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National Ataxia Foundation


A novel gene therapy-based approach with therapeutic potential in SCA3

Written by Dr. Ramya Lakshminarayan Edited by Dr. Judit M. Perez Ortiz

Cholesterol to the rescue: An alternative approach to treating SCA type 3 using gene therapy.

Spinocerebellar ataxia type 3 (SCA3) is a movement disorder that is caused by genetic mutations in a protein named Ataxin-3. Neurons in the cerebellum, striatum, and substantia nigra are important for movement, and these are affected in SCA3.

The mutant form of Ataxin-3 builds up in these neurons, eventually causing neurodegeneration and neuronal loss. The abnormal accumulation of mutant Ataxin-3 is in part due to impaired protein clearance, which is a hallmark of many other neurodegenerative diseases. Degradation (breaking down) and clearance (getting rid of) of protein aggregates are therefore crucial in the pathophysiology of neurodegeneration.

The balance between protein synthesis (creation) and degradation (destruction) is critical to the health of neurons. One of the ways in which neurons degrade proteins is called autophagy. This process is mediated by organelles called lysosomes in cells. Lysosomes employ digestive proteins to break down complex protein aggregates into simpler forms, which are eventually recycled. Hence, the transport of proteins to lysosomes is an important step in protein degradation. In a recent study, Clevio and colleagues explore the role of cholesterol in mediating protein degradation and ensuring neuroprotection in SCA3.

Photo of a researcher preparing samples, courtesy of Pixabay.

Cholesterol is a well-known biological molecule that is essential to cells for regulating various processes. However, abnormally elevated levels of cholesterol are associated with heart disease, and its production is the target of pharmacological therapies. As with proteins, homeostatic fine-tuning of cholesterol levels is maintained by a balance of production and degradation.  In many neurodegenerative disorders, such as Alzheimer’s disease and Huntington’s disease, cholesterol metabolism and turnover is impaired.  The cholesterol biosynthetic pathway facilitates production and its metabolism is mediated by an enzyme called cholesterol 24-hydroxylase (CYP46A1). CYP46A1 converts cholesterol to 24-hydroxycholesterol, a form capable of crossing the blood-brain barrier. This conversion allows the efflux of cholesterol from neurons. CYP46A1 is, therefore, necessary for cholesterol efflux and the efflux of cholesterol activates the cholesterol biosynthetic pathway. The cholesterol biosynthetic pathway produces many precursors important for protein transport and autophagy.

The role of cholesterol metabolism isn’t very well understood in spinocerebellar ataxia. In this study, Clevio and colleagues examine the role of CYP46A1 in human patients and mouse models of SCA3. They observed that levels of CYP46A1 are reduced in brain samples of patients with SCA3 when compared to samples from healthy patients. Because brain samples cannot be safely obtained from living people, the brain samples were extracted from the cerebellar region of deceased patients, which is the most affected brain region in SCA3. Interestingly, these changes were also seen in the brains of SCA3 mouse models that express mutated Ataxin-3 protein. Further, they noticed that cholesterol metabolism was impaired in SCA3 mice, suggesting that normal availability and function of CYP46A1 is important in neurodegeneration.

To more directly determine the consequence of a decrease in CYP46A1, the researchers asked how the loss of CYP46A1 would affect the brains of healthy mice with intact neurological function. To find out, the investigators used a powerful gene-silencing tool called shRNA to deplete CYP46A1 expression specifically in neurons in the striatum region of the brain.  Interestingly, they found that a 60% reduction of CYP46A1 was sufficient to impair cholesterol metabolism in the striatum.  Consequently, the mice demonstrated impaired motor performance in metrics that are affected in SCA3. These results suggest that CYP46A1 plays an important role in the neuropathology of SCA3.

To better understand the relationship between cholesterol metabolism and protein degradation in the cell in the absence of CYP46A1, the researchers examined protein transport and autophagy pathways in neurons.  Using immunofluorescence studies (a tool that makes structures “light up” to visualize under a microscope), they observed that CYP46A1 reduction alters organelles called endosomes that normally carry cargo from one destination to the other within cells. Lysosomes were also enlarged under CYP46A1 silencing suggesting that protein degradation was indeed altered. These results were confirmed in vivo in mice with CYP46A1 being essential for the activation of the autophagy pathway in neurons.

Having established a role for CYP46A1 and cholesterol metabolism in SCA3, the next steps were in testing if CYP46A1 could indeed alleviate symptoms of SCA3 in mouse models. Gene therapy is an increasingly popular tool used by scientists to deliver an essential gene to a specific region in the body. This employs the use of viral vectors such as adeno-associated viruses (AAV), which has several benefits, such as lowered immune reactivity, high delivery potential and stable expression of the gene. In this study, AAV- based viral vectors were used to deliver CYP46A1 in mouse models of SCA3 to test if restoring its levels could ensure neuroprotection in the cerebellum. SCA3 mice experience difficulty with balance, and their brains show abnormal aggregation of certain proteins and the death of key cerebellar neurons called Purkinje neurons. Strikingly, CYP46A1 gene therapy led to higher numbers of surviving Purkinje neurons, reduction of abnormal protein aggregates and overall improved motor ability. These results suggest that CYP46A1 gene therapy indeed has great therapeutic potential for patients with SCA3.

This study has significance for novel SCA3 treatment and progression as its approach is fundamental to the pathophysiology of the disease. Altered cholesterol metabolism and impaired protein degradation are common to multiple neurodegenerative disorders including Alzheimer’s disease and Huntington’s disease and CYP46A1 based gene therapy approaches might have the potential to address and rescue this neuropathology. Gene therapy is already being used to treat multiple diseases including spinal muscular atrophy (SMA), RPE65 mutation-associated retinal dystrophy, acute lymphoblastic leukemia (ALL). The challenges and the future steps of this study would be to ensure that gene therapy-based approaches are more advanced to cross the blood-brain barrier and to reach all the affected regions of the brain and have increased immune tolerance. Gene therapy viral vectors also face challenges in manufacturing and pharmacokinetic studies as new rules and terms are being put in place to measure stable gene expression over time along with the safety and efficacy of the viral vector.

Key Terms

Movement disorder: Disorders in the nervous system that affect both voluntary and involuntary movements in the body

Organelles: Compartments within cells that have distinct structure and function. Examples of organelles include mitochondria, nucleus, endoplasmic reticulum.

Enzyme: Molecules, mostly proteins that catalyze chemical reactions within cells.

shRNA: A type of nucleic acid (short hairpin RiboNucleic Acid) that has a hairpin-like structure designed to target genes and stop its expression.

Learn more about Gene Therapy in our Snapshot on the subject.

Conflict of Interest Statement

The authors and editor declare no conflict of interest.

Citation of Article Reviewed

Clévio Nóbrega et al., Restoring brain cholesterol turnover improves autophagy and has therapeutic potential in mouse models of spinocerebellar ataxia. Acta Neuropathol. 2019 Jun 14.

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