February 11, 2017
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| Susan Lindquist passed away October 27, 2016. [Courtesy of Ceal Capistrano /Whitehead Institute.] |
For the many genes and proteins involved in
Parkinson’s disease, it’s a small world. Basically, they are all at least
friends of friends. Leveraging connections between yeast genes to infer links
between human genes, researchers led by the late and beloved Susan Lindquist of
the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts,
have just made that clear. Capping Lindquist’s research focus of the past five
years of her life, her associates in academia and biotech have generated an extensive
interaction map of genetic players in various synucleinopathies using
TransposeNet, a computational algorithm they devised to “humanize” the yeast
interactome. This bird’s-eye view, published January 25 in Cell Systems,
revealed links between multiple parkinsonian risk factors and biological
processes already projected to drive the disease. In particular, the map
highlighted the importance of vesicular trafficking as well as mRNA processing
and translation in α-synuclein toxicity. In a companion study from Lindquist’s
lab, the researchers added a spatial dimension to these genetic connections by
uncovering more than 200 proteins that brush shoulders with α-synuclein in the
cell. They hope that TransposeNet and the physical interaction methods they developed
can be used to identify other disease-relevant interactomes.
“This duet of papers represents the swan song of one
of the deepest thinkers into the fundamental biology of neurodegeneration, our
recently departed friend Sue Lindquist, who sadly died a few months ago,”
commented Gregory Petsko of Weill Cornell Medical College and Scott Small of
Columbia University in New York, in a joint comment to Alzforum. “The papers
illustrate the magnificent creativeness and scope of her life’s work,
integrating yeast, genomic, and computational biology with symphonic
elegance.” Lindquist succumbed to cancer at age 67 (see New York Times
obituary).
“This body of work meant a good deal to Susan, because
it provided a glimpse of how basic questions she had worked on for decades
might play out one day to make a difference in the clinic,” former Whitehead
researchers and co-authors on the current papers Vikram Khurana, Chee Yeun
Chung, and Daniel Tardiff wrote in an accompanying tribute to their late
mentor. “A basic biologist to the core, Susan was also a deeply engaged and
empathic human being. It is thus not surprising that driving basic biological
insights for the betterment of patients and humanity became an all-consuming
goal for her in the latter part of her career.”
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Yeast screens pinpoint genes involved in α-synuclein toxicity; after that, intermeshed yeast and human interaction maps display how disease processes connect. [Courtesy of Khurana et al., Cell Systems, 2017.]
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The comprehensive interaction maps may provide a
biological framework in which to place rare genetic variants discovered in
patients, said Khurana, the first and co-corresponding author, who now heads a
lab at Brigham and Women’s Hospital, Boston. A greater understanding of how
different genetic lesions relate to mechanisms that drive the disease may then
pave the way for personalized therapies, he added.
Parkinson’s disease (PD) is the most common
synucleinopathy; however, aggregates of α-synuclein crop up in a slew of
disorders featuring parkinsonian symptoms, including dementia with Lewy bodies
and multiple system atrophy, and indeed many cases of Alzheimer’s. While
mutations or multiplications of the α-synuclein gene by itself cause
α-synuclein pathology and PD, many other genetic culprits—both common and
rare—have been identified that either cause or raise the risk of PD-like disorders,
some unrelated to α-synuclein pathology (see Jul 2014 news). Understanding
the connections between these diverse genetic factors and how they relate to
α-synuclein aggregation will be key to finding effective treatments,
Khurana said.
Genome-wide screens are a powerful way to find factors
that enhance or suppress α-synuclein toxicity, and the humble yeast, with its
supreme genetic malleability, offers a ready system in which to conduct these
screens. Lindquist spearheaded this approach long ago, implicating protein
trafficking as a driver of toxicity (see Dec 2003 news). Since
then, Lindquist’s and other labs have used the screens to pin genetic modifiers
of Aβ, TDP-43, and α-synuclein toxicity (see Oct 2011 news on
Treusch et al.,
2011; Apr 2008 news; Yeger-Lotem et
al., 2009). Next, the researchers used the yeast genetic windfall to
explore the roles of these genes in human disease and uncover other genes that
may be involved (see Oct 2013 news; Dec 2013 news;
Mar 2014
conference news; Aug 2010 news).
Alas, relying on yeast to understand neurodegeneration has its limitations.
Besides the obvious—yeast have no brains—there is the fact that there are no
yeast homologs for human PD genes such as α-synuclein and LRRK2.
Bridging the Gap
In the current study, co-first authors Khurana, Jian
Peng, now at the University of Illinois in Urbana-Champaign, and Chung, now at
Yumanity Therapeutics in Cambridge, sought to flesh out the information they
gathered from yeast screens and unearth the genes’ deeper context in humans.
First, they expanded the screening in yeast. Previously, the researchers had
overexpressed each gene of the yeast genome in yeast cells expressing
α-synuclein, and uncovered 77 modifiers of α-synuclein toxicity. This time,
they added a deletion screen, to identify genes that normally suppress
α-synuclein toxicity, and a “pooled” overexpression screen, to test various
genes simultaneously. Pooling makes screening more efficient so subtle
modifiers can be detected, too. Together, these screens yielded 332 genes that
either suppressed or enhanced α-synuclein toxicity in yeast.
To “humanize” this list, the researchers teamed up
with computational biologists Peng and co-corresponding authors Bonnie Berger
of the Harvard Stem Cell Institute, and Ernest Fraenkel of Massachusetts
Institute of Technology to develop TransposeNet. To find human homologs of a
given yeast gene, the algorithm relied on sequence and structural similarities,
and also on similarities in known interaction partners. Applying TransposeNet
to the whole yeast proteome assigned 4,923 yeast proteins to human homologs,
and conversely predicted yeast homologs for 15,200 human proteins. The
researchers used this rubric to convert their 332 yeast proteins into human
homologs, then generated an interaction network between those.
Finally, because
far fewer connections between human proteins are known compared to the wealth
of available yeast interaction data, the researchers used the yeast proteome to
fill in missing connections in the sparse human one. “It’s like giving the
human proteome a yeast roadmap,” Khurana told Alzforum. The network approach
allowed the researchers to visualize connections not just among their original
screen hits, but between human proteins that might have no yeast homolog.
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| Functional Cliques. These color-coded biological processes influence the toxicity of α-synuclein. [Courtesy of Khurana et al., Cell Systems 2017.] |
Lo and behold, the resulting genome-scale map of α-synuclein
toxicity modifiers included several genes previously implicated in PD and other
neurodegenerative diseases. Vesicular trafficking stood out as the most
prominent biological process in the network, and several genes associated with
typical PD, such as α-synuclein, LRRK2, and VPS35, were connected within these
vesicle trafficking hubs. Proteins involved in ER-to-Golgi trafficking and ER
quality control connected the two best-known PD genes, LRRK2 and α-synuclein.
Importantly, neither of these genes popped up in the network without
incorporating the yeast interactome to bolster the human one.
Other key members of this endosomal trafficking clique
included the yeast homologs of these human genes:
RAB7L1, an endosomal trafficking gene and leading candidate
for the PARK16 locus;
VPS35, aka PARK17, a member of the retromer complex
and a causal PD gene;
SYNJ1, aka PARK20, which also plays a role in
Golgi-to-endosome trafficking;
SORL1, an AD risk factor.
Deleting any of these genes in yeast that express
normal, otherwise non-toxic levels of α-synuclein triggered growth defects.
VPS35 harboring the PD-causing mutation D620N was unable to rescue deletion of
the wild-type gene. Together, these findings provided support for the
biological significance of endosomal trafficking, specifically retrograde
transport, in PD and related disorders.
Calcium signaling genes, including calcineurin and
NFAT, made an appearance smack dab in the heart of the interaction map, forming
connections with several disparate biological processes. For example,
calcineurin interacted with vesicular transport proteins as well as ATP13A2,
aka PARK9, which encodes a type 5 ATPase implicated in metal ion homeostasis.
Mutations in ATP13A2 cause a rare juvenile form of parkinsonism called
Kufor-Rakeb syndrome, and the researchers found that deleting this gene in
yeast boosted α-synuclein toxicity while overexpression suppressed toxicity.
Interestingly, only three of 77 genes that modified toxicity in α-synuclein
overexpressing cells altered toxicity in ATP13A2 deletion strains, as well. All
three genes play roles in iron and manganese homeostasis, which are disrupted
in Kufor-Rakeb syndrome. This supports the idea that metal ion transport
represents a distinct biological pathway to α-synuclein toxicity, and perhaps
to different forms of PD. Yet another set of 69 genes modified toxicity in
VPS35 deletion strains as well as in α-synuclein overexpressors, indicating
that vesicular trafficking plays a different, and perhaps more important role
in typical forms of PD.
That mRNA translation and processing emerged as
central to α-synuclein toxicity was the most intriguing outcome from the
screens, according to Khurana. The requisite genes include the translation
initiation factors EIF4G, whose role in PD is disputed, and ATNX2, implicated
in ataxia as well as ALS risk, and several ribosomal subunits. In further
support of this, the researchers found defective protein synthesis in HEK cells
overexpressing α-synuclein and in iPSC-derived neurons harboring α-synuclein
with the A53T mutation. These translation problems occurred before the
canonical ER stress response had a chance to kick in, Khurana said.
Overexpressing EIF4G1 or ATXN2 reversed these protein translation defects. The
researchers hypothesize that the involvement of so many factors related to mRNA
processing and translation implies a deep biological connection between
α-synuclein and these processes.
Touched by α-Synuclein
In their second paper, Chung and Khurana, as co-first
and corresponding author, prbrvided support for these and other associations
discovered in the genetic study. Using a technique called ascorbate peroxidase
(APEX) labeling, which was developed by Alice Ting and colleagues at
Massachusetts Institute of Technology, the researchers identified 225 proteins
in close physical proximity to α-synuclein in neurons. Older methods such as
co-immunoprecipitation require lysing of the cell and tend to identify only
stable protein complexes; in contrast, APEX identifies even transient
protein-protein interactions while the cells still live. The researchers
employed the technique by fusing α-synuclein with APEX, which oxidizes phenol
derivatives to phenol radicals. These extremely short-lived radicals then
covalently react with amino acids in their immediate vicinity, allowing
identification of labeled proteins by mass spectrometry.
Chung and colleagues transduced rat primary cortical
neurons with α-synuclein fused to APEX2, a catalytically superior version of
APEX, and then ran mass spec to unveil α-synuclein’s social network. Many of
the 225 proteins the researchers uncovered hailed from α-synuclein’s known
stomping grounds of vesicles and synaptic terminals. Biological processes that
had been well represented in the genetic networks, such as protein transport
and vesicular trafficking, once again made a showing in the APEX assay. In
addition, microtubule-associated proteins, including tau, rubbed shoulders with
α-synuclein, as did proteins involved with mRNA binding, processing, and translation.
Khurana was particularly intrigued by this last group
of interactions. “Synuclein is physically and genetically interacting with
translation and mRNA metabolism factors in a way that is surprising and
distinct from other neurodegenerative disease proteins,” he told Alzforum. “The
two papers together would suggest that there may be a physiological role for
α-synuclein in mRNA metabolism that was not previously appreciated.” He
speculated that perhaps α-synuclein physically associates with translation
factors and sequesters them. This could occur in the synapse, where synuclein
concentrates and local mRNA translation plays a key role in
synaptic plasticity.
Mark Cookson of the National Institutes of Health in
Bethesda, Maryland, was not surprised that translation appears important in
α-synuclein toxicity. He noted two recent studies implicating the PD genes
PINK, Parkin, and LRRK2 in mRNA translation (see Gehrke et al.,
2015; Apr 2014 news). Still,
Cookson favors the idea that α-synuclein and translation are most strongly
linked via malfunctions in vesicle trafficking, which halt translation.
Because the APEX2 map was generated in neurons, it
captured neuronal-specific interactions that the humanized yeast genetic
screens missed. The spatial map featured multiple neuron-specific RAB3 proteins
involved in synaptic vesicle exocytosis. Previous studies had implicated these
proteins as modifiers of α-synuclein toxicity in neurons (see Gitler et al.,
2008; Chung et al.,
2009). The researchers went on to confirm many of the physical
interactions identified in the APEX2 screen using a technique called membrane
yeast two-hybrid. MYTH is a spin-off of classical yeast two-hybrid methods; it
works well for α-synuclein, which is primarily a membrane-associated protein.
Khurana thinks it is reassuring that a number of the
genes and proteins had known ties to PD. “Visualizing how they fit together is
important and makes one wonder how many of the other genes in our network are
related to PD risk,” Khurana said. Two of the genes in the network, calcineurin
and Nedd4, are targets of available drugs that reduced α-synuclein pathology in
mouse models, and he suggested the network approach might one day lead to the
development of therapies aimed at mechanisms driving specific forms of disease
(see Oct 2013 news and
Aug 2014 news).
Cookson especially welcomed the integration of parkinsonism genes into the
APEX2 network. Should treatments beyond dopaminergic therapies pan out someday,
it could help steer patients toward therapies specifically tailored to address
the biological mechanisms that underlie their disease, he said. On the flip
side, the networks also point out that patients with different clinical
presentations may share common mechanisms of disease, such as defects in vesicular
trafficking, he added.
In their tribute to Lindquist, Khurana, Chung, and
Tardiff wrote that they believe in the potential of yeast-to-human approaches,
but were careful not to tout them too heavily until they yield effective
therapies. The three researchers co-founded Yumanity Therapeutics with
Lindquist to translate findings in yeast and human neurons into therapies for
neurodegenerative disease. “We continue to fight the good fight, spurred on by
memories of a transformative woman and the uncommonly beautiful biology she
brought back from a billion years ago,” they wrote.—Jessica Shugart
http://www.alzforum.org/news/research-news/lindquist-leaves-behind-parkinsons-interactome-her-parting-gift
