Two studies from the lab of Daryl Bosco, PhD, associate professor of neurology, offer new insights into the biochemical and molecular mechanisms that cause amyotrophic lateral sclerosis (ALS). Understanding which cellular processes are disrupted in motor neurons in ALS and how the disruption occurs will allow scientists to identify and develop potential treatments for common defects that cross the more than two dozen unique gene mutations that lead to ALS.
ALS, also known as Lou Gehrig’s disease, is a progressive, neurodegenerative disorder that involves the loss of motor neurons that control voluntary muscles. Ninety to 95 percent of cases are classified as sporadic. The remaining cases have a genetic cause linked to a family history and are known as familial ALS. An estimated 6,000 people in the United States are diagnosed each year.
Since the first gene linked to familial ALS—a protein antioxidant known as superoxide dismutase or SOD1—was identified in 1993, more than 25 other genetic mutations have been identified. It is not fully understood why motor neurons die in ALS, but this neurodegeneration is thought to involve a complex array of cellular and molecular processes.
Gelatinous FUS protein impairs nucleocytoplasmic transport
A study published in May in Nature Neuroscience by Dr. Bosco; John Landers, PhD, professor of neurology; Jeffrey Alan Nickerson, PhD, associate professor of pediatrics; and David Grunwald, PhD, associate professor of RNA therapeutics, provides insight into how mutations in the FUS gene cause ALS. FUS is an RNA-binding protein that helps shuttle molecules, such as mRNA, between the nucleus and cytoplasm of the cell. In studying motor neurons derived from ALS patient-induced pluripotent stem cells (iPSCs), the Bosco lab noticed the integrity of nuclear pore complexes, which sit on the nuclear membrane between the nucleus and cytoplasm, was compromised. Like a guarded gateway, the nuclear pore complex controls access to the genome by regulating the movement of molecules, such as RNA, between the nucleus and cytoplasm.
The Bosco lab discovered that FUS and Nups are able to bind directly to each other.
A big clue to the mechanism behind the disease-causing disruption was that in neurons, the mutant protein was mislocalized to the cytoplasm, whereas healthy FUS is normally localized to the nucleus. The environment of the cytoplasm is much different than that of the nucleus. For example, there is a much lower level of RNA molecules in the cytoplasm, a condition that favors binding between FUS and Nups in a way that changed the composition of FUS assemblies from a liquid droplet phase to a more amorphous, less dynamic gelatin-like assembly.
Bosco and colleagues believe that because Nup proteins bind to mutant FUS, it may keep them unavailable for their normal activities. Nups are some of the longest-lived proteins in the cell, sometimes persisting for years, and as such are not readily replaced, especially in nondividing cells such as neurons. If Nups are not available to properly maintain the nuclear pore complex which stands between the nucleus and cytoplasm, this may account for the impaired transportation of materials in cells with the mutant FUS protein.
“Nup62 and RNA molecules seem to compete for the same site on the mutant FUS,” said Bosco. “If we could inhibit this interaction in the cytoplasm, it would reduce the opportunity for these mutant FUS/Nup complexes to form and will free up Nups to incorporate into pores,” said Bosco. “This could potentially attenuate the toxic FUS-mediated phenotypes in animals.”
The next step for Bosco and colleagues is to map the regions within FUS that bind Nup (and vice versa), an important step toward developing inhibitors of this interaction.
Misfolded profilin dysregulates actin dynamics
In another study published in PNAS in June, Bosco; Osman S. Bilsel, PhD, associate professor of biochemistry & molecular pharmacology; and Francesca Massi, PhD, associate professor of biochemistry & molecular pharmacology, found that actin polymerization becomes dysregulated by mutant forms of the profilin protein, a protein critical for the formation and regulation of actin filaments in cells.
Thus far, eight DNA mutations in the PFN1 gene encoding the profilin protein are associated with ALS. Unlike the mutant FUS protein, the physicochemical properties of profilin are severely affected by ALS-linked mutations.
“The effects of these mutations on PFN1 conformation and stability are very robust,” said Bosco.
In a disease context, these changes in PFN1 conformation are referred to as protein misfolding.
“The prevailing thought was that misfolding of profilin due to ALS mutations would prevent profilin from binding to actin and other cytoskeletal proteins, which would then lead to motor neuron death in cases of ALS,” said Bosco. “But that’s not what we saw.”
Using mass spectrometry, researchers in the Bosco lab found that the misfolded profilin protein was still able to bind to cytoskeletal proteins, but unexpectedly, the interaction between proteins had changed. In fact, a class of cytoskeletal proteins called formins bound more actively and tightly to profilin. These altered interactions correlated with dysregulation of actin filaments in cells and in the test-tube.
“ALS is an adult-onset disease. Profilin function isn’t completely lost with disease-linked mutations, it is altered,” said Bosco.
Bosco speculated that while the motor neurons are young and healthy, they can compensate for this aberrant interaction. As the motor neurons age, however, they can no longer overcome this dysregulation and the cells further suffer from accumulation of misfolded profilin that could eventually kill the cells.
Bosco said a pharmacological intervention that can bind and stabilize the profilin protein could fix the problem. The next step for Bosco and colleagues is to search for compounds that stabilize profilin, as well as to study the effects of mutant profilin in other disease-relevant cell types such as microglia, cells in which actin dynamics play a central role.
These studies into separate ALS causing mutations offer scientists a new window through which to view the disease.
“It would be nice if all the mutations that cause ALS affected the cells in the same way,” said Bosco. “But that’s not how it works. Mutations in different genes can impact processes in different ways but still have the same end result—motor neuron death. Even within the same gene the details for how disease-linked mutations cause toxicity may be different. The hope is that we can find enough common pathways that can be targeted for multiple different genetic causes of ALS. Ultimately, it’s important to gain mechanistic understanding for precisely how disease-linked mutations alter protein function in order to develop therapeutic strategies for these devastating neurodegenerative disorders.”
Related stories on UMassMed News:
Riccio Fund for Neuroscience announces four research awards at UMMS
Mutations disrupt protein linked to ALS
