeks) with the continuous systemic administration of 3-NP using subcutaneously implanted osmotic minipumps also produced striatal degeneration. Besides the mode of delivery, the treatment dose concentration also has an impact on 3-NP toxicity.
Depending on the time period over which 3-NP is administered, and the dose administered, rodents treated with 3-NP exhibit HD-like motor disorders with hyperkinetic and hypokinetic symptoms (Borlogan et al., 1997), and rats were shown to be more sensitive to the effects of 3-NP than mice (Brouillet, 2005). In rats, the administration of 3-NP (10 mg/kg i.p.) over several days was found to induce the onset of hypokinetic symptoms (Guyot et al., 1997), while its administration in two individual doses caused hyperkinetic symptoms (Borlongan et al., 1997b). However, besides the mode of delivery, the treatment dose also has an impact on 3-NP toxicity, where a 3-NP concentration of ~20 mg/kg was found to induce the expression of an HD behavioural phenotype after two injections. Nevertheless,

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The Use of the Mitochondrial Toxin 3-NP to Uncover Cellular Dysfunction in Huntington's Disease 351 these animals did not display extra-striatal lesions, which are frequently observed in the initial stages of HD Guyot et al., 1997;Borlogan et al., 1997b). The chronic administration of low doses of 3-NP (~10 mg/kg, per day) for more than 3 weeks was found to induce sustained metabolic alterations and some other cellular features exhibited in HD patients, but did it not cause clear dyskinetic movements resembling chorea (Borlogan et al., 1997a, b;Brouillet et al., 1999).
Since there are differences in the response to 3-NP treatment among different animal strains (Ouary et al., 2000), we designed an administration plan of low concentration doses of 3-NP (15 mg/kg, i.p.) over a sub-chronic period (5 days) in C57BL/6 mice, which are known to be more resistant to 3-NP toxicity; this advantage enabled us to observe histopathological changes that mimic those found in the initial steps of the illness (Rodriguez et al., 2010), plus motor alterations such as orofacial dyskinesias and clasping behaviour (Hernández-Echeagaray et al., 2011), and spontaneous behaviours that resemblance the HD phenotype. Figure 1, shows that mice treated with low dose concentration of 3-NP displayed motor hyperactivity, as evaluated in open field tests. Hyperkinetic symptoms are exhibited in the initial steps of striatal damage in animals treated with 3-NP, whereas the hypokinetic phenotype develops later during striatal deterioration (Borlongan et al., 1997b).
The graph shows the effects of 3-NP (15 mg / kg, per 5 days) in the spontaneous ambulatory motor behaviour of mice, scored as the total distance during 10 minutes period, evaluated in the open field test (Versadata, 3.02-1E7E software, Accuscan Instruments, INC.). There was a significant increase in locomotor activity in the 3-NP treated group (t-test, *p=0.0213). Arithmetic means and standard errors are plotted. We are especially interested in discerning the cellular events that take place during the beginning of the neurodegenerative process; the understanding of early dysfunctions will help in the planning of therapeutic strategies to reduce or delay cellular damage. The neuronal damage caused by low doses of 3-NP administration is restricted to striatal calbindin-positive cells (Fig. 2), leaving sparing parvalbumin and calretinin positive interneurons as previously suggested (Ferrante et al., 1987a, 1987b, Kowall et al., 1987¸ Massouh et al., 2008. Cellular alteration can also be initiated by caspase 3-dependent apoptosis, although necrotic cell death is also present (Rodriguez et al., 2010).
Top: light micrographs of calcium binding proteins immune localization in the neostriatal tissue. A, illustrates cells immune positive to parvalbumin. Cells were homogenously distributed in the striatum in both groups. The staining of immune positive cells was located in the soma. Graph in the bottom shows that there were no differences between groups in the number of cells expressing parvalbumin (t35 =-1.519, p=0.138). B illustrates striatal cells that were immune positive to calbinding. Calbinding positive cells were homogenously distributed a long the striatum in both groups and its localization was concentrated in the cell soma. Graph in the bottom shows that in tissue of 3-NP treated group, immune positive cells were significant reduced in comparison to control group (T99=2073.5, p<0.001). C illustrates calretinin immune positive cells. These cells were localized in dorsal striatum and staining was observed in cell body, dendrites and axonal ramifications. Interestingly tissue from 3-NP treated mice exhibited a decrease in cell ramifications but as shown in the bottom graph, the immune stained cell number did not change between groups (T82= 1686.5, p=0.523). Scale bar is 100 μm. 3-NP treatment induces the abnormal production of reactive oxygen species (ROS), as well as highly reactive molecules derived from the formation of nitric oxide (NO). Succinate dehydrogenase inhibition interferes with the electron transport cascade and oxidative phosphorylation, which results in a decrease in ATP production and a cellular energy deficit (Jana et al., 2001;Lunkes et al., 2002). In studies by our group, the systemic administration of low sub-chronic doses of 3-NP did not produce a significant augmentation of NO in the brain (Rodriguez et al., 2010); however, NO and lipid peroxidation (LPO) increased in www.intechopen.com The Use of the Mitochondrial Toxin 3-NP to Uncover Cellular Dysfunction in Huntington's Disease 353 skeletal muscle (Hernández-Echeagaray et al., 2011). Previous reports looking at the involvement of NO in the toxicity of 3-NP did not draw any clear conclusions, but it has been suggested that 3-NP acts as an NO donor, increasing the levels of nitro anions (Jana et al., 2001;Lunkes et al., 2002).
Increases in NO and LPO along with signs of necrosis that manifest at the ultra-structural level in 3-NP-treated animals, like cellular oedema, are suggestive of the inflammatory process. Gene expression of the anti-inflammatory cytokine Interleukin-10 (IL-10) was found to significantly decrease whereas expression of the inflammatory cytokine Interferongamma (IFN) increased, indicating that, in low doses, 3-NP reduces the activation of antiinflammatory cytokines (Fig. 3). This may have negative effects to prevent cellular damage.
PCR products of IL-10 and INF in striatum of control and 3-NP treated groups are displayed. Data were analyzed by measuring continuously gene-specific PCR products and differences were assessed with the 2_ΔΔCT method. Data are presented as the fold increase in gene transcripts normalized to the 18S rRNA expression and relative to the control. IL-10 expression was significantly reduced in the 3-NP group (t13 = 12.904, p<0.001). INFγ exhibited a no significant increase in the 3-NP treated group (t16= 0.232, p= 0.819). Cells obtain energy from oxidative phosphorylation and from glycolysis; the glycolytic enzyme GAPDH has been implicated in neuronal degeneration (Taylor et al., 2002;Huntington's Disease Collaborative Research Group, 1993) and a reduction in GAPDH activity was demonstrated in HD patients and in a transgenic mouse model of HD (Burke et al., 1996;Matthews et al., 1997). Some studies have suggested that mitochondrial failure is secondary to striatal damage in HD (Lee et al., 2007), and then another energy source that may fail and generate damage is glycolysis. Both oxidative phosphorylation and glycolysis can be involved in the pathogenesis of neural degeneration in the striatum. However, inhibition of the glycolytic enzyme GAPDH was found to cause apoptotic damage, which is independent of the activation of caspase 3 (Rodriguez et al., 2010). Hence, alterations in glycolysis may be a critical point in neuronal death, but its inhibition activates different cellular signals than oxidative phosphorylation.

Changes in the periphery
Any deficit in energy metabolism can damage the entire physiology of an organism. Muscles metabolism is essential for locomotion and heat production (Zierath and Hawley, 2004). Patients afflicted by HD experience many deficits (Bradshaw et al., 1992;Aron et al., 2003;Abbruzzese et al., 2003), and weight loss is a characteristic feature (Robbins et al., 2006;Ciammola et al., 2011;Mochel and Haller, 2011). Transgenic mice model in an HD exhibited protein inclusions (Sathasivam et al., 1999) and mhtt aggregation in skeletal muscle (Ribchester et al., 2004). The motor impairments displayed by patients and animal models could result from central neurodegeneration, but also from alterations in muscle metabolism, reflecting peripheral disturbances as a general metabolic failure. Where metabolic collapse occurs in an organism, this could partly clarify the muscle alterations and body weight loss documented in HD patients (Stoy and McKay, 2000;Robbins et al., 2006;Ciammola et al., 2011) and transgenic HD mice (Sathasivam et al., 1999;Ribchester et al., 2004;She et al., 2011) and the alterations caused by metabolic alterations (Simoneau et al., 1995;Petersen et al., 2004).
Initially, it was thought that 3-NP did not produce major peripheral effects (Hamilton and Gould 1987). Until now, most of the studies that were carried out using 3-NP as a tool to investigate neurodegeneration focused on evaluating central damage Borlongan et al., 1997;Brouillet, et al., 1999Brouillet, et al., , 2005, even though in a number of them 3-NP was systemically administered. However, Gabrielson (et al., 2001) showed that 3-NP induced modifications in cardiac muscle physiology.
In addressing the possible changes that 3-NP might generate outside of the brain, our group documented modifications in skeletal muscle after uncoupling oxidative metabolism with 3-NP in low sub-chronic doses. Our hypothesis was that abnormal mitochondrial functioning during first stages of neurodegenerative disease is responsible for body weight loss in patients afflicted with HD or illness where there is a metabolic dysfunction (Stoy and McKay, 2000;Robbins et al., 2006). The systemic administration of low doses of 3-NP altered enzymatic activity in muscle, as well as the organization of the sarcomere (Hernández-Echeagaray et al., 2011), suggesting that energy failure exacerbates metabolic activity in the whole organism, producing high metabolic demands to counteract the failure of bioenergetics. When comparing muscle modifications in animals where glycolysis was inhibited, we found that iodoacetate IOA also alters the ultrastructure of the gastrocnemius muscle; this disorganization was more pronounced in animals that were treated with 3-NP (Fig. 4).
Electron microscopy of gastrocnemius muscle from control, IOA, and 3-NP treated animals. Control muscle shows normal sarcomere organization (white arrow) and mitochondria morphology (black arrow). However, sarcomere of IOA and 3-NP treated groups was altered; also the number of mithoxhondria and mitochondria morphology were modified. In particular muscles from 3-NP treated group were most affected than those from the IOA group. Micrographics magnification is 7000X. Scale bar is 500 nm. Oxidative phosphorylation or metabolic failur e m a y g e n e r a t e a n i n c r e a s e i n e n e r g y consumed by muscles, as has been suggested in several degenerative disorders where alterations in oxidative phosphorylation and metabolism are compromised (Ristow, 2004). It is important to mention that in models of food restriction or malnutrition, the size and body weight of animals were affected before the nervous system became involved in the behavioural phenotype (Woodall et al., 1996;Clapham, 2004). Being aware of the peripheral and central damage due to metabolic dysfunctions might help in the clinical management of the early stages of the disease. For example, treatments designed to improve energy metabolism might modify the course of the illness and delay the progression of the disease.

Conclusions
The goal of this chapter was to illustrate the fact that 3-NP in low sub-chronic doses induces cellular alterations that emulate the damage exhibited in the striatum and muscle of patients afflicted by HD in the early stages of the illness. Even though 3-NP does not reproduce all signs and symptoms displayed in HD patients, it is true that it has helped in the understanding of the cellular physiology of animal models where a general failure in bioenergetics has been presumed.

Acknowledgements
The authors wish to thank A Ruelas for carrying out the open field test evaluation. This work was partially supported by grants from CONACyT, DGAPA-PAPIIT and Institutional support from FES-I, UNAM.