A M YOT R O P H I C L AT E R A L S C L E R O S I S

Biomarkers in amyotrophic lateral sclerosis:

opportunities and limitations
Robert Bowser, Martin R. Turner and Jeremy Shefner
Abstract | Insights into the mechanisms of amyotrophic lateral sclerosis (ALS) have relied predominantly
on the study of postmortem tissue. Modern technology has improved the ability of scientists to probe
effectively the underlying biology of ALS by examination of genomic, proteomic and physiological changes
in patients, as well as to monitor functional and structural changes in patients over the course of disease.
While effective treatments for ALS are lacking, the discovery of biomarkers for this disease offers clinicians
tools for rapid diagnosis, improved ways to monitor disease progression, and insights into the pathophysiology
of sporadic ALS. The ultimate aim is to broaden the therapeutic options for patients with this disease.
Bowser. R. etal. Nat. Rev. Neurol. 7, 631638 (2011); published online 11 October 2011; doi:10.1038/nrneurol.2011.151

Introduction
Following the earliest descriptions of amyotrophic lateral
sclerosis (ALS) in the late 1800s,1 insights into the mecha
nisms of this disease were primarily based on the pathologi
cal assessment of postmortem tissue samples.2 Advances
in DNA manipulation have revealed genetic associations
with rare familial forms of the disease and have led to the
generation of transgenic animal models.36 As a rapid test is
currently unavailable, the diagnosis of ALS remains based
on clinical assessment that follows specific guidelines
(Box1). However, a major limitation of these criteria is that
the small group of patients who present with a syndrome
that affects only lower motor neurons (LMNs)termed
progressive muscular atrophy (PMA)are not considered
to have a form of ALS, even though nearly one-quarter
of these patients develop signs of upper-motor-neuron
(UMN) disease within 5years of diagnosis.7 Although
clinical diagnosis for other patients is typically accuratein
one study, only 7% of patients were re-diagnosed as having
a condition other than ALS8a persistent and notable
delay in referral to specialist neurology services has been
reported, which slows clinical diagnosis.
Despite major advances in understanding the molecular
biology of ALS, the mean delay in time from presentation
to diagnosis has remained at over 1year,9 during which
time the patient might have gone beyond the window of
therapeutic opportunity. Clinical heterogeneity is a charac
teristic yet poorly understood feature of ALS. Around 10%
of patients with this disease survive for more than 10years
after diagnosis,10,11 so biomarkers that have prognostic
value for patient survival would be of value to clinicians
to aid decision-making and care-planning. Robust bio
markers might also help us to assess drug efficacy in trials;
Competing interests
R. Bowser declares an association with the following company:
Knopp Bioscience. See the article online for full details of the
relationship. The other authors declare no competing interests.

the ultimate aim is to find biomarkers for disease activity

that that would reduce the current reliance on survival as
the main outcome, as well as reducing trial lengths and
associated costs.
Technological advancements have enabled detailed
analysis of the biological features of ALS, and monitor
ing of the disease process invivo. Such developments have
led to the discovery of candidate biomarkers for ALS in
biofluids, electrophysiological indicators of disease, and
neuroimaging markers. These and future discoveries will
change the way in which the biology of ALS is investigated
and, ultimately, how patients are treated. This Review high
lights advances in the identification and understanding
of protein-based, neurophysiological and n
euroimaging
biomarkers of ALS.

Protein-based biomarkers
Technologies that can quantify changes in protein levels
or identify abnormal post-translational modifications of
proteins have enabled detailed searches for protein-based
biomarkers for ALS. The main goal is to identify changes
at the time of symptom onset and during disease progres
sion. Efforts to discover protein biomarkers have focused
on blood and cerebrospinal fluid (CSF), but other biofluids
(such as urine) or tissues (for example, muscle) may yield
candidate biomarkers. Several reviews have described
efforts to discover protein biomarkers.1315 The evolv
ing methodologies for quantitative proteomics, which
use examples of candidate biomarkers for each method
(Box2), and the potential clinical utility of protein-based
biomarkers for ALS, are discussed in this section.

Antibody-based methods
The most common techniques for measuring protein
levels utilize antibodies that are presumed to be specific
for the protein in question. Quantitative ELISA is the most
common clinical diagnostic approach.16 This test can be

NATURE REVIEWS | NEUROLOGY

Division of Neurology,
Barrow Neurological
Institute, St Josephs
Hospital and Medical
Center, 350 West
Thomas Street,
Phoenix, AZ 85213,
USA (R. Bowser).
Nuffield Department of
Clinical Neurosciences,
Oxford University, John
Radcliffe Hospital,
Headley Way, Oxford
OX3 9DU, UK
(M.R.Turner).
Department of
Neurology, State
University of New York,
Upstate Medical
University, 750 East
Adams Street,
Syracuse,
NY132102375, USA
(J.Shefner).
Correspondence to:
R. Bowser
robert.bowser
@chw.edu

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REVIEWS
Key points
Amyotrophic lateral sclerosis (ALS) is characteristically heterogeneous in site
of onset, pattern and rate of disease progression
Candidate protein-based biomarkers have been identified in the blood and/or
cerebrospinal fluid of patients with ALS; assessment of combinations of these
biomarkers could improve diagnosis or increase prognostic ability
Physiologic biomarkers, including motor unit number estimation and electrical
impedance myography, may provide the means to improve monitoring of
disease progression in individual patients
Advanced MRI techniques have high sensitivity and specificity for detecting
ALS at group level, and along with PET can provide mechanistic insights
into disease pathogenesis
Further studies on large numbers of patients, with longitudinal follow-up, are
necessary to validate reported ALS biomarkers
The clinical utility of the biomarkers may require a combination of proteomic,
physiological and imaging-based methodologies

Box 1 | Diagnostic guidelines for ALS

The diagnosis of ALS currently remains grounded in clinical assessment
by an experienced physician, according to the following guidelines:12
Evidence of UMN degeneration by clinical examination
Evidence of LMN degeneration by clinical, electrophysiological or
neuropathological examination
Progressive spread of the symptoms within a limb or to other limbs or regions,
as determined by patient history or examination
Lack of electrophysiological or pathological evidence of other disease
processes that might explain the patients symptoms

Diagnostic certainty categories, for use in research rather than clinical practice,
are based on these features:
Possible ALS: Clinical signs of UMN and LMN degeneration in only one region, or
UMN signs alone in two or more regions, or LMN signs found above UMN signs
Probable ALS: Clinical signs of UMN and LMN deterioration in at least two
regions, with some UMN signs above LMN signs
Probable laboratory-supported ALS: Clinical signs of UMN and LMN
degeneration in one region, or UMN signs alone in one region and
electromyographic LMN signs in at least two regions
Definite ALS: Clinical evidence of UMN and LMN signs in three regions
Abbreviations: ALS, amyotrophic lateral sclerosis; LMN, lower motor neuron; UMN, upper
motor neuron.

easily performed in most laboratories, with one antibody

(indirect ELISA) or two antibodies (sandwich ELISA) to
measure protein concentrations. Among the candidate
protein biomarkers for ALS that have been most studied
by this method is phosphorylated neurofilament heavy
chain (pNfH). As estimated using a specified ALS cut-off
value, ELISA shows significantly increased levels of this
protein in CSF (sensitivity 71%, specificity 88%)17 and pos
sibly blood (sensitivity 58%, specificity 89%) of patients
with ALS.18 The high levels of pNfH in the CSF of ALS
patients could be related to the degree of axonal injury,
which results in the release of this cytoskeletal protein
into the extracellular space and its accumulation in the
CSF. Measurement of concentrations of this protein alone
may be insufficient to differentiate ALS from its mimics,19
but advances in ELISA-based technologyparticularly
in the use of electrochemiluminescencecould provide
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additional sensitivity and platform reliability that would

make this approach worth further exploration.20
Multiplex immunoassays can simultaneously measure
the levels of multiple proteins within one sample; two
basic platforms are planar arrays and microbead assays.
Planar arrays involve a number of capture antibodies that
recognize different proteins, which are spotted to defined
positions on the array. With the other method, capture
antibodies are attached to different populations of micro
beads that can be distinguished by their fluorescence
emission on flow cytometry. Both platforms, however,
have technical challenges regarding their capacity to accu
rately quantify widely varied concentrations of proteins
within the same sample, and the potential for antibody
interference limits protein detection.
Some studies using multiplex arrays have shown raised
levels of multiple inflammatory cytokines (sensitivity
88%, specificity 91% for overall panel)21 and individual
chemokines (MCP1 and IL8) 22 in the CSF of ALS
patients. However, results have been inconsistent across
laboratories, and published studies have included only
small numbers of patients, so these methods will require
further investigation in much larger cohorts of patients.
Another approach is to assess the quotient or ratio
of one protein to another; the findings can be used for
diagnostic or prognostic purposes. Two glial-derived pro
teins, namely soluble monocyte differentiation antigen
CD14 (a monocyte receptor involved in the inflamma
tory response) and astrocytic S100 (a calcium-binding
protein), can be detected in the CSF of patients with ALS.
An increased ratio of CD14 to S100 has been suggested as
a prognostic indicator in ALS patients, with a sensitivity of
75% and a specificity of 91%.23 An increased ratio of pNfH
to complement component 3 (C3) in the CSF has also been
proposed as a diagnostic and prognostic indicator for ALS;
use of this ratio increased the sensitivity for diagnosing
ALS (91%) versus pNfH alone.24 The biological relevance
of protein level ratios requires further investigation to
determine the cell types releasing these proteins and their
role in facilitating further neurodegeneration.
Future studies should combine protein measurements
with other technologies, such as electrophysiology or
neuroimaging, to assess whether the sensitivity and accu
racy for diagnosis and monitoring of disease progression
can be increased over use of these methods alone.

Mass spectrometry-based methods

The use of mass spectrometry-based proteomics has
greatly increased the yield of candidate biomarkers for
many diseases, including ALS.13 These studies have uti
lized chromatin-immunoprecipitation-based surfaces
to enrich proteins with particular DNA-binding charac
teristics, two-dimensional gel electrophoresis with sub
sequent mass spectrometry for protein identification, or
high-resolution mass spectrometry of biofluid samples to
identify protein alterations in patients with ALS. Results
from studies using chromatin-immunoprecipitation-based
mass spectrometry of CSF samples have been consistent
across laboratories, and cystatin C and transthyretin have
been identified as candidate biomarkers for ALS. 2527
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Two-dimensional gel electrophoresis and sequencing
of proteins within selected spots by mass spectrometry
has revealed a number of candidate biomarkers for
ALS in blood and CSF, including transthyretin, C3 and
fetuinA.2830 While the biological importance of these
candidate biomarkers in the pathobiology of ALS remains
unclear, both transthyretin and fetuinA were proposed to
be markers for rapidly progressive ALS.29
Liquid chromatographymass spectrometry of blood or
CSF samples also offers the potential for unbiased screen
ing of samples and for determining the sequence identity
and post-translational modifications of peptide-based
biomarkers.31 Ultimately, more-quantitative mass spectro
metry methodologies are needed to validate and translate
these results into clinically useful tests.32
Critical questions regarding protein biomarkers for
ALS remain, such as whether the levels of any candidate
protein biomarkers change over the course of disease
and whether these proteins can be useful as prognostic
indicators of ALS. Most investigations of prognostic bio
markers for ALS have involved a single prospective assess
ment of a CSF sample from each patient, with longitudinal
clinical follow-up to monitor disease progression.23,29
Longitudinal study in which CSF samples were collected
every 46months by lumbar spinal tap showed that cysta
tin C concentrations decreased over time in patients with
rapidly progressive ALS.33 Thus, serial blood samples or
spinal taps collected according to standard procedures are
necessary to determine changes in candidate biomarker
features over time.

Translation of findings to the clinic

Although early studies to identify protein-based bio
markers for ALS have produced promising results, the
findings must be further validated by multiple laborato
ries in large prospective studies, with samples collected
according to standardized protocols,34 to enable transla
tion of findings to the clinical environment. Longitudinal
studies are necessary to determine whether any proteinbased candidate biomarkers correlate with clinical features
of disease progression and, possibly, neuroimaging results.
Ultimately, incorporation of quantitative measurements of
proteins would be desirable to aid the diagnosis of ALS and
to help evaluate drug efficacy.35 Protein biomarkers may
also provide new insights into the pathogenesis of ALS and
aid the identification of targets for new drug treatments.

Physiological biomarkers
While biochemical markers may provide researchers with
insights into the specific cellular or signaling alterations
that occur during ALS, a number of global physiological
features can be assessed that might differentiate ALS from
other diseases and enable the monitoring of disease pro
gression (Box3). The motor deficits in patients with ALS
result from concurrent degeneration of neurons in both
the motor cortex (UMNs) and the spinal cord (LMNs).
LMN function is routinely assessed by nerve conduc
tion studies and electromyography. Physiological signs
of ongoing LMN degeneration or axonal loss include
the presence of fibrillation potentials and positive sharp

Box 2 | Candidate-protein-based biomarkers for ALS

Biomarkers identified with methods that measure
single-protein antibody concentrations
pNfH in CSF or blood17,18
Ratio of CD14 to S100 in CSF23
Ratio of pNfH to complement C3 in CSF24
Cystatin C levels33*
Biomarkers identified with methods that measure
concentrations of multiple antibodies
Altered levels of IL2, IL6, IL10, IL15, and GMCSF
in CSF21
Increased levels of MCP1 and IL8 in the CSF, and of
IL8, CCL11, CCL24 and CCL26 in blood22
Biomarkers identified with mass spectrometry-based
methodologies
Cystatin C, transthyretin and neuroendocrine protein
7B2 in CSF25,27
2-glycoprotein in CSF30
Complement C3 and other complement proteins
in blood28
Cystatin C and neurosecretory protein VGF in CSF26
FetuinA and transthyretin in the CSF as markers of
disease progression29
*Prognostic indicator of disease progression. Abbreviations: CCL,
CCchemokine ligand; CSF, cerebrospinal fluid; C3, component 3;
GMCSF, granulocyte macrophage-colony stimulating factor;
MCP1, monocyte chemoattractant protein1; pNfH,
phosphorylated neurofilament heavy chain.

waves on needle electromyography,36 as well as enlarged,

prolonged and polyphasic motor units, which arise as a
consequence of reinnervation.37 However, although these
abnormalities are sensitive indicators of the presence of
pathology, electromyography has a limited sensitivity
(60%) for the diagnosis of ALS,38 and the characteristics
measured during electromyographic studies (motor
unit duration, amplitude and phase) do not systemati
cally change with disease progression. Progressive motor
unit loss is a critical event that is related to disability in
patients with ALS;39 thus, this process is an attractive
target for treatment. A measure of motor unit loss that is
reproducible, noninvasive, rapidly obtained, and amen
able to repeated evaluation over time would, therefore,
be highly desirable.

Motor unit number estimation

Motor unit number estimation (MUNE) refers to a group
of techniques to estimate the number of intact motor units
that innervate a single muscle. These techniques all share
a common framework: a maximum response, which is
generated by activation of all motor units in the muscle,
is recorded, from which an estimate of individual motor
unit size is generated according to the general formula of
dividing the maximum response amplitude by the mean
of all motor unit amplitudes. The methods differ in how
the mean single motor unit amplitude is calculated, and
no clear standard for this calculation has been accepted;
however, important information has been acquired from
all techniques.

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Box 3 | Methods to measure physiological biomarkers for ALS
Motor unit number estimation
Uses surface electrodes to monitor muscle action potential and estimate the
number of motor units innervating a specific muscle, with potential uses as an
outcome measure and for long-term disease monitoring.46,52
Electrical impedance myography
Measures electrical impedance of individual muscles. An alternating current is
applied to a selected muscle, and voltage signals are recorded some distance away
from the stimulus. This technique enables the integrity of individual cell membranes
to be assessed, which acts as a marker of tissue degeneration. It can be used as
an outcomes measure and for long-term disease-monitoring.54,55
Neurophysiological index
Mathematical derivation from three combined standardized neurophysiological
measurements, representing aspects of denervation and reinnervation, although
not directly related to the number of surviving motor units. May be useful to monitor
disease progression.65
Transcranial magnetic stimulation
Electromagnetic induction that induces weak electric currents to measure activity
and function of specific brain circuits.66

In theory, ALS is perfectly suited to assessment with

MUNE, as motor unit dropout and remodeling are criti
cal aspects of the disease process. Early MUNE studies in
the hand and foot muscles of patients with ALS showed
reduced motor unit numbers but increased motor unit
amplitude, compared with healthy controls.40,41 Owing to
this compensation in amplitude, compound motor action
potential amplitudes remained within normal limits until
MUNE values dropped below 10% of normal. Clinical
atrophy was apparent when MUNE also dropped by 10%.
Longitudinal studies on patients with ALS showed that
motor unit loss was more rapid early in the disease, and
the slope of motor unit decline leveled off around 1year
after diagnosis. These patterns of compensation and
decreasing decline might complement data which shows
that muscle strength generally declines with increasing
duration of ALS,42,43 thereby improving diagnosis.
The usefulness of MUNE in clinical trials depends on
whether it declines to a greater extent than other com
monly used measurements of outcome. In a single-site,
longitudinal study that evaluated manual muscle testing,
MUNE, and the ALS functional rating scale (ALSFRS)
as outcome measures, the rate of decline over time was
similar for all three measures.44 In another single-site
study that was performed within a multicenter clinical
trial, MUNE, compound motor action potential ampli
tude, hand grip, strength testing, and vital capacity
were all measured at regular intervals over 1year.45 The
decreases in MUNE values were greater than those of the
other measures. A longitudinal study of ALS patients in
China found that the percentage change in MUNE values
over 12months was greater than the change in ALSFRS,
manual muscle testing, vital capacity, or compound motor
action potential amplitudes.46
Another important use of MUNE could be to stratify
patients according to rate of progression, which would aid
clinical trial design and assessment of prognosis. In several
studies, an increased rate of change in MUNE strongly
predicted rate of progression on functional measures47
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and survival48 in patients with ALS. By means of a single

MUNE evaluation and extrapolation to normal values that
are assumed to be present just before disease onset, MUNE
can predict survival and enable stratification of patients
according to speed of progression.49
Two multicenter clinical trials have used MUNE as a
secondary outcome measure.50,51 With extensive training
of health-care staff and validation of MUNE, testretest
variability was less than 20%. Over the duration of both
trials, MUNE declined monotonically, with an average
decline of 46% over a 12-month period. This decline was
less than that seen in other studies, and was suggested to be
due to an artifact that was intrinsic to the specific MUNE
method employed by the researchers. In a multicenter
natural history study of 71 patients with ALS, a modified
version of MUNE based on previously described methods
yielded excellent reproducibility; values declined by a
mean of 9.5% per month compared with baseline.52 Single
motor unit amplitude increased as MUNE decreased, as
expected. These attributes suggest that MUNE is a better
outcome measure for clinical trials than are previously
described methods.

Electrical impedance myography

Another promising marker of progressive LMN loss is
electrical impedance myography (EIM).53 Low-current,
high-frequency stimuli are passed through muscle, and
voltage signals are recorded some distance away from the
stimulus. Phase and reactance are recorded, either to a
single frequency or within a frequency range. In longitu
dinal studies of patients with ALS, EIM measurements
declined over time, and the technique had very low test
retest variability; power analyses suggested that the use of
EIM as an outcome measure could enable the reduction
of sample size and duration of phaseII clinical trials.54,55
Large studies are needed to further explore and validate
electrical impedance as a biomarker for ALS. A longitu
dinal natural history study of EIM in patients with ALS
is in progress.
Neurophysiological index
The neurophysiological index (NI) combines three
common measures of neuronal functioncompound
motor action potential amplitude, distal motor latency,
and Fwave frequency. Although the derived value does
not relate to the number of surviving motor units, (as it
does in MUNE), in a small single-center study of patients
with ALS, NI values had reduced by 40% after 6months
when compared with baseline values.56 No multicenter
studies have been performed to evaluate the use of NI in
the setting of a clinical trial.
Axonal excitability
Measurements of axonal excitability with thresholdtracking technology have demonstrated abnormalities
in patients with ALS compared with healthy controls.
Persistent conduction in sodium channels has been
demonstrated in mouse models of ALS and in patients
with sporadic or familial disease.5759 Values for axonal
excitability, however, vary greatly from patient to patient,
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and so far no changes in excitability have been noted with
disease progression.

biomarkers could also reveal disease mechanisms that

might aid the discovery of novel drug targets.

Transcranial magnetic stimulation

Physiological markers of UMN disease in ALS have been
sought. Use of transcranial magnetic stimulation (TMS)
has revealed several differences between patients with
ALS and healthy controls through calculation of central
conduction time or the time from cortical stimulation to
generation of action potentials in the corresponding spinal
motor neurons. In small single-site studies, central con
duction times have been found to be normal or extended
in patients with ALS,60,61 and cortical motor thresholds
were also reduced; however, no clear correlation exists
between either of these values and the severity of UMN
disease burden. In one study where two assessments of
cortical conduction time were made, 6months apart,
values did not change over time.56 Cortical silent periods,
assessed with TMS, were decreased in patients with ALS,
which potentially implies cortical hyperexcitability.6264
Interestingly, in patients with known genetic muta
tions, cortical hyperexcitability might be seen before the
development of symptoms. None of the above markers of
UMN dysfunction has been employed in a multicenter
setting, and whether these markers change with disease
progression has not been established.6264

Standard sequence
Cerebral atrophy is not consistently detectable on standard
T1-weighted clinical MRI of patients with typical ALS. A
technique known as voxel-based morphometry, can be
used to detect subtle volumetric changes in both graymatter 72 and white-matter 73 tissue compartments com
pared with healthy controls, but the changes do not equate
exactly with atrophy. A meta-analysis that investigated
changes in gray matter suggests that atrophy in the right
precentral gyrus is the most consistent finding in patients
with ALS.74 Hyperintensity in the corticospinal tract on
T2-weighted MRI was neither sensitive nor specific.

Neuroimaging
Imaging offers a noninvasive approach to biomarker
discovery and monitoring of disease progression. Major
developments in neuroimaging have been in the capacity
of MRI of the spine to exclude pathologies that mimic
ALS,67 and changes in data acquisition and processing,
which have brought this technique to the forefront of
biomarker discovery.68

Cerebral imaging
Although the clinical hallmark of ALS is simultaneous
UMN and LMN dysfunction, a study reported involve
ment of the corticospinal tract in 50% of patients with
LMN-only syndromes.69 A model of parallel cortical
UMN and spinal LMN degenerative processes in ALS
has now been postulated on the basis of rigorous clinico
pathological correlations.70 The discovery of a shared
pathological signature (namely ubiquitinated cytoplasmic
inclusions of TDP43) in ALS and frontotemporal demen
tia (FTD)71 has cemented decades of clinical observations
of cognitive involvement in ALS. Thus cerebral involve
ment, comprising extramotor cerebral lesions, must also
be included in comprehensive models of pathogenesis.
Electrophysiology can detect LMN pathology, but neuro
imaging has the unique advantage of also showing the
UMN and extramotor cerebral features.
Neuroimaging biomarkers
MRI studies have already provided clinicians with candi
date biomarkers for the diagnosis, prognostic assessment
and monitoring of progression in ALS.14 If validated, these
biomarkers could be easily integrated into routine clinical
evaluation of patients with suspected ALS. Neuroimaging

Diffusion tensor imaging

Secondary Wallerian-type demyelination of degenerate
cerebral white matter tracts has been observed in post
mortem tissue from patients with ALS.75 Diffusion tensor
imaging (DTI) is an advanced MRI application that is
sensitive to the movement of water; in intact white matter
tracts, movements are highly directional (anisotropic), but
in damaged pathways are more freely diffusible (isotropic).
Several measures can be extracted from the diffusion
tensor derived for each voxel, although the most com
monly studied parameter has been fractional anisotropy,
which decreases with increasing loss of white matter tract
integrity. Software that calculates probability can be applied
to diffusion tensor imaging (DTI) data to reconstruct the
connectivity of white matter tracts (known as diffusion
tensor tractography).76 Since DTI was first used to assess
patients with ALS,77 it has repeatedly demonstrated the
involvement of corticospinal tracts, especially the corpus
callosum,78 through fractional anisotropy changes. Effects
on the extramotor white matter tracts evolve longitudi
nally in all cases, including those categorized as PMA.79
Thus, fractional anisotropy in the brain and cervical cord
is a potentially useful biomarker for the monitoring of ALS
progression and might have prognostic value.80
Functional MRI
Functional MRI (fMRI) exploits the differential magnetic
properties of oxygenated and deoxygenated hemoglobin as
a surrogate marker of neuronal activation (blood oxygena
tion level-dependent [BOLD] fMRI). Regional cerebral
activity in response to a specific task (for example, motor
task) can be studied. fMRI data have largely confirmed the
findings from earlier PET activation studies,81 with several
fMRI experiments demonstrating expanded areas of corti
cal activation in patients with ALS.82 Resting-state fMRI
has been used to study the brain as a system of integrated
networks, and alterations have been noted in the sensori
motor network in patients with ALS.83 In a study of patients
soon after diagnosis, resting-state fMRI revealed evidence
of interhemispheric disconnection,84 which supports the
involvement of the corpus callosum.78 Combined studies of
structural and functional connectivity are now a priority,
and may provide valuable insights into the hypothesis that
ALS represents a complex neocortical system failure.

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Box 4 | Advanced applications of MRI biomarkers in ALS
Diffusion tensor imaging
Whole-brain fractional anisotropy has potential diagnostic,100 prognostic101 and
longitudinal monitoring79 functions, at group level.
Voxel-based morphometry
Whole-brain gray matter voxel-based morphometry has limited independent
diagnostic potential at group level,74 but shows improved sensitivity and specificity
as a biomarker when used in combination with diffusion tensor imaging.99
Functional MRI
Task-based functional MRI,82 particularly emerging resting-state network
analysis,83 has the potential to study the brain as a system of integrated networks
rather than searching for focal degeneration, which may be more relevant to
eventual primary prevention strategies.
Magnetic resonance spectroscopy
Primary motor cortex metabolites, in a voxel-of-interest approach, have limited
diagnostic and monitoring potential at group level,85 but emerging high field
strengths and whole-brain analyses may increase the potential of this technique
to measure relevant metabolite concentrations.

Magnetic resonance spectroscopy

Magnetic resonance spectroscopy (MRS) allows the quan
tification of proton-containing cerebral metabolites within
manually placed voxels of interest. To date, studies using
MRS have mostly studied the ratio of Nacetylaspartate to
choline or creatine, as a nonspecific surrogate marker of
neuronal damage in ALS. In one longitudinal study this
technique showed promise as a method for detecting and
monitoring disease.85 High magnetic field strengths might
improve quantification of metabolites by increasing the
metabolite spectral resolution. This might enable the iden
tification of glutamate and aminobutyric acid (GABA)
as potential surrogate markers of excitotoxicity, or myoinositol as a glial marker, thereby increasing the relevance
to ALS. MRS is, however, a highly operator-dependent
technique, and the numbers of centers that have people
with this expertise are limited.
PET
PET has led to increased understanding of ALS patho
genesis and offers unique insights into neuronal recep
tors. However, the low availability of individuals with
expertise in radiochemical production, the inherent
exposure to radiation, and the requirement for intra
venous injection are disadvantages of PET compared
with MRI. Studies using ligand PET, with flumazenil
as a marker of GABAA receptor, have shown, indirectly,
a lack of cortical inhibitory function in patients with
ALS.86 Studies with PK11195a ligand for the periph
eral benzodiazepine receptor that is expressed only by
activated microgliaprovided the first invivo demon
stration of widespread cerebral neuroinflammatory activ
ity in human patients with ALS87 and neuroinflammation
remains a proposed pathogenic mechanism involved in
this disease.88 Dramatic decreases in binding of the sero
tonin 5HT1A-receptor PET ligand WAY100635 in the
frontotemporal lobes of nondepressed ALS patients have
been reported.89 This finding has potential (although
as yet unaddressed) pathogenic importance, in light of
strikingly similar changes that are seen in FTD,90,91 with
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a clear clinicopathological spectrum between ALS and

FTD nowestablished.92
Although the emergence of PET amyloid imaging was
a major advance in the neuroimaging of patients with
Alzheimer disease, the intracellular localization of the
TDP43 aggregates that pathologically characterize spo
radic ALS71 is currently a major barrier to the develop
ment of a similar PET ligand in ALS. The development
of PET ligands for more-accessible neuronal receptors,
such as those for glutamate, might offer future novel
mechanistic insights.

Familial studies
Pathogenic genetic mutations can currently be identified in
only approximately one-third of the 5% of ALS cases with
a clear family history. When patients were systematically
tested, a small proportion of individuals with apparently
sporadic cases of disease were shown to carry dominant
gene mutations for ALS; in one series, 4% of patients with
sporadic disease also had mutations in the superoxide dis
mutase 1 gene (SOD1).93 Some ALS-related genetic muta
tions show incomplete penetrance and, therefore, whether
routine testing for mutations in the small number of genes
currently identified would be beneficial is unclear. The
benefit of testing unaffected relatives of patients with ALS
is even less clear.
Although patients with pathogenic mutations in the
SOD1 gene represent less than 2% of all cases of ALS,
neuroimaging experiments in these patients have been
revealing. Patients who are homozygous for the recessive
Asp90Ala mutation experience a considerably reduced
rate of disease progression, and studies using voxel-based
morphometry,94 DTI,95 diffusion tensor tractography 96
and flumazenil PET86 have revealed less involvement of
motor cortical regions than in patients with sporadic ALS
of similar disability. These findings suggest differences in
pathology between patients with genetic disease and those
with sporadic disease that have wider implications for
prognostication. PET showed significant changes in the
binding of flumazenil in the left frontotemporal domain
in two asymptomatic carriers of the SOD1 mutation.86 One
DTI study in presymptomatic carriers of the SOD1 muta
tion reported fractional anisotropy changes in the posterior
limb of the internal capsule.97 These findings raise the
possibility that neuroimaging may be able to detect the
earliest presymptomatic changes in this familial ALS
patient population.
Translation of findings to the clinic
The combination of multiple MRI-derived biomarkers
could potentially offer high sensitivity and specificity for
ALS (Box4).78 The greatest challenge to the viability of
neuroimaging biomarkers in ALS, however, is the successful
translation of group-level markers to individual patients.98
Large control data sets might be needed to reach this goal,
which can be achieved through multicenter collaboration,
and an international initiative that has now commenced
is a positive step in this direction.99 Longitudinal cohort
studies that assess neuroimaging and CSF biomarkers are
also needed to improve accuracy; this approach has been
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A M YOT R O P H I C L AT E R A L S C L E R O S I S
successfully applied in studies of Alzheimer disease. These
challenges notwithstanding, the concept of a brain scan for
ALS has never looked more likely.

Review criteria

Conclusions
Multiple methodological advancements have led to the
discovery of protein-based, neurophysiological, and
neuroimaging biomarkers for ALS. Importantly, many
biomarkers have provided mechanistic insights as well
as offering diagnostic, prognostic or disease-monitoring
potential. Each class of biomarker requires continued
development, but the combination of different classes
might be required for successful translation to the clinical
setting. Nevertheless, the eventual emergence of validated
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Author contributions
R. Bowser, M.R. Turner and J. Shefner contributed
equally to researching data for the article, providing
substantial contribution to discussion of the content,
writing the article, and to review and/or editing of the
manuscript before submission.

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