Spinocerebellar ataxia type 3 (SCA3) or Machado-Joseph disease (MJD) is the most prevalent autosomal dominant spinocerebellar ataxia in the world leading to severe clinical manifestations and premature death. MJD is a multisystem disorder with degeneration of specific brain regions that lead to a broad spectrum of clinical symptoms such as ataxia, postural instability, oculomotor impairments and neuropathy. Ataxia usually starts in midlife evolving to severe disability and death. Cholesterol is critical for the physiology of neurons, all through development and in adult life. Deregulation of brain cholesterol and impaired brain cholesterol turnover have been linked to several other neurodegenerative disorders, including Parkinson’s disease, Alzheimer’s disease and Huntington’s disease. Excessive brain cholesterol may be deleterious to neurons, however, cholesterol does not cross the blood-brain barrier (BBB). To be excreted out of the brain and maintain its levels equilibrated, cholesterol is converted into a molecule called 24S-hydroxycholesterol (24-OHChol), which is produced by the neuronal cholesterol 24-hydroxylase (CYP46A1) enzyme. Indeed, CYP46A1 has been recognized as the key enzyme that allows efflux of brain cholesterol and activates brain cholesterol turnover and therefore, has been considered as an attractive target to treat neurodegenerative disorders. CYP46A1 has already been shown to be decreased in the cerebellum (one of the main brain regions affected in this disorder) of SCA3 patients and SCA3 mice. Furthermore, injection into the cerebellum of adeno-associated viral vectors (AAVs) transporting CYP46A1 in SCA3 mice models, reduced mutant ataxin-3 accumulation (a hallmark of SCA3) and protected neurons, also alleviating motor impairments associated to the disease. These viral vectors have been extensively used as vehicles for gene delivery to the nervous system (the principle of the so-called gene therapy-related approach currently and widely applied in research all over the world), however, intra-cerebellar injection is an invasive procedure, involving surgical risks and with considerable limitations from a clinical translational point of view. Therefore, this project aims at investigating whether a non-invasive delivery of CYP46A1 will ameliorate the symptoms of the disease in a transgenic (Tg) MJD mouse model (MJD Q69 mouse) with established pathology. For this purpose, we will take advantage of an AAV vector with the capacity to overcome the blood-brain barrier and tropism for the central nervous system - the novel PHPeB-AAV variant, on whose backbone we have already inserted CYP46A1. The vectors will be injected intravenously in adult SCA3 mice and we will evaluate whether this strategy will be neuroprotective. In particular, we will investigate the levels of CYP46A1 in specific regions of the brain of these mice and analyse the reduction of mutant ataxin-3 protein aggregates, the effectiveness in alleviating motor impairments and the amelioration in the neuropathology of treated Tg mice. Furthermore, we will characterize lipidomic modifications in plasma and brain tissue of SCA3 Tg animals to corroborate the role of cholesterol metabolism impairment in SCA3 and demonstrate the efficacy of this novel strategy of gene therapy in this disorder. Finally, we will study autophagy (a “cell cleaning”pathway), as a possible mechanism underlying CYP46A1 role in SCA3. Overall, this project will consolidate the pivotal and beneficial role of CYP46A1 and brain cholesterol metabolism in neuronal function in a SCA3 mouse model. Furthermore, the use of a non-invasive strategy of CYP46A1 delivery will bring great promises as a relevant therapeutic approach not only for Machado-Joseph disease but also for other SCAs.
We specifically aim to answer the following questions: 1. Does PHPeB-AAV-CYP46A1 efficiently reach and transduce the brain of SCA3 Q69 mouse model after intravenous delivery and alleviate the symptomatology and neuropathology associated to the disease phenotype? 2. Do SCA3 Tg mice display lipidomic modifications in plasma and brain tissue that will be reverted by intravenous delivery of PHPeB-AAV-CYP46A1? 3. Does intravenously injected CYP46A1 induce activation of autophagy in vivo and reduce mutant ataxin-3 aggregates?