Very little is known about the mechanisms through which corticospinal axons degenerate in HSP, although numerous genes responsible for this disease have been identified in recent years (25
). We contribute here to the understanding of the pathogenic cascade underlying axonal degeneration by studying a mouse model for autosomal recessive HSP due to loss-of-function mutations in the SPG7
gene. Paraplegin-deficient mice are affected by a progressive retrograde degeneration of long ascending and descending tracts in the spinal cord and by optic neuropathy, peripheral axonopathy, and a mild muscular involvement. We provide evidence that axonal degeneration begins distally and then slowly moves proximally along the axon. We also show that axonal length is an important determinant for the selective involvement of a subset of axons in HSP. In the mouse, the corticospinal tract, being mainly polysynaptic, is composed of shorter axons (22
). Consistently its axons degenerate only in very old animals, in contrast with what occurs in humans. Finally, our findings indicate that paraplegin deficiency causes a disease mainly of axons. In both human and mouse, involvement of the skeletal muscle is very mild and in the mouse is largely dependent on the peripheral neuropathy.
The first ultrastructural feature that distinguishes the spinal cord of paraplegin-deficient mice from that of control littermates occurs at about 4.5 months of age and is represented by the appearance of abnormal mitochondria in synaptic terminals in the gray matter of the lumbar spinal cord. At this age, hypertrophic mitochondria are also present in a relevant percentage of long axons, distal to their cellular bodies. Notably, this mitochondrial phenotype correlates with the onset of a significant motor deficit on the rotarod and occurs several months prior to axonal degeneration, suggesting that it is the primary cause for axonal dysfunction and that the neurologic impairment of paraplegin-deficient mice is not directly the result of the loss of axons. This latter observation implies that there is a temporal window during which therapeutic intervention could actually prevent loss of axons.
Nerve terminals invariably contain many mitochondria, which are involved in ATP production, in regulation of cellular calcium homeostasis, and in oxyradical metabolism. All these functions may be affected in paraplegin-deficient mice and contribute to the early axonal dysfunction. One technical problem to address this issue comes from the very limited number of affected mitochondria in the spinal cord and from the inherent complexity of purifying them. We were able to measure reduced ATP synthesis in the spinal cord of very old Spg7–/– mice, suggesting that defective ATP production may play a role in the pathogenesis of HSP, at least in late stages of the disease. The establishment of neuronal cell cultures from paraplegin-deficient mice will allow measurement of the mitochondrial membrane potential and investigation of the mechanisms of mitochondrial dysfunction in the future.
With aging, there is an increase in the percentage of axons showing mitochondrial abnormalities in paraplegin-deficient mice. Furthermore, mitochondria undergo other significant changes, such as rearrangements of cristae, formation of bridges with adjacent mitochondria, and acquisition of giant dimensions. These data raise three important questions: Are the mitochondrial structural abnormalities a primary or a secondary effect of the lack of paraplegin? Why does this phenotype selectively involve only a subset of mitochondria, given the ubiquitous expression pattern of paraplegin? Finally, what is the link between the mitochondrial phenotype and the axonal dysfunction?
Paraplegin is one of the mammalian orthologues of the yeast m-AAA protease, whose role in proteolytic degradation of inner mitochondrial membrane proteins and in regulation of mitochondrial biogenesis has been extensively documented (11
). On the basis of this knowledge, structurally abnormal mitochondria in Spg7–/–
mice may be the result of the accumulation of abnormal, misfolded proteins in the inner mitochondrial membrane. Alternatively, the abnormal mitochondria could result from impaired turnover or processing of a regulatory protein involved in some aspect of mitochondrial dynamics. Mitochondrial morphology is in fact regulated by the balance between processes of fusion and fission, and many extrinsic factors, including proteases, may influence and contribute to this plasticity (26
). The identification of paraplegin substrates will provide important hints to confirm or reject these hypotheses. Finally, we cannot exclude that the abnormal and specialized mitochondrial shapes that we observe in our mice may just be secondary to impaired respiration and serve to increase the ratio of surface area to volume. However, we do not favor this possibility, because in paraplegin-deficient mice, mitochondrial swelling occurs a long time after the first morphological abnormalities are detected and is preceded by a tremendous increase in the size of the organelle.
An interesting and puzzling finding is that, although paraplegin is a ubiquitous protein, abnormalities are restricted only to a subset of mitochondria. Recent evidence suggests that mitochondria are highly dynamic organelles whose shape and function are regulated during growth and development and are dependent on cellular and tissue metabolic demands (29
). For example, mitofusin-2, one of the molecules involved in mammalian mitochondrial fusion events, is required only for selective developmental transitions in the mouse, despite a broad pattern of expression (30
). The morphological and functional heterogeneity of mitochondria raises the possibility that subpopulations of these organelles play different roles within different areas of a cell (31
). Neurons are highly specialized and polarized cells with processes that can reach a length of up to 1 meter in humans. Since the slow diffusion of ATP could limit their metabolic function, neurons meet their energy requirements by placing mitochondria near the site of demand. Accordingly, the appropriate regulation of transport and distribution of mitochondria is an essential component of the life and function of a neuron (32
). A clear understanding of the life cycle of mitochondria in postmitotic cells such as neurons is currently missing. The pattern of mitochondrial motility in axons includes bidirectional saltatory movement and prolonged stationary phases (32
). Mitochondria located very distal to the neural cell body could be required to endure for longer periods of time, and could be much less efficient in adapting to a lack of paraplegin through the recruitment of molecules involved in handling misfolded proteins or in upregulating expression of the respiratory chain components. Furthermore, regulation of mitochondrial morphology in neurons and in different regions of axons may also play a key role. Interestingly, the optic atrophy type I gene (OPA1
), the orthologue of the yeast MGM1
gene involved in regulating mitochondrial morphology, is mutated in a dominantly inherited optic atrophy (33
The appearance of structurally abnormal mitochondria in affected axons of paraplegin-deficient mice is followed in time by segmental swelling due to accumulation of neurofilaments and organelles. This finding strongly suggests impairment of anterograde axonal transport. Axonal transport is an energy-dependent process that is especially crucial to the long axons affected in HSP, which transport molecules and organelles assembled in the neuronal cell body across long distances. Disorganization of the neurofilament network is a common hallmark of toxic neuropathies (36
) and of many neurodegenerative diseases, such as amyotrophic lateral sclerosis, giant axonal neuropathy, infantile spinal muscular atrophy, and axonal Charcot-Marie-Tooth disease (37
). Axonal inclusions of intermediate filaments have been proposed to “strangulate” the axon by a chronic deficit in transport, thus leading to neurodegeneration (43
). In fact, neurofilament accumulation itself may have a toxic effect by further affecting slow axonal transport (44
) and sequestering mitochondria and other organelles. Changes in neurofilament levels also perturb the dynamics and function of microtubules, which are key organelles of intracellular transport.
We found evidence that retrograde axonal transport is delayed in paraplegin-deficient mice. This defect was found only in symptomatic mice, suggesting that impairment of axonal transport is not a primary, but rather a secondary effect. However, impairment of retrograde transport could contribute significantly to the ultimate degeneration of axons by affecting the transport of mitochondria, the trafficking of endosomes, and the internalization of neurotrophic factors. Disruption of retrograde axonal transport has already been shown to cause progressive degeneration of motor neurons in both mice and humans (45
Aberrant cellular trafficking dynamics has been suggested to underlie several other forms of HSP, based on the predicted function of the genes involved (reviewed in ref. 25
). Although involvement of HSP genes in axonal transport is purely speculative in most cases, autosomal dominant HSP linked to chromosome 12q, SPG10
, is due to mutations in a kinesin heavy chain gene, KIF5A
). Furthermore, mice lacking proteolipid protein 1 (PLP1), the only other mouse model available for HSP, display axonal degeneration associated with the accumulation of neurofilaments, mitochondria, and other organelles (48
). These data, together with our findings in paraplegin-deficient mice, suggest that chronic reduction of axonal transport is a common pathway through which axons degenerate in many forms of HSP, prompting us to search for therapeutic agents that would ameliorate it, with the notion that a timely intervention may indeed be successful in preventing axonal degeneration.