Axonal degeneration Background
Axonal degeneration may follow a traumatic, toxic, metabolic, or genetic insult to axons. Chronic processes of degeneration of axons play an important role in several common neurodegenerative diseases, occurring well before the death of the neuronal cell bodies and often accounting for the first symptoms of the patients. The mechanisms that lead to loss of axons are still largely unknown, but it is clear that they are distinct from those implicated in neuronal cell death. Research in my laboratory focuses on the pathogenic mechanisms underlying hereditary spastic paraplegia (HSP), a model disease to study axonal degeneration. HSP is an adult-onset disorder, characterized by progressive weakness and spasticity of the lower limbs and due to the retrograde degeneration of the corticospinal axons and the fasciculus gracile. HSP is genetically heterogeneous, and in recent years enormous progress has been made to map and clone several of the involved genes. Surprisingly, the protein products of the HSP genes seem to be involved into rather different processes, such as corticospinal axon development, glia to neuron signalling, mitochondrial function, and cell trafficking, outlining that the identification of a common pathway for diseases of axons may be a difficult task. The goal of our research is to address the following questions: Why ubiquitously expressed genes cause selective degeneration of a subset of axons in our body? Which is the common denominator of all these diseases? Is any of the pathogenic steps reversible? How can we intervene to block or slow down the progression of the phenotype?
Unraveling the function of spastin in HSP
SPG4 is mutated in approximately 40% of cases of autosomal dominant HSP and encodes spastin, an ATPase belonging to the AAA family. We provided the first evidence for a role of spastin in severing microtubules, leading to the attractive hypothesis that degeneration of corticospinal axons in HSP may be due to the loss of a regulatory function of spastin in the axonal cytoskeleton. Consistently, we found that spastin is enriched at the centrosome, the mitotic spindle, and the midbody. In neurons, spastin is found in the growth cones and branching regions, all regions where an active remodelling of the microtubule network is required. More recently, we identified two isoforms of spastin translated from different ATG with a different subcellular localization, exclusively cytoplasmic or cytoplasmic and nuclear, due to the presence or absence of a nuclear export signal. These isoforms are present with variable abundance in different tissues and may have specific interactors. For instance, NA14 is a centrosomal component that interacts selectively with the long cytoplasmic spastin isoform. We are interested to address the different roles of the two spastin isoforms, to investigate the cellular pathways in which spastin microtubule-severing is required, and to identify possible mechanisms of regulation of spastin activity.
The role of the mitochondrial m-AAA protease in neurodegeneration
The second major research line in the lab concentrates on paraplegin, the protein product of the SPG7 gene, which is mutated in an autosomal recessive form of HSP. Paraplegin is one of the component of the mitochondrial m-AAA protease, a high molecular weight complex in the inner membrane involved in protein quality control and in proteolytic maturation of specific substrates. Some years ago, we have developed a mouse model for lack of paraplegin, which recapitulates the main features of the human disease, and is now an invaluable tool for further studies of paraplegin function, and of the mechanisms of axonal degeneration. Paraplegin-deficient mice are affected by a late onset progressive dying-back of long spinal axons, of peripheral nerves, and optic nerves. These axons show large swellings due to accumulation of abnormal mitochondria and neurofilaments, indicating that axonal transport is impaired in paraplegin-deficient mice. Remarkably, we found that mitochondrial morphological abnormalities occur in synaptic terminals and in distal regions of axons long before the first signs of swelling and degeneration, and correlate with onset of motor impairment. We are currently investigating the cause for this mitochondrial phenotype, and for its remarkable selectivity to only a small population of organelles. In addition, we are developing mouse models of the other components of the murine m-AAA protease, Afg3l1 and Af3l2. Our aim is to use genetic mouse models to address the reason for selective neuronal and axonal degeneration in the absence of one component of the ubiquitously expressed m-AAA protease. We wish to answer the question whether this is only due to different cellular levels of paraplegin, Afg3l1, and Afg3l2, and to their different ability to homo- or hetero-oligomerize, or also entail substrate specificities. Paraplegin-deficient mice were also used in the lab to develop a therapeutic approach for HSP. We showed that AAV-mediated intramuscular delivery of paraplegin halts the progression of the neuropathological changes and rescues mitochondrial morphology in the peripheral nerves and the dorsal columns of paraplegin-deficient mice. One single injection before onset of symptoms improves the motor performance of paraplegin-deficient mice for several months, indicating that the peripheral neuropathy contributes to the clinical phenotype. This study provided a proof of principle that gene transfer may be an effective therapeutic option for patients with paraplegin deficiency, and demonstrates that AAV vectors can be successfully employed for retrograde delivery of an intracellular protein to spinal motoneurons, opening new perspectives for several hereditary axonal neuropathies of the peripheral nerves.
The parallel study of paraplegin and spastin allows us to tackle the pathogenesis of HSP from two different perspectives, and possibly to shed light on divergent and common mechanisms at the basis of this disease.
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