By David N. Leff
Though widely differing in their symptoms, the neurodegenerative diseases - notably Alzheimer's, Parkinson's, Huntington's and Lou Gehrig's - have one salient phenomenon in common: Their dying neurons, in brain or spinal cord, are choked with accumulations of aggregated mutant proteins. These testify to the failure of the central nervous system's quality control system for ridding these cells of those piled-up, polluting protein aggregates.
One glaring example of this metabolic misfeasance occurs in the failing motor neurons of amyotrophic lateral sclerosis (ALS) - Lou Gehrig's disease. It afflicts 70,000 victims worldwide, half of them in the U.S. ALS usually begins with dysfunctional hands, forearms, shoulders and feet, followed by progressive muscle weakness and wasting. Within three years of diagnosis - around age 40 - half of ALS patients are dead of their disease. Another 20 percent succumb by five years post-onset, and 10 percent by 10 years. Few survive for 30 years with the disease.
Statistics of another kind discern that 80 percent of ALS cases arise spontaneously, idiopathically - from no known cause - whereas the remaining 20 percent inherit the disease from their forebears. Cell biologist Ron Kopito, at Stanford University in California, studies this familial ALS (FALS) in transgenic mice.
Kopito said the work that he and his group have done "specifically looked at a mouse model for an inherited form of the disease, which is genetically linked to mutations in a gene called SOD - superoxide dismutase. Not all cases of FALS are SOD-linked," he said, "but the mutant SOD1 model is a very good one, because the disease in humans progresses with very similar properties to the sporadic form of the disease.
"The mouse models expressing the SOD1 mutant protein," Kopito noted, "have very similar behavior to the human disease, in terms of progression and pathology." However, he added, "the finding of mutations in SOD has been a puzzle, because SOD is a gene that encodes a protein called superoxide dismutase (SOD1), which is involved in protecting cells from damage by oxidation. So there was quite a bit of speculation at the beginning whether or not FALS was related to the loss of this oxidation protection mechanism. Many studies have failed in a variety of ways to confirm that oxidation hypothesis. There's some compelling evidence that oxidation plays a role in ALS - but it's pretty clear that it's not due to the lack of an anti-oxidant function of SOD1."
He noted that the evidence confirmed by other studies "suggests that the mutant protein acquires a toxic function. This potentially links it to many of the other neurodegenerative diseases - like the triplet codon repeats in Huntington's - in which the acquisition of the polylglutamine repeats is also associated with a toxic gain of function for the neurons."
Double-Barreled Probe Of Mystery: In Vitro, In Vivo
"The mystery for many labs," Kopito said, "has really been focusing on understanding the common features in virtually every one of these diseases - the fact that protein aggregation is almost invariably associated with their clinical onset."
To crack this mystery, Kopito conducted two kinds of experiments, as reported by the Nov. 7, 2000, Proceedings of the National Academy of Sciences (PNAS), in an article of which he is senior author. Its title: "Formation of high molecular weight complexes of mutant Cu,Zn-superoxide dismutase in a mouse model for familial amyotrophic lateral sclerosis."
"In our first experiment," he told BioWorld Today, "we worked in vitro with cultured cells, and observed the properties of mutant and wild type (WT) SOD1 proteins in those neurons. We found that the mutant, but not the WT, is unstable in cells. And it's degraded by a process called the proteosome pathway."
A proteosome, Kopito explained, "is a large degradative particle in the cell, which is endowed with many copies. They're responsible for many cellular functions, such as degrading proteins that haven't folded properly, as well as eliminating proteins that are no longer needed by the cell. Our experiment examined directly the fate of the mutant compared to the WT protein, and the role of the proteosome. It established quite convincingly that that cellular particle is responsible for the increased instability of mutant SOD1 relative to the WT."
Kopito and his co-authors generated two missense amino-acid mutations in the SOD gene - one swapping glycine 93 for alanine, the other, glycine 85 for arginine. "Those two mutations," he said, "are associated with the human FALS disease. Then, in in vivo studies with transgenic mice expressing the G93a mutation," he noted, "we developed an assay for the accumulated protein aggregates that is more sensitive than the existing microscopic assay. In the past, people have looked for aggregates only by microscopy. And they concluded that the inclusions that appear by microscopy are only visible very late in the disease."
Revealed: Earlier Onset Precursor Of FALS
"Our assay could find aggregated protein based on electrophoretic mobility. They acquire very high molecular weight, suggesting that they have linked together to form an aggregate, which we cell insoluble protein complexes (IPCs). And what we showed," he pointed out, "is that IPCs, which are composed of aggregated SOD1, appear at the earliest time point that we looked at - one month of mouse age, which is three months before the animals start to develop either symptoms of FALS, or microscopic inclusion bodies. So we propose that these ICPs are early precursors to the inclusion bodies that are seen later, and play some role in actually generating the subsequent pathological symptoms."
Kopito noted, "Our results speak only to the familial form of ALS. We have no evidence that these complexes of aggregated proteins appear in any of the much more common forms of the disease, which are not linked to mutations in SOD1."
As for ongoing research, he observed that, "All cells have a whole battery of quality-control systems that protect them from protein aggregation. One of the major ones is this proteosome pathway, which degrades proteins. So the question we are asking now is: Why - given that cells have all these protective mechanisms - why are they not functioning in the case of this disease?"