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.

Lit Up.

When added to cultured neurons, tau aggregates (green) perforate endosomes, giving markers access to the interior (red). [Image courtesy of Cell Reports, Calafate et al.]

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., 2013Wu 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

Comments

  1. It is well accepted that knowledge of the genetic determinants of AD should improve our understanding of the fundamental disease processes. However, when identified, it is often difficult to understand how genetic variants mechanistically contribute to AD pathogenesis. Two methodological possibilities can be developed to pave the way for such understanding: systematic approaches based on medium/high throughput technologies, and hypothesis-driven strategies.

    Calafate et al. chose the latter to determine how BIN1, the second strongest genetic risk factor for AD after APOE, may be involved in the AD processes. Based on the well-characterized function of BIN1 in endocytosis, they proposed, in this elegant paper that describes the use of microfluidic devices, that BIN1 may control tau aggregate entry into the cell though clathrin-dependent endocytic influx. This would lead to propagation of tau pathology from neuron to neuron after aggregates of tau permeabilize the endosome membrane. In this model, underexpression of the neuronal BIN1 isoforms would be deleterious by favoring the internalization of tau aggregates. This hypothesis is in line with the observation that expression of BIN1 may mediate AD genetic risk by modulating tau pathology (Chapuis et al, 2013; Dourlen et al., 2016). Furthermore, it also has been reported that the number of BIN1-positive pyramidal neurons in the CA1 of the hippocampus correlated with hippocampal neuritic plaque scores in AD patients as judged by CERAD criteria (Adams et al., 2016). However, it is important to note that this tau propagation is just one way BIN1 genetic variants might influence AD pathogeneis. There are others:

    (i) BIN1 has been shown to directly interact with tau in a phosphorylation-dependent manner in rat neurons (Sottejeau et al., 2016). From this observation, and additional results, we postulated that the BIN1-tau complex could be at the interface between the actin and microtubule network and that its deregulation may impact neuronal function (Sottejeau et al., 2016).

    (ii) BIN1 has been observed to be mainly expressed in oligodendrocytes (Adams, et al., 2016; De Rossi et al., 2016). This led De Rossi et al. to propose that BIN1 may deregulate myelination.

    (iii) BIN1 has been reported to increase cellular BACE1 levels through impaired endosomal trafficking and reduced BACE1 lysosomal degradation, resulting in increased Aβ production (Myagawa et al., 2016).

    Importantly, depending on the hypothesis, BIN1 overexpression may be deleterious (as proposed by Chapuis et al.) or protective (as suggested by Calafate et al.). However, there is still no consensus about the potential over- or underexpression of BIN1 in the brain even if genetic variants associated with increased AD risk have been linked to a potential overexpression of this gene in vivo and in vitro (Chapuis et al., 2013).

    In conclusion, the physiological and pathophysiological roles of BIN1 have to be better dissected since there is still little known about BIN1 in the brain. BIN1 has been mainly studied in the context of its functions in the muscle and related muscular pathology, i.e., myotonic dystrophy (Fugier et al., 2011). Even though the tau propagation hypothesis is interesting, it is important to keep in mind that several GWAS have been published on tauopathies such as Parkinson’s disease, progressive supranuclear palsy, and frontotemporal dementia. Until now, none of them reported variants at the BIN1 locus that reached genome-wide significance. This could imply that BIN1 might be involved in an AD-specific tau pathology, for example linking amyloid and tau. That is why it would be of interest to assess whether the general mechanism described by Calafate et al., is specific to AD (but potentially exacerbated by Aβ peptide), or can take place in other tauopathies or even in other proteinopathies involving neuron-to-neuron propagation.

    References:

    . Subcellular Changes in Bridging Integrator 1 Protein Expression in the Cerebral Cortex During the Progression of Alzheimer Disease Pathology. J Neuropathol Exp Neurol. 2016 Aug;75(8):779-790. Epub 2016 Jun 26 PubMed.

    . Increased expression of BIN1 mediates Alzheimer genetic risk by modulating tau pathology. Mol Psychiatry. 2013 Feb 12; PubMed.

    . Predominant expression of Alzheimer's disease-associated BIN1 in mature oligodendrocytes and localization to white matter tracts. Mol Neurodegener. 2016 Aug 3;11(1):59. PubMed.

    . Functional screening of Alzheimer risk loci identifies PTK2B as an in vivo modulator and early marker of Tau pathology. Mol Psychiatry. 2016 Apr 26; PubMed.

    . Misregulated alternative splicing of BIN1 is associated with T tubule alterations and muscle weakness in myotonic dystrophy. Nat Med. 2011 Jun;17(6):720-5. Epub 2011 May 29 PubMed.

    . BIN1 regulates BACE1 intracellular trafficking and amyloid-β production. Hum Mol Genet. 2016 May 14; PubMed.

    . Tau phosphorylation regulates the interaction between BIN1's SH3 domain and Tau's proline-rich domain. Acta Neuropathol Commun. 2015 Sep 23;3:58. PubMed.

  2. This study is a very interesting data set, one of the few that deals with potential mechanisms of tau propagation.

    BIN1 is the second-most-prevalent susceptibility gene for AD. It has been shown in a few studies in the past years that BIN1 seems to influence tau pathology (Chapuis et al., 2013). Also, strong evidence linked BIN1 as an active protein in membrane processes such as membrane curvature or clathrin-mediated endocytosis, particularly thanks to structural properties such as amphipathic helix/presence of an SH3 domain, presence of a CLAP domain, etc. These data lead the authors to hypothesize that BIN1 may be implicated in the propagation of tau proteins. In my opinion, they demonstrate this nicely, particularly showing that BIN1 overexpression reduces tau propagation while lowering intracellular levels of BIN1 increases tau propagation. The authors provide some mechanistic evidence, particularly showing that the CLAP domain is implicated in the observed effect or that this effect is probably mediated via an interaction of BIN1 with dynamin.

    We regret that no information is provided on three things: First, it is unclear from the charts provided whether the effect on tau propagation is total and if BIN1 is required in the tau propagation process or in the tau uptake via CME. Second, no information is provided on whether BIN1 directly impacts tau aggregation in their model in addition to CME. This data would be of interest as their models address tau transfer and seeding. Third, in their models the authors only see propagation of aggregates. What about propagation of soluble forms of tau, which has been demonstrated in many studies, including in vivo?

    Overall, this paper provides very interesting insights on tau propagation and now needs to be confirmed by other studies and in other models. In vivo data would be particularly interesting.

    References:

    . Increased expression of BIN1 mediates Alzheimer genetic risk by modulating tau pathology. Mol Psychiatry. 2013 Feb 12; PubMed.

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References

Alzpedia Citations

  1. Bridging integrator 1 (BIN1)

News Citations

  1. Alzheimer’s GWAS Hits Point to Endosomes, Synapses

Paper Citations

  1. . Increased expression of BIN1 mediates Alzheimer genetic risk by modulating tau pathology. Mol Psychiatry. 2013 Feb 12; PubMed.
  2. . Bridging integrator 1 (BIN1) protein expression increases in the Alzheimer's disease brain and correlates with neurofibrillary tangle pathology. J Alzheimers Dis. 2014;42(4):1221-7. PubMed.
  3. . Synaptic Contacts Enhance Cell-to-Cell Tau Pathology Propagation. Cell Rep. 2015 May 26;11(8):1176-83. Epub 2015 May 14 PubMed.
  4. . Predominant expression of Alzheimer's disease-associated BIN1 in mature oligodendrocytes and localization to white matter tracts. Mol Neurodegener. 2016 Aug 3;11(1):59. PubMed.
  5. . Heparan sulfate proteoglycans mediate internalization and propagation of specific proteopathic seeds. Proc Natl Acad Sci U S A. 2013 Aug 13;110(33):E3138-47. Epub 2013 Jul 29 PubMed.
  6. . Small Misfolded Tau Species Are Internalized via Bulk Endocytosis and Anterogradely and Retrogradely Transported in Neurons. J Biol Chem. 2013 Jan 18;288(3):1856-70. PubMed.
  7. . AMPH-1/Amphiphysin/Bin1 functions with RME-1/Ehd1 in endocytic recycling. Nat Cell Biol. 2009 Dec;11(12):1399-410. Epub 2009 Nov 15 PubMed.

Further Reading

Papers

  1. . Tau phosphorylation regulates the interaction between BIN1's SH3 domain and Tau's proline-rich domain. Acta Neuropathol Commun. 2015 Sep 23;3:58. PubMed.
  2. . Subcellular Changes in Bridging Integrator 1 Protein Expression in the Cerebral Cortex During the Progression of Alzheimer Disease Pathology. J Neuropathol Exp Neurol. 2016 Aug;75(8):779-790. Epub 2016 Jun 26 PubMed.
  3. . BIN1 regulates BACE1 intracellular trafficking and amyloid-β production. Hum Mol Genet. 2016 May 14; PubMed.

Primary Papers

  1. . Loss of Bin1 Promotes the Propagation of Tau Pathology. Cell Rep. 2016 Oct 18;17(4):931-940. PubMed.