Genetic variants in the BIN1 gene increase risk for late-onset Alzheimer’s disease. Scientists want to know why, and a paper in the October 18 Cell Reports blames tau. Researchers led by Patrik Verstreken at KU Leuven and Diederik Moechars of Janssen Pharmaceutica in Beerse, both in Belgium, suggest that the concentration of a neuron-specific form of BIN1 falls in AD and that this deficiency enhances the propagation of toxic forms of tau. The BIN1 deficit, they propose, releases the brakes on endocytosis, allowing more aggregates of tau to be taken up by cells and packaged into endosomes. Once there, the tau aggregates perforate the endosomal membrane, escape into the cytoplasm, and corrupt native tau protein to misfold.
“This is beautiful work and a surprising finding,” said Gopal Thinakaran, University of Chicago. Thinakaran explained that up to now, most scientists had believed the opposite—that an increase in BIN1 caused more tau pathology. However, he said the current set of findings is convincing, given that the authors zeroed in on a more relevant isoform of BIN1 and used mammalian cells rather than fruit flies, which were used in many prior studies.
Moechars and colleagues had previously reported higher expression of BIN1 protein in AD brain than in controls (Chapuis et al., 2013). In that study, the researchers also saw that decreasing BIN1 expression reduced tau pathology in fly eyes, so they concluded that more BIN1 was bad news. However, while others later confirmed that a ubiquitous form of BIN1 does tick up in AD, they found that a neuron-specific isoform waned (Holler et al., 2014). This changed the picture.
The neuronal isoform—dubbed BIN1V1—contains a CLAP domain that interacts with clathrin. Since clathrin-mediated endocytosis engulfs material on the cell surface, Verstreken and colleagues wondered if BIN1 might somehow regulate the uptake of tau, and hence its spread from cell to cell. Last year, these authors reported that cell-to-cell contact at synapses helps pathological tau cross between neurons (Calafate et al., 2015).
To investigate the role of BIN1 in tau propagation, first author Sara Calafate manipulated BIN1 expression in two in vitro systems designed for this purpose. In one, she cultured rat hippocampal neurons together with HEK293 cells that spew aggregates of tau P301L tagged with GFP. The neurons also expressed tau P301L, though labeled with hemagglutinin (HA) to differentiate it from the HEK293 tau aggregates. In this way, Calafate could use HA-tau P301L aggregation as an indicator of tau propagation. In the other system, she grew rat neurons that expressed tau P301L in microfluidic devices. These separate cells and growth media into three chambers that allow only axons to pass between them. Calafate added preformed tau fibrils to the first compartment to seed tau aggregation in those cells, and then tracked the spread of pathology into compartments 2 and 3.
In both systems, knocking down BIN1 with shRNA ramped up tau aggregation in recipient neurons. In contrast, overexpressing BIN1V1 in the receiving neurons reduced their accumulation of tau. However, coaxing the cells to overexpress the ubiquitous isoform 9 of BIN1, which lacks a CLAP domain, did not affect tau aggregation. These data suggest an inverse relationship between the levels of brain-specific BIN1 and tau propagation.
To test if clathrin-mediated endocytosis (CME) mediated the spread of tau, the authors examined another player in the process, dynamin. This protein, which also binds BIN1, wraps around the necks of newly forming vesicles to pinch them off from the cell membrane. Inhibiting dynamin slowed down the cell-to-cell transfer of tau. That indicates CME helps tau propagation, but other routes may contribute as well, wrote the authors.
How does this data explain how BIN1 regulates endocytosis of tau? Given that it binds dynamin, the authors tested whether BIN1 might also slow CME. When the authors overexpressed BIN1 in rat neurons, dynamin puncta at the membrane grew, suggesting it was trapped there. The authors interpret this to mean that BIN1 holds dynamin at the necks of vesicles and prevents it from pinching them off. Lowering BIN1 levels did not affect this process. In keeping with this, neurons lacking BIN1 amassed larger endosomes, while those overexpressing the protein sported fewer.
How does all this explain how added aggregates of tau can corrupt native tau inside neurons? Even if loss of BIN1 enhanced endosomal uptake of tau aggregates, they should be either recycled back to the membrane or shuttled off to the lysosome for degradation. When would they come into contact with normal tau in the cytoplasm? The authors wondered if aggregated tau somehow leaked out of the endosomes. Calafate tested this by expressing fluorescently labeled galectin-3 in the cytoplasm of mouse neurons. This protein binds sugars on the inner face of the endosome, and can’t reach them if the endosome membrane is intact. Galectin-3 stayed outside endosomes in control neurons, but once Calafate added tau aggregates to the neurons, galectin-3 lit up their endosomes. This indicates that tau aggregates may disrupt the endosomal membrane to allow proteins to pass through.
All told, the findings may explain BIN1 GWAS hits, Verstreken said, although he cautioned that BIN1 may contribute to AD pathology in other ways, as well. For example, Thinakaran and others have found that most BIN1 in the brain is expressed by oligodendrocytes (De Rossi et al., 2016).
The study also explains one way in which toxic proteins enter the cell, avoid degradation in lysosomes, and corrupt healthy proteins in the cytoplasm, Verstreken told Alzforum. He plans next to assess whether this occurs in vivo, as well, and if it works with the wild-type version of tau. Thinakaran added that it would be useful to see where in the cell BIN1 is located.
The data complement other evidence that different forms of endocytosis are instrumental in tau propagation (Holmes et al., 2013; Wu et al., 2013), wrote the authors. The results also reinforce BIN1 as a risk factor for AD, said other scientists. Claudia Almeida, CEDOC-NOVA Medical School, Lisbon, Portugal, recently reported that the protein affects APP processing by slowing BACE1 trafficking out of endosomes and making these vesicles larger (Nov 2015 news). The new data fit, she said, and suggest that BIN1 contributes to AD in multiple ways. However, she speculated that instead of affecting clathrin-mediated endocytosis, BIN reduction may delay endosomal recycling of aggregated tau back to the extracellular space. That could cause the endosomes to swell. Almeida pointed to a previous study assigning human BIN1 to that part of the trafficking process (Pant et al., 2009).
Jean-Charles Lambert, Institute Pasteur de Lille, INSERM, France, pointed out that BIN1 has not emerged as a risk factor in GWAS of other tauopathies, such as progressive supranuclear palsy and frontotemporal dementia. It would be interesting to test whether the mechanism described here is specific to AD, perhaps exacerbated by Aβ, or if it could occur in other tauopathies or even other proteinopathies, he said.—Gwyneth Dickey Zakaib
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