26 July 2008. Starting from an unexpected finding that human blood contains abundant α-synuclein, a protein implicated in both hereditary and sporadic Parkinson disease, a trio of collaborators has unearthed an unlikely regulator of α-synuclein gene (SNCA) expression. In a paper published in this week’s PNAS Early Edition, Clemens Scherzer of Harvard Medical School, Michael Schlossmacher of the University of Ottawa in Ontario, Canada, and Emery Bresnick of the University of Wisconsin, Madison, present evidence that GATA family transcription factors, best known for their role in the blood cell development, also regulate the activity of the SNCA gene both in blood and in brain. That makes the transcription factors prime targets for therapies aimed at lowering α-synuclein levels. The result should also pique the neurobiologists’ interest in the latest work from Stuart Kim and colleagues at Stanford University. In a paper out in this week’s Cell, they show that GATA-like transcription factor activity increases during aging in C. elegans, and that the factors regulate a large number of age-related genes. Their results implicate the continued activity of developmental pathways as possible drivers of aging.
Also of interest to α-synuclein enthusiasts is a report from Virginia Lee and colleagues at the University of Pennsylvania in Philadelphia indicating that synuclein expression and neuroinflammation act synergistically to cause neuronal death. Using mouse models expressing solely human α-synuclein, they show that the presence of the protein renders neurons susceptible to the harmful effects of inflammation, and that nitric oxide and superoxide induce oxidation and nitration of the synuclein, which then appears in inclusions. That result, appearing in the July 23 issue of the Journal of Neuroscience, provides a direct link between inflammation (a known risk factor for PD) and the abnormal modification of α-synuclein and PD pathology.
Increased expression of the α-synuclein gene (SNCA) has been tied to PD (see ARF related news story), and data from patients with SNCA amplifications suggest that even a small increase in α-synuclein over many years is sufficient to cause disease (see ARF related news story). Because of that, finding out how SNCA expression is normally regulated could give clues to disease etiology and provide targets for treatment. Scherzer and Schlossmacher were working on PD biomarkers in blood when they found a surprisingly high level of α-synuclein mRNA and protein in red blood cells. That suggested to them that blood and neuronal cells might have a common regulatory mechanism for gene expression, and Scherzer began to explore that idea using transcriptional array data and looking for genes whose expression might be correlated, and thus co-regulated, with α-synuclein. He found, in fact, that α-synuclein expression in blood closely correlated with several classic erythroid genes, including three involved in heme biosynthesis.
One of the genes was ALAS2, which Bresnick had previously shown was regulated by the transcription factor GATA-1. It was a simple experiment, Bresnick told ARF, to use GATA-1-null erythroid cells and do an add-back GATA-1 to show α-synuclein expression depended on that factor. To see if the regulation was direct or indirect, Bresnick performed chromatin immunoprecipitation assays, and found that GATA-1 protein was sitting on a GATA recognition site in the first intron of the α-synuclein gene, but was not present on 10 other nearby sites with a similar sequence. “That was strong evidence for direct control,” Bresnick said.
But what about the regulation of α-synuclein in brain? GATA-1 is not expressed in brain, but its relative, GATA-2, is. Conditional knockouts of GATA-2 show it is involved in development of interneurons, but its target genes are unknown. In hematopoietic cells lacking GATA-1, the researchers found GATA-2 occupying the SNCA intron-1 site, suggesting that it was a candidate for a brain regulator of α-synuclein. Consistent with this idea, Scherzer and colleagues detected mRNA for GATA-2 in the substantia nigra and frontal cortex of postmortem human brain and in human neuroblastoma cells. When they knocked down GATA-2 expression in the neuroblastoma cells, they found what they call a “small, but biologically plausible” reduction of α-synuclein mRNA (by 28 percent) and protein (by 46 percent).
All the data together, they write, “suggest that in dopaminergic cells relevant to PD, SNCA expression is regulated by GATA-2 via occupancy of the intron-1 site.” That does not prove it happens in mammalian brain, Schlossmacher told ARF, but they are now doing experiments using GATA-2 heterozygote knockouts (homozygous knockouts are embryonic lethal) to look at SNCA regulation and α-synuclein levels. There are likely other factors at play, too, he said, since the RNAi experiments only resulted in partial reduction of α-synuclein.
The co-regulation of α-synuclein and heme-producing enzymes may provide a missing link between α-synuclein and iron deposition, which Schlossmacher calls “two unreconciled, alleged culprits in PD.” He told ARF, “In PD and the related disorder MSA (multiple systems atrophy, where α-synuclein accumulates in oligodendrocytes), we and others have found a lot of iron deposition in the substantia nigra in PD and in the putamen in MSA. Where there is too much synuclein, there is too much iron, and it makes us ask if this is by chance, or on purpose that we are seeing α-synuclein and three heme genes synchronously expressed.”
The co-regulation of synuclein with other hematopoietic genes may also give clues into the normal role of the protein. The investigators show that α-synuclein is induced during hematopoietic differentiation, and that is something Bresnick plans to follow up. “If we know what synuclein is doing in blood, we may derive some understanding of its function in brain,” he said. Moreover, he said, it may be easier to study synuclein function in blood.
Finally, the idea that GATA-2 regulates α-synuclein expression offers a possible new option for therapies to reduce SNCA expression. Bresnick reports they are now doing small molecule screens to look for inhibitors of GATA function. “These results offer a productive approach to reducing α-synuclein, compared to trying to neutralize the mass of α-synuclein that is already present, which might already be too late,” he said. As Schlossmacher points out, 70 percent of Alzheimer disease patients show signs of too much synuclein, too, so the protein may be an interesting target beyond PD.
Aging the GATA Way
In the C. elegans work, first author Yelena Budovskaya and colleagues looked at the transcriptome changes in worms during aging, and identified a set of 1,294 age-regulated genes. Three GATA-related factors were responsible for the regulation of many of these genes, they found. Two of the factors (ELT-5 and ELT-6) increased with age, and suppressed the third factor (ELT-3), which regulated many of the target genes. Knocking down ELT-5 and ELT-6 increased longevity. Importantly, the aging-related changes in the GATA pathway did not seem to be a response to environmental stress, insulin signaling, or caloric restriction, which have been linked to cellular damage over time. Rather, Kim and colleagues speculate, changes in GATA factor activity could represent an “age-related drift of an intrinsic developmental program that becomes imbalanced in old age.”
There have been reports on age-related increases in α-synuclein in humans and monkeys (Chu and Kordower, 2007), and the link between GATA-related factors and aging in worms raises the question of whether changes in GATA factors during human aging might be linked to the increasing synuclein risk for PD.
GATA expert Bresnick says, “No one right now is looking at GATA in human aging, and Kim’s paper will stimulate an interest in that area in our field.” However, he cautions, there is little relationship between the C. elegans GATA factors and mammalian GATAs. “The DNA binding domains are similar but key functional regions outside of that are entirely different. It would be nice if there was a precise relationship between ELT-3 and the mammalian GATA factors, but there is not. There is no mouse ortholog of ELT-3.”
Schlossmacher says he does not know if humans show age-related changes in GATA factors, but, he says, “The whole concept of Kim’s paper is really exciting, and we’d like to know if overactive GATA-2 plays a role in driving PD. Now we are looking at all six GATA family factors in the brain, but we do not know yet if there are age-related changes in humans. GATA-2 is important in embryonic development, but maybe its job is not done there.”
An Inflammatory Situation
Once synuclein accumulates, how does it kill the dopaminergic neurons that disappear in PD? A new clue to that process comes from the Lee lab, where colleagues induced inflammation in the substantia nigra of human synuclein transgenic mice and found that there was a synergistic interaction between the overexpression of human α-synuclein and inflammation to cause dopaminergic cell death.
To look at the effects of inflammation on synuclein, first author Hui-Ming Gao and colleagues developed mice that expressed human α-synuclein, either wild-type or with the Parkinson-associated A53T mutation, in the absence of mouse α-synuclein. When they injected lipopolysaccharide directly into the substantia nigra of the mice to induce local inflammation, they found that loss of dopaminergic neurons was only detected in α-synuclein expressing mice, not in the synuclein-null parental strains. The damage was worse in the mice expressing mutant α-synuclein. LPS-induced inflammatory response was similar in all mice, but the effect on neurons was not. Using in vitro cell cultures, they showed that the ability of LPS to kill the neurons depended on the expression of α-synuclein and the presence of glia, but that glia from any of the mice could support cell killing. These results suggested that neuronal expression of synuclein rendered the neurons susceptible to LPS-induced inflammation and degeneration.
Using inhibitors, the researchers identified the responsible inflammatory factors as superoxide and NO. Turning to the fate of synuclein, they found that LPS injection resulted in production of synuclein aggregates, and that the inclusions contained oxidized and nitrated synuclein. The results suggest that glia-derived free radicals drive the nitration of SYN, and that promotes the formation of inclusions. Thus, the authors formulate a “two-hit” hypothesis, where overexpression of human α-synuclein made the dopaminergic neurons more vulnerable to LPS-induced inflammation, and inflammation led to the accumulation of insoluble synuclein aggregates and cytoplasmic synuclein inclusions in nigral neurons.
As Michael Schlossmacher put it, “Dosage is everything when it comes to synuclein and Parkinson disease,” and the results of Lee and colleagues fit with this idea by tying together the expression of α-synuclein in dopaminergic neurons with a higher vulnerability of those neurons to inflammatory stress.—Pat McCaffrey.
Scherzer CR, Grass JA, Liao Z, Pepivani I, Zheng B, Eklund AC, Ney PA, Ng J, McGoldrick M, Mollenhauer B, Bresnick EH, Schlossmacher MG. GATA transcription factors directly regulate the Parkinson’s disease-linked gene alpha-synuclein. 2008 July 21. PNAS Early Edition. Abstract
Budovskaya YV, Wu K, Southworth LK, Jiang M, Tedesco P, Johnson TE, Kim SK. An elt3/elt5/elt6 GATA transcription circuit guides aging in C. elegans. Cell. 2008 July 25;134:291-303. Abstract
Gao, HM, Kotzbauer PT, Uryu K, Leight S, Trojanowski JQ, Lee, VMY. Neuroinflammation and oxidation/nitration of alpha-synuclein linked to dopaminergic neurodegeneration. J Neurosci. 2008 Jul 23;28(30):7687-98. Abstract