SALT LAKE CITY—When trying to understand neurologic disease, finding a causative mutation is an important first step, but it’s only the beginning. At the 133rd Annual Meeting of the American Neurological Association, William T. Dauer, MD, detailed his recent work deciphering the etiologies of dystonia and Parkinson’s disease, which he described as prototypes of neuronal dysfunction and neurodegeneration, respectively.
“We intensively study the most common genetic forms of each disease, on the premise they may provide unique and complementary insights into the motor system,” he said. Like a clinical neurologist attempting to localize a lesion and then understand etiology when examining a new patient, Dr. Dauer said his work is aimed at first identifying the molecular pathway or organelle in which the disease protein operates, and then understanding the effect of the mutation on the normal function of that pathway or organelle.
Many cases of early-onset generalized dystonia are due to mutations in the DYT1 gene, which encodes a protein called torsinA. Studies have shown that torsinA associates with the nuclear membrane, and “neurons have a unique requirement for this function,” explained Dr. Dauer, Assistant Professor of Neurology at Columbia University in New York City. Unlike the wild-type protein, the mutant protein persists at the membrane, rather than shuttling from it to the far reaches of the cell. In genetically modified mice, this mutation leads to the disruption of nucelar membrane morphology selectively in neurons, despite its widespread expression in other cell types.
Recently, Dr. Dauer’s lab has identified a protein that interacts with torsinA, termed LAP1. In normal mice, knocking out LAP1 causes abnormalities similar to DYT1 mutation, indicating a functional relationship between the two proteins. This, he said, begins to identify “a torsin-related pathway at the nuclear membrane.”
TorsinA’s function at the membrane is still unknown, but its localization there links it at least topographically with other nuclear membrane proteins responsible for other neurologic diseases, including Emery-Dreifuss muscular dystrophy, rare forms of Charcot-Marie-Tooth disease, certain lipodystrophies, and even the accelerated aging condition progeria.
“The fact that these diseases, resulting from nuclear membrane mutations, are clinically different tells us that a range of essential functions are localized and controlled at the nuclear membrane, and we can exploit these diverse phenotypes to help decipher how specific dysfunction leads to specific clinical diseases,” he said. “Likewise, the cellular similarity between these diverse diseases raises the question of whether there may be unrecognized clinical similarities between them.”
A Death Pathway in Parkinson’s Disease
One of the most extraordinary developments in Parkinson’s disease research has been the discovery of the LRRK2 gene, Dr. Dauer noted. It is far more common than any other genetic cause of Parkinson’s disease, accounting for 5% to 10% of all cases, and a much higher percentage in certain populations, due to a founder effect. LRRK2 Parkinson’s disease is clinically and pathologically similar to sporadic Parkinson’s disease, “giving us confidence that what we find about this disease will relate to common disease we are all interested in,” he said. The gene codes for a “leucine-rich repeat kinase,” a protein of unknown function that phosphorylates other proteins using its kinase domain. Multiple mutations have been found, all of which are dominantly inherited and most of which occur in the enzymatic core of the protein. The protein’s domains are well understood, he indicated, suggesting that experiments to elucidate its function may be more tractable than for some other genes.
Dr. Dauer’s lab has shown in cell culture that the mutant protein provokes apoptotic cell death in neurons, an effect that requires kinase function. Inserting a second mutation to inactivate the kinase completely eliminates the toxicity of the first mutation.
“This led to the straightforward hypothesis that the mutation may enhance kinase activity,” he said, but in fact, the kinase function is enhanced by only one of the known mutations, suggesting an additional disease mechanism. “We were quite struck by finding that in some percentage of cells, LRRK2 would form very complex filament strings throughout the cell.” Turning to the literature, he found this feature was shared by other proteins that also shared a similar structure. The filaments arise from homo-oligomerization, or multiple LRRK2 proteins linking with one another. “This is an important aspect to the normal function of these proteins,” he pointed out. These other proteins are also intimately related to cell death.
“This intrigued us and led us to ask whether oligomerization might also play a role in the LRRK2 signaling mechanism and perhaps its ability to cause neurotoxicity,” he said. To explore this, he tagged copies of the protein with two different fluorescent tags, to see if they linked together. Several of the mutations did indeed enhance oligomerization. Interestingly, the mutation that enhances kinase activity did not, indicating the possibility that different mutations may be acting through different disease mechanisms.