Polyglutamine, Inclusion Bodies and the Proteasome—The Good, the Bad, and the Innocent?

Garbage in–garbage out is a common mantra among computer programmers. In biology, it could easily apply to the proteasome, which pulls in waste proteins, chews them up, and gets them out of circulation. So if instead the garbage is piling up in intracellular inclusion bodies, such as those found in Parkinson and Huntington diseases, is it the fault of the proteasome? Current papers from Ron Kopito's lab at Stanford University in Palo Alto, California, and Huda Zoghbi’s lab at Baylor College of Medicine, Houston, have addressed this issue but have come up with different answers. While Kopito concludes that the proteasome is indeed impaired, Zoghbi finds that the garbage disposal is working fine.

To measure activity of the ubiquitin proteasome system (UPS), both groups used a similar strategy—a degradable green fluorescent protein (GFP) chimera easily quantified by fluorescence microscopy. Put this chimera into a cell along with fodder for inclusion bodies, say, a polyglutamine protein such as huntingtin or SCA7, and you have the makings of a system that reveals how inclusion formation affects the proteasome.

Reporting in the February 4 Molecular Cell, Kopito and colleagues describe how they used, in cultured HEK 293 cells, two such reporters. One was targeted to the nucleus to measure proteasome activity there, while the other was fitted with a nuclear export signal to measure proteasome activity in the cytosol. What first author Eric Bennett and colleagues found most surprising was a “cis-trans” relationship. When the researchers expressed these reporters along with either nuclear (ataxin 1) or cytosolic (huntingtin) protein, they found that polyglutamine proteins in one compartment could affect the levels of the GFP chimera in the other, and vice versa. For example, expressing ataxin 1 with an expanded stretch of 82 glutamines led to a near-doubling of the GFP chimera in both the nucleus and the cytoplasm. Expression of the cytosolic huntingtin with 103 glutamine repeats had the same effect. “Protein aggregation can impair nuclear and cytoplasmic UPS function when the aggregates accumulate in cis or in trans,” the authors conclude.

But is the proteasome really impaired? Do levels of the reporter truly reflect proteasomal degradation, or could levels of protein synthesis account for at least some of it, as well? This relationship was addressed by Huda Zoghbi’s group. In a January 20 advanced access publication from Human Molecular Genetics, first author Aaron Bowman and colleagues described results of using a GFP reporter to measure UPS activity in SCA7 “knock in” mice, where the mutant protein is expressed under endogenous regulatory control (see ARF related news story).

Similar to Kopito’s group, Bowman and colleagues found that expression of an expanded SCA7 with 266 glutamines caused about a 1.5- to threefold increase in levels of the GFP reporter in the outer nuclear layer of the retina once the animals reached about 13 weeks (neurons in this region are most sensitive to SCA7 damage). Yet when Bowman and colleagues looked at expression of the reporter, they found that mRNA levels were significantly increased, as well. So is the increase in UPS reporter protein due to increased expression, impaired proteasome activity, or both?

To answer this question, Bowman and colleagues turned to a different method. Measuring the chymotrypsin proteasome activity from extracts taken from wild-type and SCA7-damaged retinas, the scientists found no difference between them. That leaves elevated expression as the reason for the increase in the GFP reporter in this in-vivo study, a finding that would fit with reports of widespread alterations in gene expression caused by polyglutamine proteins (see ARF related news story).

In fact, some of the data in the Kopito paper would support the idea that the UPS is not inactivated, at least not by sequestration of UPS components, protein aggregates, simple substrate competition, or inclusion bodies. For example, Bennett and colleagues found no significant redistribution, i.e., sequestration, of proteasome subunits in cells with nuclear or cytoplasmic inclusions. They found that polyglutamine proteins had no effect on isolated proteasome activity, even at 100-fold molar excess. And they found that overexpressing other UPS substrates, such as ornithine decarboxylase, had no effect on levels of the GFP reporter, i.e., substrate competition didn’t slow down the proteasome. They also found that inclusion bodies weren’t necessary to get elevated levels of the UPS reporter—when just diffuse huntingtin was present in the cytosol, they found that the GFP was elevated.

So where does all this leave us with respect to inclusion bodies and the UPS? Given that UPS reporter was elevated in the absence of inclusion bodies, Kopito and colleagues concluded that “intermediate forms of protein aggregates may be able to inhibit UPS function.” (See ARF related news story.) This team adds that the observations support “the hypothesis that sequestration of aggregates into IBs may be a protective, rather than a pathogenic, response.” Others have made similar suggestions in the past (see, for example, ARF related news story).

The Zoghbi group came to a similar conclusion on the role of inclusion bodies. Bowman and colleagues found that levels of the GFP reporter were highest in those cells with the most neurologic damage—those with downregulation of photoreceptor transcripts, such as rhodopsin, for example—and that those cells with the most inclusion bodies had the lowest levels of the reporter.

As for whether or not the proteasome is affected by polyglutamine proteins, it may be affected in some models, such as those overexpressing proteins in transfected cells. At the same time, the Zoghbi group concluded that “significant UPS impairment is not required or detected in an authentic model of SCA7 polyglutamine neuropathology.” This leaves open the possibility that the UPS is impaired in some specific neurodegenerative diseases, but suggests that protein aggregation per se does not always impair the UPS in the course of pathology.—Tom Fagan

Comments

  1. This paper reports additional evidence that nuclear inclusions of polyglutamine-containing proteins may actually be protective because they may segregate and inactivate the free mutant proteins (for example, huntingtin), which may be more toxic to the cell than are their aggregates. Thus, protein aggregation may not necessarily be a toxic process and may actually play a cell-protective role in Huntington disease and spinocerebellar ataxias. The similarities to the Alzheimer disease (AD) field are clear because this field is finally accepting the notion that the amyloid-β aggregates are not the main neurotoxic agent that drives the AD neurodegeneration (see Neve and Robakis, 1998).

    References:

    Neve RL, Robakis NK. Alzheimer's disease: a re-examination of the amyloid hypothesis. Trends Neurosci. 1998 Jan;21(1):15-9. PubMed.

    View all comments by Nikolaos K. Robakis

  2. Please access article in Nature news July 2004 re "Lithium may fend off Alzheimer's disease". It is available to Premium Plus subscribers to news@nature.com, available to institutions or at a rate of 15.99 per month.

    Both my deceased brother and myself were prescribed lithium. My father at age 84 is showing Alzheimer's symptoms. Those of us still alive would gladly give research scientists access to our blood and in my own case (under the direction of my doctors) other scientific tests if this could lead to a cure for Alzheimer disease.

    View all comments by Susan R Saslaw

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References

News Citations

  1. New Insights and Strategies for Treating PolyQ Disorders
  2. Transcriptional Activators Kidnapped by Huntingtin
  3. Polyglutamine—Gristle for the Proteasome Mill?
  4. New Microscope Resolves Role of Huntington Inclusions—Neuroprotection

Further Reading

No Available Further Reading

Primary Papers

  1. Bennett EJ, Bence NF, Jayakumar R, Kopito RR. Global impairment of the ubiquitin-proteasome system by nuclear or cytoplasmic protein aggregates precedes inclusion body formation. Mol Cell. 2005 Feb 4;17(3):351-65. PubMed.
  2. Bowman AB, Yoo SY, Dantuma NP, Zoghbi HY. Neuronal dysfunction in a polyglutamine disease model occurs in the absence of ubiquitin-proteasome system impairment and inversely correlates with the degree of nuclear inclusion formation. Hum Mol Genet. 2005 Mar 1;14(5):679-91. PubMed.

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