amyotrophic lateral sclerosis (ALS) kills people has long been mysterious. Even
for cases driven by known gene mutations, scientists still don’t have a
complete picture of how those mutations lead to illness.
for want of trying. Long before the Ice
Bucket Challenge, ALS research was a busy field, funded amply relative to the number of patients with the disease. In
fiscal year 2013, it received about $3,000 in NIH funding per patient, compared
to about $100 per patient for Alzheimer’s.
That ALS has
been such a strong draw for researchers and research funds may be due, in part,
to the keen sense of injustice it creates among patients and their loved ones.
No other disease on its scale strikes seemingly healthy people in the prime of
life and kills them so quickly, so implacably.
news is that decades of ALS research, helped by broad advances in research
technology, are showing signs of paying off. Scientists seem to be zeroing in
on the once-elusive mechanisms of the disease, and are starting to design and
test therapies that target those mechanisms.
“We now understand
much more about the pathogenic process than we did ten years ago,” says Don W. Cleveland, a long-time ALS researcher at the University of
California—San Diego and member of the Dana Alliance for Brain Initiatives
ALS has been
in medical literature since 1869, but almost all the advances in
understanding it have come since the development of modern molecular biology
techniques in the 1980s.
In 1992, for
example, researchers reported finding that ALS patients’ brain and spinal cord tissue have defects in
the management of glutamate, the principal neurotransmitter that motor neurons
use to signal each other. In particular, the scientists found evidence of reduced
“glutamate transport,” which normally helps keep glutamate concentrations at
safe levels in the synapses of motor neurons. Too much glutamate can kill
neurons via a mechanism called excitotoxicity.
trial soon showed that riluzole, a compound that
(among other things) inhibits glutamate signaling, extended the lives of ALS
patients by a few months on average. Riluzole was approved by the US FDA in
1996 and remains the only officially sanctioned therapy for ALS. However, its benefit
appears to be very modest at best, and other trials of drugs with anti-glutamate effects, including ceftriaxone, have failed in clinical trials with ALS patients.
researchers made another apparent breakthrough in the early 1990s by finding that a small proportion of inherited, “familial” ALS cases are caused by
mutations in SOD1—a
gene that codes for an antioxidant enzyme called superoxide dismutase.
only a single mutant copy of the gene to cause ALS. That fact, and studies in SOD1-mutant mice, soon established that SOD1 mutations cause disease by altering the SOD1
enzyme in a way that somehow makes it toxic to cells.
mechanism of that toxicity hasn’t yet been found. But scientists have shown
that in both “SOD1 mice”
and in humans with SOD1-ALS, clumps
or “inclusions” of the mutant enzyme form within motor neurons in disease-affected parts of the brain and spinal
studying SOD1-ALS eventually found evidence that these clumps of mutant-SOD1 contain
particularly tough aggregates known as amyloids. This suggested that ALS, or at
least SOD1-ALS, might be part of the wider family of amyloid-aggregate diseases
that includes Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and Creutzfeldt-Jakob disease.
have begun to appreciate that the amyloid aggregates in these diseases can be self-propagating: They can make new copies of their
aggregate structures, or enlarge existing copies, by co-opting individual
that these aggregates can spread their corrupted state if they happen to be secreted
from one cell (or released from a cell as it dies) and taken up by another—and
that phenomenon might explain the progress of cell death and symptoms that is
seen in ALS. Indeed, in 2011, researchers in the group of Anne Bertolotti at the Laboratory of Molecular
Biology in Cambridge, UK reported that mutant SOD1 aggregates can spread from cell to cell in
the lab dish.
there was strong opposition, but since then some of the most skeptical people
have seen this phenomenon in their own labs,” says Bertolotti.
Helper cells stop helping, start
SOD1 mice helped provide another important clue to ALS. As Cleveland’s
laboratory found, for example, in studies beginning in the
late 1990s, mutant SOD1 hits helper
cells called astrocytes particularly hard—and thereby harms the motor neurons
that depend on these cells’ support. “We
were able to double the survival time of SOD1 mice after disease onset by
lowering synthesis of mutant SOD1 just in astrocytes,” says Cleveland.
years ago, researchers began to find evidence that astrocytes also drive ALS more directly, by
somehow becoming toxic to nearby motor neurons—possibly by their production of
SOD1 or other aggregates. This evidence initially came from studies of
different types of SOD1-ALS, but in a pivotal finding in 2011, researchers showed that astrocytes from patients with
common, sporadic ALS also have this toxic effect: healthy motor neurons
cultured with them start to die off within days.
another “helper cell” type, are the brain’s resident immune cells—cousins of
the pathogen-gobbling macrophage cells of the ordinary immune system. In the
early 1990s, researchers began noting that microglia in ALS-affected parts of the brain and spine are
activated and surrounded by other signs of inflammation. Since then, studies
have found that these inflamed microglia are not just there to clean up the
damage caused by the disease, but, like astrocytes, directly worsen the disease process. Exactly how they do that isn’t yet clear,
but there is already some evidence from ALS mouse models that one can block their harmful influence and slow the disease process significantly.
In 2012 came
a report that oligodendrocytes, helper cells
that build and maintain the myelin-protein insulation around neuronal axons,
have a similar adverse role in ALS. Scientists found that oligodendrocytes in a
commonly used type of SOD1 mouse die early in the disease course, and new
replacement cells fail to mature normally—leading to a progressive loss of
myelin insulation from the axons of motor neurons. The researchers found similar
changes, including the loss of myelin from axons, in autopsied tissue from familial
and sporadic ALS patients.
Affected motor neurons are big and
One of the
central questions about ALS has always been: why does it kill motor neurons (and
nearby helper cells, we now know) but have much less—albeit some—effect on other types of neuron?
A likely factor
is that the motor neurons most affected by the disease are gigantic in
comparison with most other neurons—indeed with most other cell types. “They are
5,000 times the volume of a typical human cell,” says Cleveland.
larger means having much greater needs for energy as well as general repair and
maintenance systems. It also likely means having less leeway when those systems
recent years, as ALS research has focused increasingly on protein aggregation,
scientists have found that this is a factor against which motor neurons lack
strong defenses. “Although they’re enormous, these motor neurons seem to have
relatively poor mechanisms for dealing with protein aggregates and associated
stresses,” says Cleveland.
course like almost all other neurons in mammals, motor neurons are virtually
irreplaceable once they have matured and formed connections—connections that in
some cases extend for more than a meter, from spine to feet.
percent of ALS cases do not feature SOD1
gene mutations or SOD1-containing inclusions in motor neurons and support
cells. Yet virtually all these non-SOD1 cases do feature inclusions of some
type of aggregate. In 2006, University of Pennsylvania researchers John Q. Trojanowski
and Virginia Lee, both DABI members, reported in Science that they and their colleagues had
identified the major constituent of these non-SOD1 aggregates: a protein called
studies, the Trojanowski/Lee laboratory and others found these TDP-43
aggregates in the vast majority of ALS cases examined. Moreover, in 2008 researchers
made a key finding
that mutations in the TDP-43 gene account for some cases of familial ALS—as
well as some cases that had been deemed sporadic.
that TDP-43 pathology is almost always found in areas of degeneration, and the
fact that TDP-43 mutations can cause ALS, strongly suggest that TDP-43 is
central to ALS,” Trojanowksi says.
How could TDP-43
drive ALS? The protein’s putative cell-harming role, and its normal function in
cells, have both been hard to study, in part because of early difficulties
in creating mice that have no TDP-43 (the mice die) or that successfully model
ALS-like TDP-43 pathology.
know that normal TDP-43 has its primary role in the cell nucleus, where it
binds to both DNA and RNA, and—broadly speaking—helps in the handling of
RNA-based gene transcripts, which will often go on to be translated into
proteins. In ALS, however, TDP-43 proteins in affected cells tend to be found
not in the nucleus but instead in the main compartment (cytoplasm) of the cell,
where they form abnormal, granular or threadlike aggregates.
“The loss of
TDP-43’s nuclear functions when it is trapped in the cytoplasm leads to changes
in the levels and the splicing patterns of about 1,500 RNAs, which can’t be
good—in fact we know that chronic loss of TDP-43 from the nucleus is lethal to
mice,” says Cleveland.
possible too,” says Trojanowski, “that TDP-43 aggregates become toxic by, for
example, blocking molecular traffic within neurons and displacing organelles.”
some evidence that TDP-43, like mutant SOD1 and other neurodegenerative disease
proteins, can form amyloid-type aggregates, which may then spread from cell to
cell, thus helping to explain the typical progression of neuronal
loss and symptoms in ALS. Work by Trojanowski and
colleagues suggests that TDP-43 only rarely forms large, fibrous, insoluble
amyloids like those seen in Alzheimer’s and Parkinson’s. But other researchers
have reported that TDP-43 can form smaller, soluble
aggregates (oligomers), which may be more relevant to the disease
notes that many labs now are trying to establish that this aggregate-mediated
spread really does happen in ALS. “It would astonish me if it weren’t
occurring,” he says.
question for TDP-43 research is whether the protein causes neuronal death
mainly by moving from its usual functional location in the nucleus, or alternatively
by acquiring some toxic property, involving aggregates or not, when it gathers
in the cytoplasm.
seem tantalizingly close to being able to resolve this question. Lee, Trojanowski, and their colleagues recently developed a mouse model
in which human TDP-43 is produced in brain and spinal cord neurons, in a form
that prevents it from localizing to the nucleus. Both the human TDP-43 and the
mice’s own TDP-43 drift out into the cytoplasm, with the result that the mice develop
a fast-moving degenerative condition remarkably like ALS, including TDP-43
inclusions and muscle weakness. However, their disease essentially stops
progressing if the human TDP-43 production is shut off and mouse TDP-43 returns
to its normal location in the cell nucleus.
In 2009, two
teams of researchers reported that mutations in a gene called FUS are yet another cause of ALS. As in
the case of TDP-43, the protein encoded by the FUS gene had been studied hardly at all, but was known as an
RNA-binding protein that normally works in the cell nucleus. And like the mutant
TDP-43 that causes ALS, the mutant FUS protein turned out to be depleted in the
nucleus and aggregated in the cytoplasm. As one of the research teams noted in describing its discovery, “Neuronal cytoplasmic protein aggregation and defective RNA
metabolism thus appear to be common pathogenic mechanisms involved in ALS and
possibly in other neurodegenerative disorders.”
developments in ALS research were now coming more frequently. In October 2011,
another two teams of researchers reported that they had linked many cases of familial ALS
to a particular mutant gene on chromosome 9. Again, almost nothing was known
about the gene in question, called C9ORF72.
But while a normal copy of the gene contains a few dozen repeats of a six-nucleotide
sequence, GGGGCC, the two teams found that people with ALS often have hundreds
of GGGGCC repeats. Genetic repeat expansions are known to underlie many
disorders, including the neurodegenerative Huntington’s disease.
research has found the C9ORF72 repeat expansion in about 40% of familial ALS cases from a
worldwide sample, and 7% of more common, non-familial cases—making it by far the
largest single known cause of ALS. Remarkably, genetic analysis suggests that
the C9ORF72 repeat-expansion arose
only about 1,500 years ago, in Finland, probably in a single individual, and that
it was disseminated to other parts of Europe—perhaps via Viking raids and conquests—and later made its way even to parts of Asia.
how C9ORF72 repeat expansions harm
cells seem broadly consistent with the idea that pathological aggregates and
defective RNA processing drive ALS. For example, in a study published in Nature
last year, Jiou Wang and colleagues at Johns Hopkins
School of Medicine found that the mutant C9ORF72
gene forms abnormal loops of DNA, that it is transcribed into RNA that also
forms abnormal loops, and that these DNA and RNA loops stick together and grab
other gene transcripts and proteins in their vicinity, thus probably preventing
them from functioning normally.
abnormal structures appear to affect cells at many levels, from the
transcription of DNA into RNA, to the translation of RNA into proteins,” says
One of the trapped
proteins they identified as nucleolin, named for its important role in the
nucleolus, where the essential RNA-to-protein-translation machines called
ribosomes are constructed. A subsequent study from the University of Texas Southwestern concluded that
nucleolin is also—or alternatively—bound and trapped by the abnormal protein encoded
by mutant C9ORF72.
are working to determine more precisely and conclusively how the C9ORF72 mutation kills motor neurons and
helper cells. They also want to know why TDP-43 aggregates show up in
“A number of
genetic mutations, involving genes other than TDP-43, culminate in the
production of TDP-43 aggregates or inclusions; and that’s still totally
mysterious,” says Trojanowksi.
An emerging hypothesis
some researchers have a hypothesis for why these aggregates form. The idea is
that there is initially some stress—presumably exacerbated by aging in most
cases—that leads to an “unfolded protein response” in affected cells.
protein response in a cell includes a drastic reduction in protein synthesis
and in the RNA processing that supports that synthesis. This is thought to help
the cell by de-cluttering it and taking the load off systems that handle abnormal
aggregates and other “unfolded” or “misfolded” proteins. But the response,
while it needs to be robust, is apparently meant to be only temporary—among
other reasons because the long-term shutdown of protein production would leave
the cell unable to maintain itself in good repair, and thus in effect would
There is some
evidence that TDP-43, at least, plays a role in this stress response by rapidly aggregating and moving out of the nucleus, in order to facilitate the
shutdown of RNA processing and protein production. It then shows up in the
cytoplasm—in conglomerations of nucleus-originating proteins and RNA that are known
as “stress granules” because they tend to appear during episodes of cell stress.
researchers now suspect that this process, meant to be temporary, lasts longer
than it should in certain situations, such as when the TDP-43 gene is mutated,
or when there are other long term stresses on a cell—from aggregating mutant SOD1,
for example, or from the various derangements caused by mutant C9ORF72, or perhaps even from chronic
inflammation following head injury.
“I would say
that as a consequence of some provoking event, TDP-43 forms these cytoplasmic
stress granules, which then convert from a reversible form by some mechanism
into an irreversible form, so that now you have a cascading disease,” says
A continuum of neurodegenerative
Solving the ALS
mystery will likely provide the keys to other neurodegenerative mysteries. In
fact, the more that scientists study ALS and other neurodegenerative diseases,
the less distinct they appear.
merely share its broad pathological features, such as protein aggregation, with
other neurodegenerative diseases. It also shares at least three key proteins. Lee
and Trojanowski and colleagues reported in 2006 that TDP-43 aggregates are found in many cases of fronto-temporal
lobar degeneration (FTLD), which as the name suggests, kills neurons in the
frontal and temporal lobes.
discovery prompted us to start referring to these diseases as TDP-43-proteinopathies,
even though they were perceived as distinct disorders,” says Trojanowksi.
mutations also were linked to FTLD, as—later—were FUS mutations and aggregates, and C9ORF72 repeat expansions.
cases that initially seem like ALS, TDP-43 aggregates and associated cell death
are often seen in the fronto-temporal lobes and
other parts of the brain, with accompanying clinical signs of cognitive and behavioral problems. About one in five ALS
patients gets a diagnosis of “ALS-FTLD.”
researchers have since found TDP-43 aggregates in association with the
head-injury-related degenerative syndrome Chronic Traumatic Encephalopathy (CTE), which also features tau aggregates. As
Trojanowski suggests, the most important defining factors in these diseases may
not be the clinical signs and symptoms but the abnormal structure-forming
molecules, such as TDP-43, that drive the disease process.
and colleagues have been looking into the possible use of antibodies that grab aggregated (but not
normal) TDP-43, which in principle—even if they only blocked the cell-to-cell
spread of TDP-43 aggregates—would slow the course of ALS as well as TDP-43-linked
FTLD and CTE.
Other laboratories, including Cleveland’s, are using gene-silencing nucleotide or small-protein
strategies to try to reduce the expression of ALS-causing genes and the
production of their harmful protein products, and thereby slow or stop or
prevent the disease. “Already in trial are designer DNA drugs to silence
ALS-causing mutations in the SOD1
gene,” Cleveland says. “We expect the clinical trial of our C9ORF72-targeting therapy to start by
the third or fourth quarter of 2016.”
are targeting the unfolded protein response. Working from the hypothesis that
ALS is caused by an unfolded protein response that
stays “on” too long, shutting down the synthesis of essential proteins, a group
from the University of Pennsylvania, including Trojanowski, reported last year that inhibiting the response with a
small-molecular compound seemed to block or slow the disease process in fly and
cell models of ALS.
On the other
hand, Bertolotti’s laboratory reported in Science this
April that a compound designed to enhance
the protein synthesis shutdown in the unfolded protein response—and thus clear
mutant SOD1 more effectively—successfully protected SOD1 mice from the deaths
of motor neurons as well as physical deficits, and without noticeable
side-effects. The same compound also worked in mice that model a different misfolded-protein
disease, Charcot Marie Tooth 1B. Whether this approach will work in non-SOD1
ALS remains to be seen, but Bertolotti hopes to answer that and other questions
before long. “While there is still much more to do, I am pleased to say that
our work receives a lot of interest from pharma companies and investors who may
help in developing this further,” she says.
approach, now being pursued by several groups, is to inject neural stem or
progenitor cells into the spines of ALS patients, in hopes that they will
mature into neuron-nourishing helper cells that help prevent the loss of motor
neurons. DABI member Eva Feldman of the University of Michigan heads one such group. “To date we
have injected up to 16 million such cells directly to the spinal cords of ALS
patients, and a subset of participants are experiencing a stabilization in the
progression of their disease,” she says. “Our application for a broader
trial is under way and we are cautiously optimistic about future results.”
“It is a
time of optimism,” says Cleveland. “Not optimism for solutions tomorrow
necessarily, but for success in the next decade, at least against ALS caused by
mutant genes such as SOD1 and C9ORF72. For other types of ALS I’m
optimistic that there’ll be a successful or partly successful therapy in my
SOD1 [ital.] = the human gene
= the protein encoded by the gene
mice = conventional term for transgenic mice that express human mutant SOD1 and develop ALS-like disease.
= conventional term for ALS caused by mutant SOD1.