Collaborators Harry Orr (above left) and Michael Koob

The pioneering molecular biologist Sydney Brenner famously said that scientific progress depends on new techniques, new discoveries, and new ideas, “probably in that order.”  

A recent collaboration among LMP faculty and laboratory scientists studying the neurodegenerative disease spinocerebellar ataxia type 1 (SCA1) is showing how technical advances can reveal previously unknown aspects of the disease and help guide long-term therapeutic strategies.  It is a model for how medical discovery and innovation proceed through teamwork and tools.

A singular partnership 

First, some background.  LMP professor Harry Orr’s path-breaking SCA1 research collaboration with Baylor College of Medicine’s Huda Zoghbi, initiated more than three decades ago, is a singular partnership in neuroscience.  The collaboration culminated in their winning the prestigious Kavli Prize for Neuroscience last year.  Orr and Zoghbi discovered the gene mutation that causes loss of balance and coordination in SCA1 patients on the very same day in the early 1990s.  They reported their discovery in a paper published in Nature Genetics in 1993.  They used genetic linkage analysis to map the gene, called ATXN1, to chromosome 6’s HLA (human leukocyte antigen) locus and then cloned the ATXN1 mutation, which is a repetition of a cytosine-adenine-guanine (CAG) trinucleotide DNA sequence.  The buildup of mutant ATXN1 protein is responsible for progressive neural degeneration in SCA1 patient cerebellums, brain stems, and spinocerebellar tracts and the resulting motor and cognitive deficits.  Patients typically die 10-20 years following diagnosis. 

Within two years of reporting the ATXN1 gene mutation, Orr and Zoghbi and their colleagues had created a SCA1 transgenic mouse that mimicked SCA1 pathogenesis in humans.  In recent years, they’ve used induced pluripotent stem cells (iPS cells) created from SCA1 patient biopsies to pin down specific steps in the disease’s progression.  They’ve also employed their mouse model to show that ATXN1 “gene silencing” with oligonucleotides – short synthetic nucleic acid sequences – can ameliorate SCA1-like symptoms in mice. 

These developments set the stage for an Orr-Zoghbi collaboration with LMP associate professor Michael Koob and his lab.  Koob has pioneered human gene replacement in mice using a recipe involving mouse embryonic stem cells, intricate genetic recombination and editing processes, cell cloning, DNA sequencing, and delicate handling.  Transgenic mice normally are made by manipulating several thousand DNA base pairs. Koob’s technological approach can involve several hundred thousand DNA base pairs, a substantial section of a chromosome.  Plus, Koob is able to place the human gene sequence with its flanking regulatory elements in the exact chromosomal location where the comparable mouse gene once resided, something transgenic technologies are unable to do.  His precision genome engineering approach for developing superior mouse models for studies of Alzheimer disease, frontotemporal dementia and other dementias, plus Parkinson’s disease now includes the ataxias with the Orr and Koob-led study “Regional vulnerability in a neurodegenerative disease: Delineating SCA1 CNS and muscle therapeutic targets using a conditional mutant ATXN1 mouse.”    For the study, ATXN1 mouse coding regions were replaced with human mutant ATXN1 coding regions bearing the repetitive CAG sequence characteristic of SCA1.  

Lifting the curtain

Orr said the mouse study revealed two dimensions of SCA1 previously unknown: cells called medium spiny neurons found in the brain’s striatum, an interior structure, are involved in SCA1 pathology in addition to Purkinje cells in the cerebral cortex, long the focus of investigation; and the presence of extensive skeletal muscle pathology in the mutant ATXN1 mice.

“We think that a big part of the pathology in the striatum is due to the specific expression of the mutant gene in those striatal cells,” Orr said.  “The extent to which that disrupts circuitry between the respective tissues or regions in the brain, that’s pure speculation but we envision there would be some disruption. In other regions, too.  Fairly broadly throughout the brain.  That’s what our data say.”

Orr finds it intriguing that that activity in the striatum “is similar to what’s going on in the striatum in Huntington’s disease. Yet the stratum is affected very early in Huntington’s disease, whereas it looks like both in humans and our mouse model it doesn’t become dysfunctional until much later in SCA1. So why do we see all these similarities, but the diseases are quite distinct?”

The pervasive muscle-wasting observed in the mutant ATXN1 mice is not related to neurodegeneration in the brain, Orr said.  “Our data indicates that the mutant gene has a direct effect on muscle tissue.  The pathology we see is not due to motor neuron degeneration.”   He said some have speculated that “because these are neurodegenerative diseases that the muscle-wasting you see is due to the degeneration of motor neurons.  Our data says a big part of the muscle pathology is a direct pathological factor of the mutant gene on the muscle itself.” 

To confirm that what they observed in mice could occur in SCA1 patients if their lifespan were to be extended through therapeutic advances, Orr’s team plans to test that hypothesis by creating iPS cells from SCA1 patient skin biopsies, directing the differentiation of the iPS cells into muscle cells, and examining the muscle cells for evidence of myopathy or muscle disease.   If evidence of myopathy is sufficient in these experiments, the next step will be to proceed with patient muscle biopsies, which are more invasive than skin biopsies.  What the novel finding in the mutant ATXN1 mouse model suggests, Orr said, is that “to fully treat the effects of SCA1 we’re going to have to target peripheral tissue as well as the brain itself.”

Orr has distributed Koob’s mutant ATXN1 mice to other investigators studying SCA1.  He cites two major advantages to using them in SCA1 research: “One is that it’s the human gene we replaced in the mouse.  Therefore, the therapies that are specifically targeting the human gene like the ASOs [allele-specific oligonucleotides] and interfering RNAs, you can use these mice for those types of tests.  Also, from a research standpoint, because we put in recombination sites flanking the ATXN1 gene, now we can ask and perhaps answer the questions:  Where does the pathology that causes symptoms begin?  What brain regions are contributing to symptoms?  Which of the symptoms are due to pathology and in what regions?  Sometimes I refer to it as understanding function from dysfunction.

Practice pays off

Orr is highly complimentary of Koob’s mouse work. “Mike’s expertise and skills have not just impacted us, but they’ve quite broadly impacted the University’s and the scientific community’s ability to study these human neurodegenerative diseases more efficiently using a mouse model,” Orr said.

Koob said discussions that led to the recent collaboration with Orr began around 2014 or 2015.   “I wanted to do human gene replacement in mice, but I didn’t know how to do it yet, so we needed some practice.  We needed to figure out how to make this work.”  By 2018, he and his team had figured out how to make human gene replacement in mice work.

"Harry’s been a pioneer in SCA1 research,” Koob said. “I’d been familiar with his work ever since I came to Minnesota and was peripherally involved.  He had a transgenic model system, but there are some limitations to that, particularly the limitation of just having expression of the transgene in the cerebellum.  His group was really interested to see what the effects might be outside the cerebellum, and they want to see some dynamics of the gene that you wouldn’t see with a transgenic model.”

Mouse models like the one Koob and his laboratory colleagues created for SCA1 “will help us look beyond where the immediate degeneration is,” Koob said.  “Cerebellum degeneration has been the focus in SCA1.  I think you can’t just limit your attention to that primary tissue. Our study points up that you need to look at other tissues to see if the pathology has a broader scope.” 

Koob notes that MODEL-AD (model organism development & evaluation for late-onset Alzheimer’s disease), a National Institute on Aging-funded consortium, “is shifting toward our approach” in mouse model development for studying neurodegenerative diseases.

SCA1 research advances reflect and reinforce the importance of teamwork, as the Orr-Zoghbi partnership has demonstrated through the years.  They also reflect and reinforce Sydney Brenner’s dictum that scientific progress depends on new techniques.  That’s where Koob comes in.

Background image above:  Neuroscience Intramural Research Program.