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.

Reference:
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

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  1. Gnerer and colleagues present evidence to suggest that accumulation of dihydroxyacetone phosphate (DHAP), by virtue of the wasted away mutation of triosephosphate isomerase in Drosophila, may be a mechanism linked to neuronal toxicity. Increased accumulation of DHAP results in generation of methylglyoxal (MG), a highly reactive α-oxoaldehyde that is a precursor to generation of advanced glycation end-products (AGEs). Experimental evidence suggests that AGEs accumulate in human neurodegenerative disorders such as Alzheimer disease, amyotrophic lateral sclerosis (ALS), and Parkinson disease. The possibility that increased accumulation of AGEs is linked to neurodegeneration—especially given biochemical data in human neurodegenerative disease—is compelling.

    Experimental evidence suggests that AGEs may not solely be “biomarkers” of diseases such as diabetes and renal failure. Rather, by their ability to interact with and activate signal transduction receptors, chief among them being receptor for AGE (RAGE), AGEs may impart toxic effects to neurons, including, ultimately, cell death. Although Gnerer and colleagues suggest that aberrant protein misfolding may not be a key mechanism driving neurological phenotypes, it is important to note that among the properties of AGEs are their ability to cross-link and significantly modify protein structure and conformation. Thus, the findings of Gnerer and colleagues may place AGEs at the fore of pivotal mechanisms that trigger a cascade of receptor-dependent and perhaps receptor-independent pathways that may lead to advanced neural toxicity. Does such neural toxicity yield or contribute to frank neurodegeneration? Studies testing the role of RAGE in neurodegeneration in mammalian models suggest that this receptor is part of the problem. In a murine model of accumulation of mutant amyloid precursor protein (mAPP), our laboratory group has shown that transgene-driven expression of neuronal RAGE exacerbated neuronal dysfunction in the mutant mice; in contrast, introduction of a signal transduction deficient RAGE mutant, selectively in neurons in the mAPP background, attenuated neuronal stress.

    The previously held narrow view that AGE biology is restricted to hyperglycemia is now greatly expanding. Does accumulation of AGEs in chronic neurodegeneration focus neuronal and inflammatory stress, in part via RAGE, in the injured brain? These considerations suggest that probing the role of AGEs and their signaling receptors in neurodegeneration may uncover novel targets for therapeutic intervention in these highly refractory disorders. Indeed, simple lessons from simpler organisms such as Drosophila may uncover universal roles for AGEs in mediating toxicity in cells such as neurons of the central nervous system.

  2. Enzymes involved in glucose metabolism emerge as key players in the pathogenesis of a range of neurodegenerative disorders. Gnerer and coworkers identify a role for triosephosphate isomerase in age-related neurodegeneration in Drosophila, possibly due to a build-up of methylglyoxal. Interestingly enough, we have shown by using Affymetrix chips that glyoxalase I, a detoxifying enzyme preventing the formation of advanced glycation end-products (AGEs) is increased in mice with an Alzheimer-related tau pathology (Chen et al., 2004).

    Extending these studies, we were able to show by proteomics that other enzymes involved in glucose metabolism such as pyruvate kinase isozymes M1/M2 or phosphoglycerate mutase 1 are differentially regulated in Aβ-treated tau-transgenic mouse and tissue culture models (David et al., 2005; David et al., 2006, in press).

    References:

    . Role for glyoxalase I in Alzheimer's disease. Proc Natl Acad Sci U S A. 2004 May 18;101(20):7687-92. PubMed.

    . Proteomic and functional analyses reveal a mitochondrial dysfunction in P301L tau transgenic mice. J Biol Chem. 2005 Jun 24;280(25):23802-14. PubMed.

References

News Citations

  1. GLOing Reports from Gene Profiling of Mouse Models

Paper Citations

  1. . Age- and stage-dependent glyoxalase I expression and its activity in normal and Alzheimer's disease brains. Neurobiol Aging. 2007 Jan;28(1):29-41. PubMed.
  2. . wasted away, a Drosophila mutation in triosephosphate isomerase, causes paralysis, neurodegeneration, and early death. Proc Natl Acad Sci U S A. 2006 Oct 10;103(41):14987-93. PubMed.

Further Reading

Papers

  1. . Temperature-sensitive paralytic mutants are enriched for those causing neurodegeneration in Drosophila. Genetics. 2002 Jul;161(3):1197-208. PubMed.
  2. . wasted away, a Drosophila mutation in triosephosphate isomerase, causes paralysis, neurodegeneration, and early death. Proc Natl Acad Sci U S A. 2006 Oct 10;103(41):14987-93. PubMed.

Primary Papers

  1. . wasted away, a Drosophila mutation in triosephosphate isomerase, causes paralysis, neurodegeneration, and early death. Proc Natl Acad Sci U S A. 2006 Oct 10;103(41):14987-93. PubMed.