5.2. Consequences of SLC25A46 dysfunction on mitochondrial metabolism

Disorganization of cristae leads to disruption of the assembly of the respiratory supercomplexes that mediate oxidative phosphorylation, which reduces the activity of their components (i.e., respiratory complexes I–V) and, thus, diminishes respiration efficiency [46, 47]. Mitochondrial metabolism is disrupted in both patients with mutant SLC25A46 alleles and animal models of disrupted SLC25A46. Fibroblasts from patients harboring mutant SLC25A46 showed varying extents of decreased oxygen consumption rate and glycolytic shift (decreased oxygen consumption rate-to-extracellular acidification rate ratio), consistent with the increased lactate levels indicated by magnetic resonance spectroscopy (MRS) [17]. Moreover, siRNA-mediated suppression of SLC25A46 in control fibroblasts phenocopied the basal oxygen consumption defect observed in the cells of patients with mutant SLC25A46, confirming a specific function of SLC25A46 in respiration [20]. Given the specificity of each tissue and each cell type in responding to physiological stresses and mutations, mitochondria isolated from the cerebellum of SLC25A46 knock-out mouse displayed a remarkable decrease in the activities of respiratory complexes I–IV, and thus a dramatically reduced ATP production potential [39]. Taken together, these observations support the hypothesis of an energetic defect being a principal cause of SLC25A46-related neurological disease.

### 5.3. SLC25A46 dysfunction in pathology

Mitochondrial pathobiology has long been linked to the pathogenesis of neurodegenerative diseases, in part because neurons are highly dependent upon mitochondrial metabolism. Autopsy on a pair of deceased siblings who died due to SLC25A46 mutations showed a very small cerebellum, as well as degenerative changes in the inferior olive nucleus. Histology of the cervical spinal cord illustrated loss and ongoing degeneration of spinal motoneurons in the

medial anterior horn at all levels from the cervical to the lumbar region. Rectus femoris muscle biopsies showed severely atrophied muscle fibers of all types, without type grouping [48].

dramatically by the loss of SLC25A46, indicating that SLC25A46 provides direct coupling of lipid flux between the ER and mitochondria at outer-inner mitochondrial membrane contacts [20]. Meanwhile, endoplasmic reticulum chaperone BiP (a.k.a. 78 kDa glucose-regulated protein), which acts at the ER-mitochondria interface under stress conditions and is considered as a major regulator of the ER, was down-regulated in SLC25A46 knock-out mice [43]. These findings support the notion that the facilitation of lipid flux at ER contact sites may be a

Studies implicating OPA1 and the MICOS complex in the maintenance of cristae architecture are compelling, but it is unclear how they may interact [45]. The observation that SLC25A46 interacts with OPA1 and MIC60, the major MICOS organizer, provides a molecular link that

It now seems likely that SLC25A46 may possess multiple homeostatic functions in mitochondrial dynamics. Further studies are expected to reveal more refined details of the pathophysiological functions of SLC25A46, such as which domain interacts with dynamic proteins and

Disorganization of cristae leads to disruption of the assembly of the respiratory supercomplexes that mediate oxidative phosphorylation, which reduces the activity of their components (i.e., respiratory complexes I–V) and, thus, diminishes respiration efficiency [46, 47]. Mitochondrial metabolism is disrupted in both patients with mutant SLC25A46 alleles and animal models of disrupted SLC25A46. Fibroblasts from patients harboring mutant SLC25A46 showed varying extents of decreased oxygen consumption rate and glycolytic shift (decreased oxygen consumption rate-to-extracellular acidification rate ratio), consistent with the increased lactate levels indicated by magnetic resonance spectroscopy (MRS) [17]. Moreover, siRNA-mediated suppression of SLC25A46 in control fibroblasts phenocopied the basal oxygen consumption defect observed in the cells of patients with mutant SLC25A46, confirming a specific function of SLC25A46 in respiration [20]. Given the specificity of each tissue and each cell type in responding to physiological stresses and mutations, mitochondria isolated from the cerebellum of SLC25A46 knock-out mouse displayed a remarkable decrease in the activities of respiratory complexes I–IV, and thus a dramatically reduced ATP production potential [39]. Taken together, these observations support the hypothesis of an energetic defect being a principal cause of

Mitochondrial pathobiology has long been linked to the pathogenesis of neurodegenerative diseases, in part because neurons are highly dependent upon mitochondrial metabolism. Autopsy on a pair of deceased siblings who died due to SLC25A46 mutations showed a very small cerebellum, as well as degenerative changes in the inferior olive nucleus. Histology of the cervical spinal cord illustrated loss and ongoing degeneration of spinal motoneurons in the

may integrate their functions in modulating cristae architecture [18, 20].

5.2. Consequences of SLC25A46 dysfunction on mitochondrial metabolism

which domain recognizes and communicates with the ER.

primary function of SLC25A46.

82 Recent Advances in Neurodegeneration

SLC25A46-related neurological disease.

5.3. SLC25A46 dysfunction in pathology

For now, most anatomical and histological analyses for SLC25A46-related diseases are based on animal models. Four SLC25A46 mutant mammalian models have been discovered or designed, including a calf model and 3 mouse models with different genetic backgrounds (Table 2). Turning calf syndrome, a neurodegenerative disease, is characterized during massive inbreeding in cattle. Genetic study determines that this disease resulted from a single substitution in the coding region of the SLC25A46 [43]. All affected calves manifested early onset of ataxia, especially of hind limbs, and paraparesis (2–6 weeks old). Despite symptomatic care, nervous symptoms progressed over the next months, leading to repetitive falls and ultimately resulting in permanent recumbency and inevitably euthanasia. Characteristic degenerative microscopic lesions were found in both gray matter (brainstem lateral vestibular nuclei and spinal cord thoracic nuclei) and white matter (dorsolateral and ventromedial funiculi of the spinal cord) regions of the central nervous system, as well as demyelination of some peripheral nerves [49]. Electron microscopy confirmed this neuropathy phenotype, revealing discrete demyelinating lesions and some enlarged nodes of Ranvier.

Three SLC25A46 knock-out mouse models with different genetic backgrounds, including FVB/ N, C57BL/6J, and B6D2, were generated, respectively (Table 2). In spite of various mutation positions and sizes, three mouse lines displayed very similar phenotypes, including growth delay, progressive ataxia, optic atrophy, short life span, which recapitulated the pathological state in human. Further histopathologic studies have shown tissue- and cell-specific lesions in both the central nervous system and peripheral nervous system (Table 2).

Although macroscopic examination showed no overt abnormalities in the gross anatomy of mutant brain, histological staining revealed markedly reduced cerebellums, with Purkinje cells (PCs) that had stunted dendrites and were reduced in number. Degeneration (evidenced by Fluoro-Jade C dye) was selectively present in mutant PCs [39]. Examination via electron microscopy (EM) revealed that degenerated PC dendrites exhibited disorganized cytoskeleton, often containing remnants of mitochondria and other organelles. Numerous atypical mitochondria with cytoplasmic inclusions were found both in the soma and dendrites of PCs. In addition, a significant reduction in vGlut1 and vGlut2 immunoreactivity both in PCs and molecular layer indicated a paucity of glutamatergic synapses in mutant mice [42]. Apart from PCs, degenerative signals were also aggregated in the vestibular nucleus of brainstem, deep cerebellar nuclei, the striatum, the corpus callosum, and the spinal cord, but not in other parts of the brain [39]. The neurodegeneration was associated with astrogliosis and microgliosis in the cerebellum and spinal cord, indicating high levels of neuroinflammation [39, 42]. These observations suggest that although SLC25A46 mRNA is transcribed in most neural tissues, SLC25A46 may have a tissue- or cell-specific function. Interestingly, mice with conditional knock-out MFN2 or DRP1 mutations showed a similar phenomenon, with major changes in cerebellar Purkinje cells, whereas granule cells seemed to be spared [50]. It is not yet known why specific neurons are selectively affected by mitochondrial dysfunction. It could be that a large neuronal size makes particular neurons susceptible; Purkinje cells are one of the largest neuron types in the brain with long axons and extremely extensive dendritic branches. Their complex architecture requires the transport and distribution of highly organized organelles, including mitochondria.

optic atrophy, peripheral neuropathy, progressive ataxia, Leigh syndrome, and lethal congenital pontocerebellar hypoplasia. In SLC25A46-related neurodegenerative diseases, phenotype

Neurodegenerative Diseases Associated with Mutations in *SLC25A46*

http://dx.doi.org/10.5772/intechopen.79992

85

This work was supported by the Center for Pediatric Genomics at the Cincinnati Children's Hospital, a grant from the National Institutes of Health (1R01EY026609-01) to Taosheng Huang, and a grant from the National Natural Science Foundation of China (81470299) to

, Lingqian Wu<sup>2</sup> and Taosheng Huang1,3\*

1 Division of Human Genetics, Cincinnati Children's Hospital Medical Center, Cincinnati,

2 Center for Medical Genetics, School of Life Sciences, Central South University, Changsha,

[1] Kann O, Kovacs R. Mitochondria and neuronal activity. American Journal of Physiology-

[2] Chen H, Chan DC. Mitochondrial dynamics–fusion, fission, movement, and mitophagy– in neurodegenerative diseases. Human Molecular Genetics. 2009;18(R2):R169-R176

[3] Twig G, Hyde B, Shirihai OS. Mitochondrial fusion, fission and autophagy as a quality control axis: The bioenergetic view. Biochimica et Biophysica Acta. 2008;1777(9):1092-1097

3 Human Aging Research Institute, Nanchang University, Nanchang, Jiangxi, China

severity correlates strongly with the magnitude of SLC25A46 level deficit observed.

Acknowledgements

Conflict of interest

Author details

OH, USA

Hunan, China

References

Zhuo Li1,2, Jesse Slone1

The authors declare that they have no conflicts of interest.

\*Address all correspondence to: taosheng.huang@cchmc.org

Cell Physiology. 2007;292(2):C641-C657

Zhuo Li.

Aged SLC25A46 mutant mice displayed enhanced hind limb clasping reflex and muscle atrophy, suggesting potential peripheral neuropathy. Acquiring compound muscle action potentials (CMAPs) reduced in mutant sciatic nerve measured by electromyography (EMG) in vivo [39]. Mutant peripheral nerves exhibited sporadic degenerative lesions with local macrophages containing lipid debris and signs of demyelination. In addition, the size of individual endplates in mutant mice was significantly reduced. Retarded neuromuscular junction maturation and improper innervation, early hallmarks of CMT2D, were also documented in mutant muscle [42]. All of these alterations are indicative of peripheral neuropathy.

Optical coherence tomography (OCT) scanning on retina for live mice revealed that although the optic discs were grossly normal in terms of retinal appearance, retinas were thinner in aged SLC25A46 mutant mice [39]. Further quantitative measurements indicated that ganglion cell complex (GCC) thicknesses, which includes the nerve fiber layer (NFL), ganglion cell layer (GCL), and inner plexiform layer (IPL), were significantly reduced in adult mutant mice. Retinal and these reductions were associated with retinal ganglion cell loss and atypically small optic nerve axons with reduced neurofilament expression, as well as some axons that exhibited signs of degeneration and demyelination [39, 42]. Pax6+ and GAD65+ GABAergic amacrine cells—both of which form synapses with retinal ganglion cells—were also significantly reduced. These pathological changes are in line with the phenotypic features of ADOA.

Ultrastructural studies revealed dysmorphic mitochondria in both the central and peripheral nervous systems. Numerous enlarged and round mitochondria with abnormal cristae were found in Purkinje cell dendrites, while ring- or C-shaped mitochondria were commonly observed in soma. Peripheral nerve axons also had abnormal round, fused, and aggregated mitochondria in myelinated and non-myelinated fibers [39, 43].

Given the degeneration in long peripheral axons and distal optic nerves of SLC25A46 knockout animal models, the aforementioned findings support the idea that neurons with long axons or complicated dendrites are more sensitive to abnormal mitochondrial dynamics. Similar to the findings in mutant Purkinje cells, this sensitivity could also be due to the impaired transport of hyperfused mitochondria along axons and dendrites, probably due to their abnormal size and/or reduced ATP availability in the distal portions of long axons secondary to mitochondrial dysfunction. Further studies are needed to clarify this point.
