01 Aug 2013

In neurodegenerative diseases, certain brain regions fall victim to toxic proteins faster than others. Scientists have long wondered what explains this selective vulnerability. The answer could be the speed of protein breakdown, at least that is what a new in vitro study suggests. Researchers led by by Steven Finkbeiner, University of California, San Francisco, developed a way to measure protein clearance rates in individual neurons and applied it to the protein that causes Huntington's disease (HD). Published in the July 21 Nature Chemical Biology, their data suggest that the ability to clear mutant huntingtin (Htt) and regulate its levels to maintain protein homeostasis defines a neuron’s susceptibility the toxic protein.

"The basis for selective cellular susceptibility in neurodegenerative disease has been an enduring mystery," Finkbeiner wrote to Alzforum in an email. "The observation that cells exhibit the necessary diversity in protein homeostasis to at least partially account for the susceptibility is rather surprising, since most people assume that protein homeostasis is highly conserved."

Scientists typically measure protein clearance by briefly introducing a radioactive amino acid into its structure and then watching how quickly it disappears. They usually sample groups of lysed cells over a series of time intervals to quantify the loss of radioactivity. This pulse-chase method cannot measure protein degradation rates in single cells. Spontaneous cell death also confounds the results, as it leads to an overestimation of protein breakdown.

Finkbeiner's group developed an optical pulse chase method to measure protein clearance. The researchers fuse mutant Htt with Dendra2, a fluorescent protein that changes color from green to red when hit with blue light. They flashed a brief pulse of light onto cells expressing the fusion protein, and then used a microscope to measure the decline in red florescence from the same individual cells for up to several weeks. Neurodegeneration does not obscure the results, nor does synthesis of new, green Htt.

Using this method, first author Andrey Tsvetkov and colleagues compared the clearance of mutant Htt in striatal, cortical, and cerebellar cells isolated from either rat embryos or pups. Striatal neurons eliminated the protein about twice as slowly as cortical neurons, which in turn lagged behind cerebellar neurons. The longer the protein stuck around, the shorter the cells' lifespan, meaning striatal neurons died first. This mirrors the selective vulnerability observed in HD, which primarily affects neurons in the striatum. The researchers boosted autophagy by transfecting striatal neurons with the transcription factor Nrf2, which regulates stress response genes, including the autophagy-related p62. This shortened the half-life of mutant Htt in a given striatal neuron and lengthened the cell's lifespan. "The findings would suggest that interventions that promote protein clearance could be helpful therapeutically," proposed Finkbeiner.

What causes the differences in protein metabolism between neurons? Cells rid themselves of proteins via highly regulated processes whose activation likely varies depending on the substrate and cell type, wrote Finkbeiner. It is too early to know whether differences in protein homeostasis underlie selective vulnerability in other neurodegenerative diseases, but Finkbeiner plans to explore the possibility. His group has found that neuronal subtypes are differentially vulnerable to mutant ataxin-1, involved in spinocerebellar ataxia type 1, and is also probing the benefits of enhancing neuronal clearance of TPD-43 and α-synuclein, implicated in amyotrophic lateral sclerosis and Parkinson’s disease, respectively.

Complimenting the study, Jeffrey Johnson, University of Wisconsin-Madison, said it will be important to test whether its results hold up in vivo. Johnson was not involved in this work. He is trying to work out the mechanism of the beneficial effects of Nrf2. Does the general stress response kindled by Nrf2 treatment simply keep cells healthy and allow clearance to continue? Or does Nrf2 change the expression of particular proteins that somehow enhance metabolism? Further studies will help answer those questions, Johnson said.

Finkbeiner’s results support previous research that proposes an autophagy-stimulating route to treating HD, said Ralph Nixon, Nathan Kline Institute in Orangeburg, New York, who was not involved in the study. For instance, small-molecule enhancers of autophagy reduce neurodegeneration in a Drosophila model of HD (see ARF related news story on Sarkar et al., 2007). However, simply enhancing autophagy might not work for AD and PD, as the offending proteins in those diseases disrupt the process (for a review, see Nixon and Yang, 2012). In that case, stimulating autophagy may be detrimental, Nixon said.—Gwyneth Dickey Zakaib

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References

News Citations

1. New Targets for Neurodegenerative Diseases: Autophagy and More 11 May 2007

Paper Citations

1. Sarkar S, Perlstein EO, Imarisio S, Pineau S, Cordenier A, Maglathlin RL, Webster JA, Lewis TA, O'Kane CJ, Schreiber SL, Rubinsztein DC. Small molecules enhance autophagy and reduce toxicity in Huntington's disease models. Nat Chem Biol. 2007 Jun;3(6):331-8. PubMed.
2. Nixon RA, Yang DS. Autophagy and neuronal cell death in neurological disorders. Cold Spring Harb Perspect Biol. 2012;4(10) PubMed.

Further Reading

Papers

1. Sarkar S, Perlstein EO, Imarisio S, Pineau S, Cordenier A, Maglathlin RL, Webster JA, Lewis TA, O'Kane CJ, Schreiber SL. Small molecules enhance autophagy and reduce toxicity in Huntington's disease models. Nat Chem Biol. 2007 May 1;3(6):331-8.
2. Williams A, Sarkar S, Cuddon P, Ttofi EK, Saiki S, Siddiqi FH, Jahreiss L, Fleming A, Pask D, Goldsmith P, O'Kane CJ, Floto RA, Rubinsztein DC. Novel targets for Huntington's disease in an mTOR-independent autophagy pathway. Nat Chem Biol. 2008 May;4(5):295-305. PubMed.
3. Nixon RA, Yang DS. Autophagy and neuronal cell death in neurological disorders. Cold Spring Harb Perspect Biol. 2012;4(10) PubMed.

News

1. Synuclein Resists and Disrupts Autophagy 12 Apr 2013
2. An Extra Strain on the Brain—α-Synuclein Seeds Tau Aggregation 8 Jul 2013
3. “Huntingtin Holiday” Helps Mice Back to Health 22 Jun 2012
4. Australia Report: Protein Aggregation, Selective Vulnerability, Spreading 7 Oct 2011
5. Nrf2 Pumps Up Astrocyte Protection of Motor Neurons in ALS Model 13 Dec 2008
6. New Targets for Neurodegenerative Diseases: Autophagy and More 11 May 2007
7. Eat 'Em Up Early—Autophagy Might Delay Huntington's Disease 24 May 2004