You might be inclined to wonder about your refuse collector if garbage was piling up in your neighborhood. What about in your brain? For years scientist have looked to cellular recycling mechanisms to explain the accrual of toxic protein aggregates, such as the senile plaques and neurofibrillary tangles found in Alzheimer disease, or the Lewy bodies found in Parkinson’s. Two papers in this week’s Journal of Neuroscience may bring us a few steps closer to understanding the relationship between the cell’s protein recycling machinery—the ubiquitin proteasome system—and AD.

Led by Paul Lombroso at Yale University, New Haven, Connecticut, researchers report that a protein phosphatase accumulates in mouse models of AD and mediates Amyloid beta (Aβ) toxicity. The buildup of the phosphatase, called striatal-enriched protein tyrosine phosphatase 61—or STEP61—is not due to overproduction, but failure of degradation by the ubiquitin proteasome system (UPS), the researchers show. In a second paper, a group led by Andrea LeBlanc at McGill University, Montreal, Canada, report that a ubiquitin-dependent ATPase that is crucial for the UPS is cleaved by caspase-6, a protease that LeBlanc’s research implicates in AD pathology. One cleavage fragment of the ATPase acts as an inhibitor of the UPS in cells, and the researchers show that this fragment is elevated in AD brains, hinting that it might impair the UPS in the disease as well. “The papers agree with the idea that an efficient working UPS is very relevant for preventing and/or delaying AD,” suggested Fred van Leeuwen, University of Maastricht, in an email to ARF.

Lombroso’s work connects STEP61 with Aβ-induced blockage of long-term potentiation, a form of synaptic plasticity. The phosphatase is localized to the post-synaptic compartment and is thought to act as a counter balance to synaptic strengthening by promoting internalization of glutamate receptor subunits. Previously, Lombroso and colleagues discovered that Aβ activates the phosphatase (see ARF news) and other researchers reported that the protein is elevated in J20 mice expressing mutant human amyloid precursor protein, or APP (see Chin et al., 2005). Now, joint first authors Pradeep Kurup, Yongfang Zhang and colleagues extend that observation to the widely-used Tg2576 mouse model and to the human brain. They found that between three and 12 months of age, STEP61 accumulates in the frontal cortex of the mice, and it seems elevated the AD brain as well. Comparison of postmortem cortical tissue extracts from five AD patients and five controls, revealed elevated levels of the phosphatase in the former. The findings suggest that STEP61 might play a role in AD pathology.

Testing this idea, Kurup and colleagues exposed rat and mouse cortical neurons to conditioned medium (CM) from Aβ-producing 7PA2 cells. The CM, previously shown to contain toxic forms of Aβ that block synaptic plasticity (see Walsh et al., 2002 and related ARF news), not only decreased the number of NR2B glutamate receptor subunits on the neurons, but increased the cellular content of STEP61. On the other hand, the conditioned medium had no effect on glutamate receptor subunits when added to neurons from STEP61 knockout mice. Overall, the findings indicate that the phosphatase is required for Aβ-induced internalization of glutamate receptors.

Curiously, neither inhibitors of transcription nor translation prevented Aβ-induced elevation of STEP61, which would seem to eliminate increased synthesis as an explanation for Aβ’s effects. “One other possible explanation for elevated STEP61 would be reduced degradation,” said Lombroso. That is, in fact, what the researchers found. By blocking the proteasome, the researchers increased STEP61 in cortical cells. They found that the phosphatase was ubiquitinated when cells were treated with Aβ. And they found increased levels of ubiquitinated STEP61 in the brain of 12-month-old Tg2576 mice compared to wild-type controls. These results point to a failure of the proteasome to clear STEP61 under an Aβ onslaught.

These discoveries would predict that reducing STEP61 levels in AD mouse models will prevent the loss of glutamate receptors and reverse cognitive deficits. Lombroso told ARF that he is investigating this possibility using a genetic approach. His lab is also screening for small molecules that might inhibit STEP61 phosphatase activity.

LeBlanc and colleagues’ study of the UPS was more direct. In a previous screen the scientists identified the valosin-containing ATPase, p97, which helps drive protein ubiquitination, as a potential substrate for caspase-6. Now, first author Dalia Halawani and colleagues report that caspase-6 cleaves both N- (residues 1-480) and C-terminal (residues 481-806) fragments of the ATPase in vitro. The N-terminal half was cleaved into at least four different polypeptides, and the C-terminal half at least two. Caspase-6 also cleaved the native protein, which forms a hexamer that is generally resistant to proteolysis.

To identify the exact sites of cleavage, the researchers separated caspase-6 cleaved fragments using liquid chromatography and analyzed the peptides using mass spectrometry. The size of the protein fragments was consistent with five major cleavage sites, the most consistent being the VAPD motif at residues 176-179. The researchers raised an antibody to the N-terminal fragment of the protein generated by proteolysis at this position and found that it specifically recognized the truncated protein, p97(1-179) but not full length. The antibody (anti-p97D179) stained hippocampal sections taken postmortem from AD patients and some MCI patients, indicating that caspase-6 cleavage of p97 occurs in the disease and possibly starts early in the progression of pathology. Of 15 AD hippocampal samples, anti-p97D179 immunoreactivity was least intense in those taken from patients with the highest MMSE scores and most intense in those with severest disease (MMSE LeBlanc thinks that since p97 is cleaved in AD, it could explain the high level of ubiquitin in Alzheimer’s neurons. “Some of it is on tau, but there are many other proteins that are ubiquitinated that accumulate in neurons in the Alzheimer’s brain and nobody really knows how it happens,” she said. Halawani found that expressing an ATPase-deficient mutant of p97, or the caspase-6 truncated ATPase, p97(1-179), caused accumulation of UPS substrates in N2a neuroblastoma cells, supporting the idea that dysfunctional p97 or caspase-6 cleavage of the protein jams up the UPS recycling system. Interestingly, anti-p97D179 staining in AD hippocampal tissue was mostly granular, “which is very similar to when you look at ubiquitinated products in Alzheimer’s,” said LeBlanc

What activates caspase-6? “We haven’t nailed that yet,” said LeBlanc. One possibility is through activation of the death receptor 6 by the N-terminal of APP, a pathway recently uncovered by Marc Tessier-Lavigne and colleagues at Genentech Inc., San Francisco, California (see related ARF news). DR6 can activate caspase-6 in axons. LeBlanc thinks caspase-6 activation might be the result of inflammation. “We found that if we block caspase-1, then caspase-6 is blocked as well. Since it has been known for a long time that interleukin 1-β is increased in Alzheimer’s, and caspase-1 is the IL1- β converting enzyme, then caspase-1 may be elevated as well,” she said.—Tom Fagan


  1. Time for the Ubiquitin-Proteasome System #3
    These papers look very interesting and show the relevance in Alzheimer disease of an efficient working protein quality control by the ubiquitin-proteasome system (UPS) and by autophagy, two systems that show crosstalk. I am organizing a satellite on this subject soon at a FENS meeting.

    The papers agree with the idea that an efficient working UPS is very relevant for preventing and/or delaying AD; see our recent mini-review in Neurodegenerative Diseases (Dennissen et al., 2010). These two papers add again pieces of the puzzle to aid in dissecting AD pathogenesis, particularly the Halawani paper in MCI (see also my two previous comments Time for the ubiquitin-proteasome system and Time for the Ubiquitin-Proteasome System #2: Dendritic Spines).

    The paper by Halawani reports on the ubiquitin-escort protein valosin (p97) as a new substrate for caspase 6 in AD, and as a novel mechanism of UPS impairment in AD. Caspase-6 expression is activated in AD and other substrates of caspase-6 are cytoskeletal proteins, which are critical for axonal function. In this regard, there is a link with a paper showing that misframed ubiquitin (UBB+1) causes neuritic beading (see Tan et al., 2007). P97 facilitates UPS-mediated degradation in several pathways, including the ubiquitin fusion degradation pathway (UFD). Moreover, UBB+1 is the first-reported naturally occurring UFD substrate (see Lindsten et al., 2002). The present data open an avenue for further research, e.g., colocalization of Caspase-6 cleaved P97, UBB+1 and caspase-6 in AD.

    The paper of Kurup et al. provides biochemical evidence that the enzyme “striatal-enriched protein tyrosine phosphatase 61(STEP 61)” is post-translationally modified by ubiquitin after Aβ-mediated NMDA receptor endocytosis in a mouse model of AD of Karen Hsiao (Tg2576) and supported by studies in postmortem tissue of AD prefrontal cortex. This group indicates that decreased STEP 61 turnover is an important consequence of a dysfunctional UPS, and links directly to an impairment of normal NMDAR trafficking at synapses. Thus, here again a link between Aβ and ubiquitin is provided, and the present report is in agreement with previous studies (see Song et al., 2003). Since a unique model for neuronal proteasomal inhibition and a behavioral phenotype became recently available via Jackson Laboratories (see also Fischer et al., 2009), relevant experiments are now possible.


    . Mutant ubiquitin found in Alzheimer's disease causes neuritic beading of mitochondria in association with neuronal degeneration. Cell Death Differ. 2007 Oct;14(10):1721-32. PubMed.

    . Mutant ubiquitin found in neurodegenerative disorders is a ubiquitin fusion degradation substrate that blocks proteasomal degradation. J Cell Biol. 2002 Apr 29;157(3):417-27. PubMed.

    . Essential role of E2-25K/Hip-2 in mediating amyloid-beta neurotoxicity. Mol Cell. 2003 Sep;12(3):553-63. PubMed.

    . Long-term proteasome dysfunction in the mouse brain by expression of aberrant ubiquitin. Neurobiol Aging. 2009 Jun;30(6):847-63. PubMed.

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News Citations

  1. Amyloid-β Zaps Synapses by Downregulating Glutamate Receptors
  2. Earliest Amyloid Aggregates Fingered As Culprits, Disrupt Synapse Function in Rats
  3. Copper Mountain: Death and Trophin Receptors—New Insight, New Drugs?

Paper Citations

  1. . Fyn kinase induces synaptic and cognitive impairments in a transgenic mouse model of Alzheimer's disease. J Neurosci. 2005 Oct 19;25(42):9694-703. PubMed.
  2. . Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature. 2002 Apr 4;416(6880):535-9. PubMed.

Further Reading

Primary Papers

  1. . Abeta-mediated NMDA receptor endocytosis in Alzheimer's disease involves ubiquitination of the tyrosine phosphatase STEP61. J Neurosci. 2010 Apr 28;30(17):5948-57. PubMed.
  2. . Identification of Caspase-6-mediated processing of the valosin containing protein (p97) in Alzheimer's disease: a novel link to dysfunction in ubiquitin proteasome system-mediated protein degradation. J Neurosci. 2010 Apr 28;30(17):6132-42. PubMed.