The classic scientific method requires a hypothesis—but sometimes it can be refreshing to dive in without one. The authors of two screens, published this week, took just that approach and came up with pathological pathways linked to two different neurodegenerative conditions.

A multisite team led by senior author Ilya Bezprozvanny, of the University of Texas Southwestern Medical Center in Dallas, studied a compound that slowed progression of motor symptoms in animal models for Huntington’s disease. The authors determined that the drug worked by blocking the store-operated calcium (SOC) entry pathway, and that the pathway is overactive in HD neurons.

The work, published in the June 24 issue of Chemistry & Biology, is “a breath of fresh air in the Huntington’s literature,” said Grace Stutzmann of Rosalind Franklin University in North Chicago, Illinois. The study of HD has been focused on genetics and histone deacetylases, she said, and the current work “opens up a new way to think about disease mechanisms at a cellular level.” In addition, calcium dysregulation is not specific to Huntington’s, so the SOC pathway might be relevant to other neurodegenerative conditions such as Alzheimer’s, she said.

Researchers led by Lee Rubin at Harvard University sought to find pathways that are involved in spinal muscular atrophy (SMA). Since people who have this disease usually have low levels of the protein survival of motor neuron (SMN), the researchers used a visual screen to identify compounds that boost SMN levels in fibroblasts. They discovered a slew of hits, published online June 24 by Nature Chemical Biology. While the motor neurons affected by the disease may not respond to the same treatments, they hope that the study points to biological pathways involved in the pathology. “Very little is known about regulation of SMN cellular levels,” said Claudia Fallini of Emory University in Atlanta, Georgia, who was pleased to see the study identify specific pathways. Following up on one hit, the authors discovered that blocking glycogen synthase kinase-3 increased SMN levels by stabilizing the protein.

Calcium Key to Huntington’s?
The Huntington’s screen began at EnVivo Pharmaceuticals in Watertown, Massachusetts, in a fruit fly project led by one of the study’s joint first authors, Hsin-Pei Shih, now at Vertex Pharmaceuticals, Inc. in Cambridge, Massachusetts. The EnVivo scientists screened drugs in a fly HD model and discovered that the quinazoline derivative EVP4593 allowed older animals to climb better than age-matched, untreated HD flies.

EVP4593 is a known inhibitor of the transcription factor nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB; Tobe et al., 2003). But standard NF-κB inhibitors failed to reproduce its positive effect on the flies, suggesting that EVP4593 does not protect neurons via NF-κB. The company turned to Bezprozvanny for help in deducing the real the mechanism of action. EnVivo has since dropped its Huntington’s project, but Bezprozvanny continued the study.

Bezprozvanny and joint first author Jun Wu at the Texas Medical Center confirmed that EVP4593 was beneficial in their Huntington’s mouse model. Looking for a mechanism, they noted a previous report that EVP4593 could inhibit the store-operated calcium entry (SOC) pathway in T cells (Choi et al., 2006). The SOC pathway is activated in response to low calcium levels in the endoplasmic reticulum (ER). When the ER runs out of calcium, cellular signals open up SOC channels, which allow more calcium to flow into the cell.

Traditionally, SOC has been the domain of immunologists, not neuroscientists, but Wu and colleagues decided to look for SOC activity in medium spiny neurons (MSNs), the type most affected by Huntington’s disease. Compared to wild-type MSNs, an MSN line expressing the disease-linked, polyglutamine-expanded protein huntingtin accumulated more calcium, suggesting upregulation of the SOC pathway. EVP4593 treatment restrained the SOC overactivity.

Bezprozvanny also collaborated with joint first author Vladimir Vigont, in the laboratory of Elena Kaznacheyeva at the Russian Academy of Science in St. Petersburg, to test the electrophysiological effects of EVP4593 in HD neurons. Vigont discovered that when he induced SOC in a human neuroblastoma cell line, the current across the cell membrane shot up to five times normal when the cells expressed the polyglutamine-expanded huntingtin.

How might the drug interrupt SOC? Wu and Bezprozvanny made an educated guess that the transient receptor potential channel 1 (TRPC1) might be the target. TRPC1 is thought to be a SOC component (Wenning et al., 2011), and it is expressed in neurons (Riccio et al., 2002). The scientists transfected neuroblastoma cells with a short interfering RNA against TRPC1, and found that it reduced the high currents associated with expanded huntingtin expression.

EVP4593 may block TRPC1, but it will require more work to deduce the exact relationship among huntingtin, SOC, and the compound. However, Bezprozvanny has a theory: He recently found that mutant huntingtin binds and activates an intercellular calcium release channel, the type 1 inositol 1,4,5-triphosphate receptor (IP[3]R1; Bezprozvanny, 2011). In so doing, huntingtin would cause the release of calcium from the ER. This would then activate the SOC pathway, flooding the cytoplasm with even more calcium. “You basically have continuous influx of calcium into the cell, which creates continuous calcium stress,” he said.

Young neurons, Bezprozvanny suspects, can handle the stress—for example, by storing excess calcium in mitochondria. But with age, those stress-response pathways weaken, calcium levels creep up, and the cells degenerate.

SOC might also be involved in “any neurological syndrome that has been associated with aberrant calcium signaling,” said Murali Prakriya of the Northwestern University Feinberg School of Medicine in Chicago, Illinois. That list includes Alzheimer’s, Parkinson’s, and amyotrophic lateral sclerosis, he noted, adding that the paper should motivate many scientists to take a look at this calcium pathway.

Multiple Mediators of SMN Levels
Insufficient SMN protein in neurons is one cause of spinal muscular atrophy. Mutations in the SMN gene usually produce alternative splice forms that are unstable and rapidly degraded. “Theoretically, anything that elevates the level of the protein should be therapeutic,” said Lee Rubin, who led the SMN screen with first author Nina Makhortova.

Many researchers have screened for drugs that influence the splicing or transcription of SMN, using reporter constructs in quick, high-throughput assays. Makhortova and Rubin took a different approach, using an automated microscope to visually screen cultures stained with an SMN antibody. This technique allowed them to observe not only total protein levels, but also to distinguish SMN in the cytoplasm from that in the nucleus or in nuclear gems, protein complexes where SMN participates in the genesis of small ribonuclear proteins. Wilfried Rossoll, at Emory University, called it a “high-content screen” because it offers more information than a high-throughput screen with reporter genes.

The scientists collected fibroblasts from carriers of the defective gene, that is, parents of children with SMA. They tested 3,500 different compounds on the cultures and came up with 188 hits.

Fibroblasts are strongly affected by SMA, so the group is also running a screen with motor neurons derived from iPS cells from SMA mice. “There was almost no overlap in terms of compounds that worked on the fibroblasts and worked on the motor neurons,” Rubin said. For example, platelet-derived growth factor (PDGF) upregulated SMN in fibroblasts, but is unlikely to do so in motor neurons as they lack PDGF receptors. Thus, Rubin figured that the fibroblast screen would not reveal promising treatments so much as general pathways related to SMN levels.

The researchers categorized their hits into groups including those that affect ion channels, the cytoskeleton, kinases, and nearly two dozen others. Several hits were compounds that inhibit sodium, potassium ATPases. Blocking these enzymes produces an influx of sodium and calcium cations; indeed, other treatments to promote cation entry were also effective at amplifying SMN levels.

The ATPase inhibitors in the study are thought to work by modulating the activity of receptor tyrosine kinases (Wasserstrom and Aistrup, 2005). Makhortova found that several tyrosine kinase ligands, including PDGF, upped SMN levels.

PDGF activates several cellular pathways. The scientists tested out different inhibitors on the PDGF-treated cells and discovered that a PI3K inhibitor abolished the growth factor’s effects on SMN protein levels. That led them to GSK-3, which sits downstream of PDGF and PI3K. GSK-3 was phosphorylated, and thus inhibited, in PDGF-treated cells.

Next, the authors sought to inhibit GSK-3 by chemical means. Alsterpaullone and several other GSK-3 inhibitors increased SMN amounts. They apparently did so by stabilizing the protein, because there was no change in SMN splicing and little effect on levels of SMN RNA. The authors suggest GSK-3 may normally destabilize SMN by phosphorylating it.

Finally, the scientists confirmed that alsterpaullone also led to more SMN protein and better cell survival in a cell type more relevant to SMA, mouse motor neurons depleted of SMN. Even so, “There is no way general protease inhibitors could be used as a drug,” Rossoll said, because it would likely stabilize all cellular proteins. Rubin’s motor neuron screen may yield more clinically applicable hits, or researchers may find ways to prevent SMN degradation.—Amber Dance


  1. This is a fascinating piece of translational work that provides a new line of support for the hypothesis that an alteration in calcium homeostasis is important in HD pathogenesis. The identification of a new small molecule that protects striatal neurons expressing mutant huntingtin is a key step toward the development of a viable neuroprotective strategy for this devastating disease. One of the most interesting aspects of the study is the suggestion that the expression of mutant huntingtin leads to the upregulation of a protein that enhances calcium entry into intracellular stores, but that normally plays little or no role in this process (the compounds identified in the fly screen had little or no effect in wild-type neurons). In the event that the quinazoline-derived compounds are not suitable for use in humans, the identification of the protein they target could be extremely valuable in developing alternative strategies for neuroprotection.

    The study also provides a beautiful model of how to conduct unbiased translational studies for neurological diseases.

    View all comments by D. James Surmeier

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Paper Citations

  1. . A novel structural class of potent inhibitors of NF-kappa B activation: structure-activity relationships and biological effects of 6-aminoquinazoline derivatives. Bioorg Med Chem. 2003 Sep 1;11(18):3869-78. PubMed.
  2. . Nuclear factor-kappaB activated by capacitative Ca2+ entry enhances muscarinic receptor-mediated soluble amyloid precursor protein (sAPPalpha) release in SH-SY5Y cells. J Biol Chem. 2006 May 5;281(18):12722-8. PubMed.
  3. . TRP expression pattern and the functional importance of TRPC3 in primary human T-cells. Biochim Biophys Acta. 2011 Mar;1813(3):412-23. PubMed.
  4. . mRNA distribution analysis of human TRPC family in CNS and peripheral tissues. Brain Res Mol Brain Res. 2002 Dec 30;109(1-2):95-104. PubMed.
  5. . Digitalis: new actions for an old drug. Am J Physiol Heart Circ Physiol. 2005 Nov;289(5):H1781-93. PubMed.

Further Reading


  1. . Converging pathways in the occurrence of endoplasmic reticulum (ER) stress in Huntington's disease. Curr Mol Med. 2011 Feb;11(1):1-12. PubMed.
  2. . Calcium dysregulation and homeostasis of neural calcium in the molecular mechanisms of neurodegenerative diseases provide multiple targets for neuroprotection. Antioxid Redox Signal. 2011 Apr 1;14(7):1275-88. PubMed.
  3. . Multiprotein complexes of the survival of motor neuron protein SMN with Gemins traffic to neuronal processes and growth cones of motor neurons. J Neurosci. 2006 Aug 16;26(33):8622-32. PubMed.
  4. . Deletion of murine SMN exon 7 directed to skeletal muscle leads to severe muscular dystrophy. J Cell Biol. 2001 Mar 5;152(5):1107-14. PubMed.
  5. . Mitochondrial calcium uptake capacity as a therapeutic target in the R6/2 mouse model of Huntington's disease. Hum Mol Genet. 2010 Sep 1;19(17):3354-71. PubMed.
  6. . A novel association of the SMN protein with two major non-ribosomal nucleolar proteins and its implication in spinal muscular atrophy. Hum Mol Genet. 2002 May 1;11(9):1017-27. PubMed.

Primary Papers

  1. . A screen for regulators of survival of motor neuron protein levels. Nat Chem Biol. 2011 Aug;7(8):544-52. PubMed.
  2. . Neuronal store-operated calcium entry pathway as a novel therapeutic target for Huntington's disease treatment. Chem Biol. 2011 Jun 24;18(6):777-93. PubMed.