**4. Mitochondrial structural damage in ALS and experimental motor neuron degeneration**

The death process involved in the motor neuron loss characteristic of ALS is not yet fully understood. Several functional alterations present in both human disease and experimental models have been reviewed in the previous sections, but several studies have shown also morphological and ultrastructural changes in motor neurons that may be associated with apoptosis and/or necrosis.

Postmortem examination of ALS patients tissues has revealed morphological and ultrastructural abnormalities in mitochondria. Atypical mitochondrial aggregates were found in skeletal muscle subsarcolemmal region and in intramuscular axons (Afifi et al., 1966; Atsumi, 1981), and morphological abnormalities were also detected in proximal axons, as well as dense clusters of mitochondria in the ventral horn of spinal cord SALS patients (Hirano et al., 1984a; b; Sasaki & Iwata, 1996). Giant mitochondria with intramitochondrial inclusions were observed in the liver of some ALS patients and these alterations were disease specific (Nakano et al., 1987). Further, mitochondria with increased volume and with high Ca2+ concentration were found in motor nerve terminals in muscle biopsies of alive ALS patients, which were not observed in patients with other neuropathies or in control subjects (Siklos et al., 1996). Ultrastructural damage of mitochondria, characterized by swelling and rounding, was recently described in platelets of ALS patients (Shrivastava & Vivekanandhan, 2011; Shrivastava et al., 2011a,b).

The main problem with pathological studies in human ALS is the difficulty in determining whether the alterations observed are a cause or a consequence of the disease. This highlights the importance of developing experimental models of motor neuron death to study the temporal progress of the morphological changes, including the alterations of mitochondrial structure. With this objective, we have recently studied the ultrastructural changes of mitochondria in both our acute and chronic models of spinal motor neuron death described above. In the acute model we observed motor neurons with mitochondrial swelling as soon as 2 h after AMPA perfusion, followed in a few hours by the rupture of mitochondrial, nuclear and plasma membranes, which led to total neuronal disruption. These ultrastructural alterations are characteristic of a necrotic process. In contrast, in the chronic

Role of Mitochondrial Dysfunction in Motor Neuron Degeneration in ALS 209

The mitochondrial damage seen in our models is similar to those observed in the human disease and also in muscle and spinal cord of mSOD1 rodent models, namely mitochondrial fragmentation, enlargement, vacuolization, rearrangement of the cristae and swelling (Bendotti et al., 2001; Kong & Xu, 1998; Martin et al., 2009; Menzies et al., 2002b; Wong et al., 1995). The observed rearrangement of the inner membrane to form small vacuoles has been associated with an alteration in the MPTP permeability and also with the trigger of intrinsicapoptosis pathway by release of proapoptotic factors, such as cytochrome c (Bendotti et al., 2001; Martin, 2010; Martin et al., 2009; Ohta et al., 2008) followed by the cleavage of caspases (Li et al., 2000; Pasinelli et al., 2000) Fig. 2 illustrates the ultrastructural mitochondrial damage and shows a schematic representation of the mechanisms associated with these

Altogether the foregoing data suggest that mitochondrial respiratory chain damage is a relevant event in ALS pathogenesis, although it is still unknown if mitochondrial abnormalities are the cause of the disease process or if they are consequence of neuronal degeneration, However, it is clear from the evidence reviewed here that mitochondria definitely play a central role in determining the fate of motor neurons and in their degeneration process. This evidence comes from studies in several tissues of ALS patients, both from biopsies or from postmortem observations, and from direct measurements of mitochondrial function in experimental models of motor neuron degeneration, both in vitro and in vivo. These experiments clearly point to energy deficits and disruption of Ca2+

Integrity of the mitochondria morphology and structure is pivotal for their function and for cellular health. It is interesting that similar structural alterations have been observed in ALS tissues and in in vitro and in vivo models of motor neuron degeneration, including transgenic mSOD1 rodents and excitotoxicity. Clearly, this damage can be associated with the mitochondrial functional deficits, which trigger deleterious process resulting in cellular death by apoptosis, necrosis or a combination of these mechanisms. Although there is biochemical evidence of an apoptotic process involving the mitochondria, no ultrastructural evidence of classic apoptosis, such as apoptotic bodies, has been found. Rather, mitochondrial swelling and membrane disruption are frequently observed, suggesting the

The evidence for a role of calcium homeostasis disruption in the induction of neuronal death is vast, and the involvement of mitochondria in this mechanism seems determinant. The advances in the elucidation of this process should help to understand the importance of the preservation of mitochondrial structure and function, which hopefully can lead to the

This work and the authors' experiments cited were supported by Dirección General del Personal Académico, Universidad Nacional Autónoma de México, (project IN215610) and Consejo Nacional de Ciencia y Tecnología (CONACYT), México (project 128229). L.D.S.C.

alterations.

**5. Conclusions** 

homeostasis and axonal transport.

predominance of a necrotic process.

**6. Acknowledgement** 

design of preventive and therapeutic measures for ALS.

and U.N.R.J. are recipients of a scholarship from CONACYT.

model we observed by day one swelling of the endoplasmic reticulum and only later progressive alterations in mitochondrial internal and external membranes that generated mitochondrial swelling. So, the initial mitochondrial integrity might indicate an apoptotic process, although motor neurons eventually probably die by a slow necrotic process (Fig. 2; Ramírez-Jarquín and Tapia, in preparation). The mitochondrial swelling observed in both models may be associated with energy failure, which as discussed above causes ATP depletion, oxidative stress and inflammatory events, leading to cell death.

Fig. 2. Role of mitochondrial damage in motor neuron excitotoxicity. The electronmicrographs show normal mitochondria and endoplasmic reticulum in a spinal motor neuron of a control rat (left), and swollen mitochondria with altered cristae observed 2 h after perfusion of AMPA by microdialysis (right) (Ramírez-Jarquín and Tapia, unpublished). Bottom: proposal of the involvement of mitochondrial damage in the apoptosis and necrosis processes leading to motor neuron death. The symbols are the same as in Fig. 1. Description in the text.

The mitochondrial damage seen in our models is similar to those observed in the human disease and also in muscle and spinal cord of mSOD1 rodent models, namely mitochondrial fragmentation, enlargement, vacuolization, rearrangement of the cristae and swelling (Bendotti et al., 2001; Kong & Xu, 1998; Martin et al., 2009; Menzies et al., 2002b; Wong et al., 1995). The observed rearrangement of the inner membrane to form small vacuoles has been associated with an alteration in the MPTP permeability and also with the trigger of intrinsicapoptosis pathway by release of proapoptotic factors, such as cytochrome c (Bendotti et al., 2001; Martin, 2010; Martin et al., 2009; Ohta et al., 2008) followed by the cleavage of caspases (Li et al., 2000; Pasinelli et al., 2000) Fig. 2 illustrates the ultrastructural mitochondrial damage and shows a schematic representation of the mechanisms associated with these alterations.
