Being a segregase, it’s the job of valosin-containing protein (VCP) to straighten out a myriad of poorly folded proteins. We can now add tau to that list. In a paper published in Science on October 1, researchers led by Edward Lee at the University of Pennsylvania, Philadelphia, report that a rare mutation in the VCP gene causes an autosomal-dominant form of frontotemporal dementia. Autopsy samples—and even a brain biopsy—from carriers of the D395G mutation revealed neurons riddled with vacuoles, and also with neurofibrillary tangles of tau that appear to be much like those found in Alzheimer’s disease. The researchers christened this neuropathology “vacuolar tauopathy.” Biochemical studies, as well as findings from mutant knock-in mice, indicated that the mutation derails VCP’s “disaggregase” activity, allowing a build-up of tangles.
- The pAsp395Gly mutation in VCP causes autosomal-dominant FTD.
- The mutation disrupts its ability to break up tau aggregates.
- Carriers accumulate endocytic vacuoles and tangles in their brains.
“The identification of a new mutation in a chaperone protein that causes a novel tauopathy is very exciting,” commented Laura Blair of the University of South Florida in Tampa. “Links between VCP and tau have been made over the last 10 years by a number of groups, and these data significantly strengthen this connection.”
Manuela Neumann of the German Center for Neurodegenerative Diseases in Tübingen thinks that while this VCP mutation might be very rare, the realization that VCP is a tau disaggregase could have implications for other tauopathies.
As an ATPase Associated with diverse cellular Activities (AAA+), VCP harnesses the power of ATP hydrolysis to perform diverse functions. It yanks proteins out of membranes, sorts them for interaction with other proteins, and unfolds them to hasten their destruction by proteases (for review, see van den Boom and Meyer, 2018). When VCP malfunctions, numerous biological pathways get disrupted (Abisambra et al., 2013; Mar 2013 news).
Mutations in VCP have been implicated in a range of diseases, including multisystem proteinopathy (MSP), which afflicts bone, muscle, and neurons; amyotrophic lateral sclerosis; and frontotemporal dementia (Watts et al., 2004; Dec 2010 news; Mar 2013 news). In ALS/FTD, inclusions of TDP-43 and ubiquitin are the predominant underlying pathology associated with VCP variants (Neumann et al., 2007).
That was what Lee, a neuropathologist, expected to find when he autopsied the brain of a VCP mutation carrier. Instead, he found something entirely different: neurofibrillary tangles of tau, and neurons overloaded with vacuoles. Nary a TDP-43 aggregate.
It turned out that this particular mutation meddles with VCP in a different way than do its other known variants. Co-first authors Nabil Darwich and Jessica Phan and colleagues found the D395G variant in three siblings who had behavioral-variant FTD. One unaffected parent did not carry the mutation, and the other, for whom genetic data was not available, had died around the age of 65 with a diagnosis of FTD. The aspartate-to-glycine swap appeared to be inherited in an autosomal-dominant fashion; all carriers developed FTD even though only two generations are available in this pedigree. The D395G mutation was absent from several large genomic databases, including gnomAD, the Alzheimer’s Disease Genetics Consortium, and the 1000 Genomes Project, suggesting that it is exceedingly rare. The researchers had been following the family for several years when one sibling died at age 55.
Alzheimer-Like Tau. Antibodies to multiple forms of tau present in AD bound inclusions in the brain of a DG-VCP mutation carrier with FTD. Electron microscopy (bottom right) revealed a tau fibril. [Courtesy of Darwich et al., Science, 2020.]
Upon autopsy, the researchers found tau tangles that resembled those seen in AD. Immunohistochemistry, biochemical studies, and electron microscopy indicated that the tangles consisted of paired helical filaments of phosphorylated 3R and 4R isoforms of tau (see image above).
However, unlike in AD, and much like in FTD, the carrier’s tangle burden was highest in the frontal cortex. Some also appeared in the temporal and parietal cortices. Their distribution correlated strongly with neuronal loss, which was consistent with the typical pattern for frontotemporal lobar degeneration. Gliosis also tracked with the distribution of tangles and neurodegeneration. Subsequently, a tau-PET scan of one of the living affected siblings revealed tau tangles that co-localized with atrophy detected by MRI.
Where were the vacuoles? Oddly, these seemingly empty spaces had opened in brain regions mostly spared from tangles, for example in the occipital neocortex. The vacuoles crowded near neuronal membranes and expressed the marker EEA-1 on their membranes, suggesting they were endocytic. Unlike the notorious vacuoles detected in prion disease, these vacuoles did not coincide with regions ravaged by neurodegeneration.
Lee told Alzforum that he does not know what causes this vacuolization. He suspects it could arise from a malfunction in sorting endosomes, since VCP is known to play a role in this process via its interaction with EEA-1. In the brain of the mutation carrier, VCP did not associate with vacuoles. The findings suggested that the VCP mutation could lead to vacuolization and tauopathy via two distinct mechanisms.
As Lee’s group was writing their paper, a case of FTD with the same VCP mutation emerged from a genetic study in Greece co-led by Sokratis Papageorgiou (Ramos et al., 2019). Lee reached out to the Greek researchers, who had the patient’s family medical history. There, too, the mutation was associated with what appeared to be an autosomal-dominant pattern of inheritance, over three generations. It was absent in the proband’s two unaffected siblings, and though genetic data was unavailable on the parents or grandparents, one parent and one grandparent had died with dementia with FTD-like behavioral symptoms around age 50.
An MRI scan revealed a pattern of hyperintensity outlining cortical gyri, called cortical ribboning. Because this is a known feature of a vacuolized cortex and can flag prion disease, the Greek patient underwent a frontal biopsy. The researchers shared some of the sample with Lee, who confirmed the tissue was laden with tau tangles.
How might this mutation affect VCP function? D395 lies within the D1 ATPase domain (see below), and in silico analysis indicated the aspartate-to-glycine swap would destabilize it. Indeed, in vitro, recombinant D395G VCP had one-third less ATPase activity than normal VCP. In contrast, another VCP mutation known to cause MSP and ALS/FTD had the opposite effect, raising ATPase activity. This gain of function has been documented for several VCP mutations that cause MSP (Manno et al., 2010; Blythe et al., 2019).
How might this loss of a VCP function goad tau pathology? A clue emerged from postmortem study. In brain samples from a person with AD, their wild-type VCP mingled with aggregated tau in dystrophic neurites around plaques. In samples from an FTD patient with the DG-VCP mutation, mutated VCP steered clear of aggregated tau. Perhaps wild-type VCP serves as a disaggregase, unfolding tangles? In AD, this activity may ultimately be insufficient to suppress tangles, the scientists believe.
Subsequent biochemical experiments supported this hunch. The researchers mixed insoluble tau aggregates extracted from an AD brain with recombinant wild-type VCP, and its co-factors UFD1L and NPLOC4, and measured thioflavin S fluorescence to gauge subsequent changes to tau fibrillization. When ATP was added to the reaction, tau fibrillization dropped by a third compared to reactions that included a non-hydrolyzable form of ATP. This suggested that active, wild-type VCP detangled tau fibrils. Even in the presence of ATP, D395G-VCP was a poor tau detangler, leading to about half the drop in tau aggregation as wild-type VCP. In line with previous reports that VCP recognizes poly-ubiquitin-tagged substrates, ubiquitin was required for wild-type VCP’s disaggregase activity. Biosensor cell lines using FRET to report tau aggregation told a similar story.
To learn how the D395G mutation would affect tau aggregation in the brain, the scientists used CRISPR gene editing to generate knock-in mice expressing one or two copies of D395G-VCP in place of their normal gene. VCP and tau levels in the knock-ins were on par with wild-type controls, and they had no overt neurodevelopmental problems or neurodegeneration. They also had no tau tangles, in keeping with the idea that VCP does not initiate tau aggregation, but untangles it. Since mouse tau does not fibrillize spontaneously, the researchers had to introduce human pathological tau to test the effect of the mutation on detangling. They injected tau aggregates extracted from AD brain into the dorsal hippocampi and overlying cortices of both wild-type and knock-ins. Three months later, hyperphosphorylated, aggregated tau had crept into neuroanatomically connected brain regions. Mice expressing one or two copies of DG-VCP had more severe pathology in several of these regions, including the retromammillary nucleus, entorhinal cortex, and ventral hippocampus.
Together, the data suggested that VCP acts as a tau disaggregase and that D395G thwarts this, promoting tau pathology.
Lee thinks there are other possible consequences. For example, altered VCP trafficking and substrate binding could explain why wild-type VCP associated with tangles in the AD brain, but D395G-VCP did not in the two FTD patients who carry the variant.
The role of VCP in other tauopathies, including AD, is also unclear, said Lee. A previous study found slightly less VCP in AD brain than in controls, as well as a build-up of phosphorylated tau in VCP-deficient cells (Dolan et al., 2011).
“It’s hard to wrap your head around how many things this protein does,” said Lee, complicating it as a potential therapeutic target, he said. If VCP activity could be enhanced to specifically turn over poly-ubiquitinated substrates, it is possible that a workable therapeutic range might be found, he said. If D395G causes tau pathology in humans, why do other VCP mutations that increase its ATPase activity lead to neurodegeneration with TDP-43 pathology? Lee said these mutations could interfere with other VCP functions and/or disrupt specific interactions between VCP and TDP-43.
“I find it particularly surprising that other proteins known to aggregate in a poly-ubiquitinated form, such as α-synuclein or TDP-43, seem not to be affected by this VCP impairment,” wrote Neumann. “While it would have been informative to include preparations of ubiquitinated forms of α-synuclein and TDP-43 in the assays, dissection of the potentially specific mode of action of VCP on tau aggregates will be very interesting in the future,” she wrote.
Rita Guerreiro of the Van Andel Research Institute in Grand Rapids, Michigan, noted that it is rare to see such an in-depth study of one mutation. “This finding clearly indicates the need to fully study different mutations in the same gene and not assume all have the same effects on disease” she wrote (full comment below).—Jessica Shugart
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No Available Further Reading
- Darwich NF, Phan JM, Kim B, Suh E, Papatriantafyllou JD, Changolkar L, Nguyen AT, O'Rourke CM, He Z, Porta S, Gibbons GS, Luk KC, Papageorgiou SG, Grossman M, Massimo L, Irwin DJ, McMillan CT, Nasrallah IM, Toro C, Aguirre GK, Van Deerlin VM, Lee EB. Autosomal dominant VCP hypomorph mutation impairs disaggregation of PHF-tau. Science. 2020 Oct 1; PubMed.