Toxic forms of tau come in many shapes and sizes, ranging from tiny fragments to large oligomers to hyperphosphorylated forms entrenched within neurofibrillary tangles. According to new findings published in the Journal of Neuroscience on July 9, the calpain proteases may play a part in creating all of them. What’s more, blocking calpain staved off neurodegenerative disease and prolonged survival in a mouse model of frontotemporal lobar degeneration (FTLD) that expresses human mutant tau. Led by Ralph Nixon at New York University, the study may breathe new life into the idea of using calpain inhibitors to thwart neurodegenerative disease. Nixon hopes the findings will help revive efforts to craft an inhibitor specific enough to do the job safely.

The study “highlights the importance of proteolytic dynamics in neurodegeneration, placing the calcium-dependent protease, calpain, in the center of the universe,” wrote Rodney Guttmann of the University of West Florida in Pensacola, in an email to Alzforum.

Calpain proteases, which switch on in response to elevated calcium, alter the activity of their substrates by trimming them. Of more than a dozen calpain proteases, calpains I and II are expressed in the central nervous system. The enzymes first raised eyebrows in the neurodegeneration field in 1993, when Nixon’s lab reported high calpain activity in the brains of people with Alzheimer’s disease (see Saito et al., 1993). In 2008, the group reported that the endogenous calpain inhibitor calpastatin was depleted in AD brains (see Rao et al., 2008). Studies implicated the proteases in several disease-associated processes, including Aβ-mediated destruction of synapses and tau hyperphosphorylation (see Dec 2009 news story and Jul 2008 news story). Researchers subsequently reported that calpain inhibitors protected against Aβ and α-synuclein pathology in AD and PD mouse models, respectively (see Medeiros et al., 2012, and Diepenbroek et al., 2014). Nixon’s group decided to test whether calpain’s effects on tau was sufficient to drive neurodegenerative disease. 

Calpains Sweat the Small Stuff.
A mixture of small and large-diameter motor neuron axons line lumbar cross sections in normal mice (left). In mice expressing mutant tau (center), small axons have degenerated, but they remain unscathed in mice that also express calpastatin (right). Image courtesy of Rao et al., 2014, Journal of Neuroscience.

First author Mala Rao and colleagues studied JNLP3 mice, a model of FTLD. These overexpress a single copy of human tau with the P301L mutation and start developing symptoms of neurodegeneration—including motor deficits—as they age. They die about three months earlier than normal mice. Armed with a battery of tau antibodies that recognize different species of the protein, the researchers western-blotted brain extracts and looked at cortical sections with immunofluorescence. They found that the mutant mice expressed high levels of several species of tau that may cause neurotoxicity, including small fragments and larger oligomers and hyperphosphorylated oligomers. Using an antibody that recognizes the active form of calpain II, the researchers stained cortical sections and found higher protease activity in the brains of JNLP3 mice than in wild-type. Conversely, the level of calpastatin paled in comparison to that in normal mice. The researchers also identified high levels of a calpain cleavage product, the constitutively active p25 subunit of cdk5. This kinase is known to hyperphosphorylate tau. Levels of hyperphosphorylated tau were far higher in the mutant mice. 

To determine whether calpain activity was responsible for the potentially toxic tau species, the researchers crossed the JNLP3 animals with mice expressing high levels of calpastatin. Strikingly, the double transgenics had fewer tau fragments and hyperphosphorylated oligomers. The researchers also observed a halving in the number of neurons in the cortex and pons that react with an antibody against neurofibrillary tangles.

Could this reduction in myriad tau forms affect the course of disease? The double transgenic mice lived nearly as long as wild-type. However, the animals still succumbed to the disease, albeit later than the JNPL3 mice. While the JNLP3 mice performed normally on a maze test that measures hippocampal memory, they started slipping in their ability to build nests—an activity that involves many parts of the brain—at 17 months of age. The double transgenic mice, on the other hand, still still built proper nests at that time, and showed less decline with age than the JNLP3 mice did. At 23 months of age—when small-caliber motor neuron axons wither in JNLP3 mice and their motor control wanes—mice that overexpressed calpastatin boasted a full complement of such axons and moved normally.

Nixon hypothesized that mutant tau somehow triggers elevated calcium levels, which turns on calpain activity. Calpains then tamp down levels of calpastatin by activating caspases, which have been reported to cleave the inhibitor (see Wang et al., 1998). Overzealous calpain then modifies tau directly, by cleaving it into fragments, and indirectly, by activating cdk5, which hyperphosphorylates tau. “These combined activities generate the full range of tau pathologic species speculated to be neurotoxic,” Nixon said.

Peter Davies of Albert Einstein College of Medicine in New York liked the paper, with one reservation: “Perhaps the only issue I see is that the authors choose to use a very mild pathology model, the heterozygous P301L mouse,” he wrote in an email to Alzforum. While the survival boost from calpain inhibition was significant, it did not occur in more aggressive models of neurodegeneration (see full comment below). Nixon argued that the mouse better models human disease. “Even though it takes longer to study than the homozygous P301L or other similarly aggressive models, we are more closely mimicking the human condition,” he said.

Nixon imagines that calpains could play a role in any neurodegenerative disease that involves elevated levels of calcium, as the proteases have been reported to wreak havoc in tau-independent ways as well. Efforts to develop calpain inhibitor therapies have been stymied by lack of specificity and an uncertainty about the pathological mechanisms the proteases mediated. AbbVie of North Chicago is testing a small molecule calpain inhibitor in a Phase 1 trial for AD.—Jessica Shugart

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Comments on News and Primary Papers

  1. This is a nice paper, and perhaps the only issue I see is that the authors choose to use a very mild pathology model, the heterozygous P301L mouse. The effects of calpain inhibition were significant in this model, but I would argue that to compare the effectiveness of inhibition with that reported by other groups who used the homozygous model (more common) is pushing matters a little.

    As regards therapy, one wonders how a lifetime of expression of an anti-tau antibody would do compared to lifelong expression of calpastatin.

    View all comments by Peter Davies
  2. This is a nice article. One could ask why calpastatin is downregulated. Since proteasome inhibition may modulate calpain activity, what is the status of the proteasome in JNLP3 mice?

    References:

    . Proteasome inhibition and Tau proteolysis: an unexpected regulation. FEBS Lett. 2005 Jan 3;579(1):1-5. PubMed.

  3. The work by Rao and colleagues highlights the importance of proteolytic dynamics in neurodegeneration, placing the calcium-dependent protease, calpain, in the center of the universe. Evidence for calpain involvement in neurodegeneration has grown in the last 20 years as further demonstrated in this P301L tauopathy model. This timely research builds a strong case for deeper investigation of how tau proteolysis contributes to the neurodegenerative process, including as it relates to AD. While other proteases are involved in tau processing, calpains indeed play a central role and the present work provides support for selective targeting of calpains to attenuate tau-centric neurodegeneration. One hurdle to overcome in the development of a calpain strategy will be to allow normal calpain activity to take place. Like virtually all enzymes linked to disease, calpain has a physiological role to play and potent inhibition has potentially negative consequences. One of the beauties of the calpain protease is that the highly active form detected in this study is more closely associated with pathology. Therefore, targeting this form may provide a unique opportunity to decrease the pathological actions while maintaining the physiological function. As noted by Rao et al., targeting calpains has been a challenge, but I believe it can be overcome.

    View all comments by Rodney Guttmann

References

News Citations

  1. Enzyme Essential to Brain Development Found to Hyperphosphorylate Tau, Kill Neurons
  2. Target Practice: A Trio of Papers to Ponder for Potential Therapies

Research Models Citations

  1. JNPL3(P301L)

Paper Citations

  1. . Widespread activation of calcium-activated neutral proteinase (calpain) in the brain in Alzheimer disease: a potential molecular basis for neuronal degeneration. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2628-32. PubMed.
  2. . Marked calpastatin (CAST) depletion in Alzheimer's disease accelerates cytoskeleton disruption and neurodegeneration: neuroprotection by CAST overexpression. J Neurosci. 2008 Nov 19;28(47):12241-54. PubMed.
  3. . Calpain Inhibitor A-705253 Mitigates Alzheimer's Disease-Like Pathology and Cognitive Decline in Aged 3xTgAD Mice. Am J Pathol. 2012 Aug;181(2):616-25. PubMed.
  4. . Overexpression of the calpain-specific inhibitor calpastatin reduces human alpha-Synuclein processing, aggregation and synaptic impairment in [A30P]αSyn transgenic mice. Hum Mol Genet. 2014 Aug 1;23(15):3975-89. Epub 2014 Mar 11 PubMed.
  5. . Caspase-mediated fragmentation of calpain inhibitor protein calpastatin during apoptosis. Arch Biochem Biophys. 1998 Aug 15;356(2):187-96. PubMed.

Further Reading

Primary Papers

  1. . Specific calpain inhibition by calpastatin prevents tauopathy and neurodegeneration and restores normal lifespan in tau P301L mice. J Neurosci. 2014 Jul 9;34(28):9222-34. PubMed.