24 September 2006. A genetic screen for mutations causing age-related neurodegeneration in fruit flies has uncovered a neuroprotective role for the glycolytic enzyme triosephosphate isomerase (Tpi). A new paper from Barry Ganetzky and colleagues at the University of Wisconsin in Madison shows that mutations in the enzyme lead to neuron loss, paralysis, and shortened lifespan in the flies. The mechanism of neuronal damage is not clear, but the researchers hypothesize that the build-up of reactive methylglyoxal as a result of Tpi deficiency leads to the overproduction of toxic advanced glycation end products (AGEs), which damage neurons.
The results provide a model for a novel type of neurodegeneration which is also observed in humans with Tpi mutations. In addition, AGEs have been linked in several ways to neurodegenerative diseases, including Alzheimer disease (AD). Their modification of amyloid-β residues may contribute to plaque formation, while the receptor for AGEs (RAGE) is involved in Aβ transport in the brain. The methyglyoxal-degrading enzyme glyoxylase also emerged as the only gene that was significantly upregulated in a microarray analysis of tau mutant mice, and the protein was found to be increased in AD brain (see ARF related news story), but subsequently decreased with aging and advancing disease (Kuhla et al., 2006).
To find the unusual mutant, Ganetzky, with first author Joshua Gnerer and Robert Kreber, took advantage of a collection of temperature-sensitive paralytic fly mutants, which previously led researchers to many genes involved in synaptic function. At normal temperature, the flies are fine, but when the temperature is elevated to 38 degrees centigrade, they rapidly become paralyzed and die within minutes. The researchers found that the mutant population was enriched for flies that underwent age-dependent neurodegeneration, and thus might be a good place to look for “neurodegeneration suppressor” genes.
The paper, out this week in PNAS Early Edition, describes a recessive, hypomorphic mutant, wasted away, that displays progressive motor impairment, vacuolar neuropathology, and reduced lifespan. In the mutant, the paralysis that accompanied the temperature shift became worse with age: more flies were paralyzed at the restrictive temperature, the time required for them to become paralyzed after the temperature shift became shorter, and their recovery period became longer as they got older. Even at a normal temperature, the flies are shorter lived and their lifespan gets even shorter at higher temperatures. The flies showed neurodegenerative vacuolar lesions in the brain that became worse with age.
The investigators identified the wstd gene, which turned out to be the essential and highly conserved glycolytic enzyme triosephosphate isomerase. In four wstd alleles, they found mutations that caused either early termination or amino acid changes. Rescue experiments with wild-type gene confirmed that loss of Tpi was responsible for the wasted away phenotype.
What is Tpi doing in the flies? During glycolysis, glucose first gets converted to fructose-1,6-bisphosphate, which is then broken down into the three-carbon products dihydroxyacetone phosphate (DHAP) and glyceraldehydes-3-phosphate (GAP). Tpi converts DHAP to GAP, which feeds into the tricarboxylic acid cycle. Despite its critical role in glycolysis, Tpi deficiency in flies did not decrease ATP levels, consistent with the situation in humans with Tpi mutations. These results suggest that neuronal loss was not caused simply by an energy deficit. In addition, the researchers found no evidence for protein misfolding problems, as the mutants do not confer dominant or gain-of-function phenotypes.
Another explanation for the neurodegeneration phenotype which the authors favor is related to the build-up of DHAP in the absence of Tpi, a biochemical lesion also observed in humans. DHAP spontaneously converts to highly reactive methyglyoxal, which modifies proteins and DNA to produce advanced glycation and produces (AGEs), which are toxic to neurons and other cells.
Normally, cells are protected against methyglyoxal by the enzymes glyoxylase I and II, which convert it to lactic acid with the help of glutathione. Under conditions of oxidative stress, and lowered glutathione levels, methyglyoxal levels rise. The model could explain the temperature sensitivity of the wstd mutants: rather than the Tpi protein itself undergoing temperature-dependent inactivation, the increased stress of higher temperatures would overwhelm the ability of the cells to detoxify elevated levels of methyglyoxal. Null mutants of Tpi, they showed, are lethal, consistent with the elevation of methyglyoxal at normal temperatures. “From this perspective, Tpi can be considered not just a glycolytic enzyme but also a component of a protective pathway that limits the potentially deleterious accumulation of DHAP, a toxigenic compound.” That could give Tpi, and other glycolytic enzymes downstream of DHAP, a role in a wide variety of neurodegenerative diseases, the authors conclude.—Pat McCaffrey.
Gnerer JP, Kreber RA, Ganetzky B. wasted away, a Drosophila mutation in triosephosphate isomerase, causes paralysis, neurodegeneration, and early death. PNAS Early Edition; 2006 September 19. Abstract