Review Article on Toward Precision Medicine in Neurological Diseases

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Toward precision medicine in amyotrophic lateral sclerosis

Zhang-Yu Zou, Chang-Yun Liu, Chun-Hui Che, Hua-Pin Huang

Department of Neurology, Fujian Medical University Union Hospital, Fuzhou 350001, China
Contributions: (I) Conception and design: ZY Zou, HP Huang; (II) Administrative support: ZY Zou, HP Huang; (III) Provision of study materials
or patients: ZY Zou, CY Liu, CH Che; (IV) Collection and assembly of data: ZY Zou, CY Liu, CH Che; (V) Data analysis and interpretation: All
authors; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors.
Correspondence to: Zhang-Yu Zou, MD, PhD. Department of Neurology, Fujian Medical University Union Hospital, No. 29, Xinquan Road, Gulou
District, Fuzhou 350001, China. Email: pumczzy@gmail.com; Hua-Pin Huang, MD, PhD. Department of Neurology, Fujian Medical University
Union Hospital, No. 29, Xinquan Road, Gulou District, Fuzhou 350001, China. Email: hh-p@163.com.

Abstract: Precision medicine is an innovative approach that uses emerging biomedical technologies to deliver
optimally targeted and timed interventions, customized to the molecular drivers of an individual’s disease. This
approach is only just beginning to be considered for treating amyotrophic lateral sclerosis (ALS). The clinical and
biological complexities of ALS have hindered development of effective therapeutic strategies. In this review we
consider applying the key elements of precision medicine to ALS: phenotypic classification, comprehensive risk
assessment, presymptomatic period detection, potential molecular pathways, disease model development, biomarker
discovery and molecularly tailored interventions. Together, these would embody a precision medicine approach,
which may provide strategies for optimal targeting and timing of efforts to prevent, stop or slow progression of ALS.

Keywords: Amyotrophic lateral sclerosis (ALS); precision medicine; customized therapies

Submitted Dec 18, 2015. Accepted for publication Jan 11, 2016.
doi: 10.3978/j.issn.2305-5839.2016.01.16
View this article at: http://dx.doi.org/10.3978/j.issn.2305-5839.2016.01.16

Introduction population is 2–3 people per 100,000, and the overall

lifetime risk of developing the condition is 1:400 (3-5).
Precision medicine is an innovative approach that applies
Approximately 5–10% of patients have familial ALS (FALS)
recently developed biomedical technologies to optimize
and show a Mendelian pattern of inheritance; the remaining
and individualize treatment to the molecular drivers of an
90–95% of patients have sporadic ALS (SALS) (6). Over
individual’s disease. It involves not creation of treatments
60% of patients die within 3 years of presentation, usually
that are unique to a patient, but rather classification from respiratory failure and about 10% survive for more
of individuals into subpopulations that differ in their than 10 years (7). There is no disease-modifying therapy
susceptibility to a particular disease, in the biology and/or for ALS, though riluzole slows the rate of progression and
prognosis of the disease they develop or in their response prolongs survival by 2 or 3 months (8).
to a specific treatment (1,2). This approach of using ALS is a complex, multifactorial disease with variations
tailored, mechanism-based therapies has been applied to in individual susceptibility and phenotype. The clinical and
cancer care and gained progressively greater impact, but it biological complexities of ALS have hindered development
is only beginning to be considered in amyotrophic lateral of effective therapeutic drugs. In this review we envision
sclerosis (ALS). applying the key elements of precision medicine to ALS.
ALS is a neurodegenerative disease characterized by
progressive deterioration mainly involving the corticospinal
Comprehensive risk assessment
tract, brainstem and anterior horn cells of the spinal cord.
Patients develop focal and then generalized weakness Genetic and environmental factors that influence
leading to paralysis. The incidence of ALS in the European susceptibility to ALS depend on multiple gene–gene and

© Annals of Translational Medicine. All rights reserved. www.atmjournal.org Ann Transl Med 2016;4(2):27
Page 2 of 12 Zou et al. Toward precision medicine in ALS

gene–environment interactions and epigenetic effects, all of environmental exposures all contribute to the development
which also drive phenotypic individuality. It is important to of ALS (52). Environmental exposure as a risk factor for
identify these modifying factors, as they could be targets for ALS, though weak, is likely to be cumulative over time,
therapeutic intervention. exceeding a genetic-environmental threshold in those who,
Over the last two decades, a great deal of new knowledge at some later time, develop ALS (53). Because ALS is a rare
has been gathered on ALS, especially on its underlying disease, single-center studies usually lack sufficient statistical
genetics. To date, about 23 genes have been implicated power to assess the environmental risk in ALS with known
in FALS (Table 1). Mutations in these genes account for genetic backgrounds, and thus test for gene–environment
approximately two thirds of the genetic etiology of FALS interactions. Future studies of environmental risk should
and 10% of SALS (40). Chromosome 9 open reading enroll a large sample of patients from multiple centers
frame 72 (C9orf72), Cu/Zn superoxide dismutase1 (SOD1), stratified by age of onset and known genetic risk factors.
fused in sarcoma (FUS), and TAR DNA binding protein
(TARDBP) are the most common mutated genes in both
Phenotypic classification
FALS and SALS in various patient populations (41). Thus,
there is consensus that these four genes play a causal role High phenotypic variability of ALS is observed, with
in ALS, whereas further evidence is required to support the regard to site of onset, age at onset, familial occurrence,
roles of the other genes. Multiple genome-wide association type of motor neuron involvement, extent of extramotor
studies (GWAS) have been performed, identifying several involvement and rate of progression. Primary muscular
candidate susceptibility genes for ALS, including DPP6 (42), atrophy, which involves pure lower motor neurons (LMN),
ELP3 (43), UNC13A (44,45), ZNF512B (46), ITPR2 and and primary lateral sclerosis, which involves pure upper
SUNC1 (47). Some of these risk genes were proposed motor neurons (UMN), constitute the ends of a spectrum.
to modify phenotype, for example, age at onset (48) and Intermediate phenotypes, such as UMN-predominant ALS,
survival (49,50). However, these associations should classical ALS and LMN-predominant ALS, are considered
be interpreted cautiously, as attempts to replicate the to be different expressions of ALS. Regionally isolated
observed effects have led to either conflicting or negative variants of ALS include flail arm syndrome, which involves
results. Future GWAS should involve larger case-control bilateral proximal and typically predominant LMN arm
cohorts and should stratify GWAS data based on different weakness, and the flail leg syndrome, characterized by often
populations and well-defined clinical categories to maximize asymmetric and primarily distal LMN involvement in the
the statistical power and minimize the false discovery rate. lower limbs.
Genome sequencing will continue to drive research in Advances in ALS genetics have greatly broadened the
ALS genetics forward, yielding even greater insight into known phenotype of this disease. The discovery of TARDBP
the genetic architecture of ALS by providing a complete and FUS enabled recognition that ALS and frontotemporal
catalog of rare variants. It will also allow exploration of the dementia (FTD) represent overlapping clinical syndromes
role of noncoding and intergenic genetic variation in the (13,14,18,19) and this convergence has been strengthened
pathogenesis of this disease. by the discovery of C9orf72, Ubiquilin 2 (UBQLN2)
Non-genetic factors, including environmental exposure and other genes (27,33,34). It is now accepted that ALS
to toxins, smoking, excessive physical activity, occupation, constitutes a continuum with FTD, with pure ALS and
dietary factors and changes in immunity, have been pure FTD at the ends of this spectrum of motor neuron
proposed as increasing risk of developing SALS. These and frontotemporal neuron involvement. Intermediate
factors may drive epigenetic changes over many years, phenotypes include ALS with behavioral impairment, ALS
which then induce disease onset and progression. However, with cognitive impairment, and ALS-FTD (7). In addition,
the only established risk factors so far are old age and male discovery of mutations in valosin-containing protein
gender. A pooled analysis of five large cohorts found that (VCP), Sequestosome-1 (SQSTM1), heterogeneous nuclear
smoking is the only probable environmental risk factor for ribonucleoprotein A2B1 (HNRNPA2B1) and heterogeneous
ALS, but no dose-response relationship with either pack- nuclear ribonucleoprotein A1 (HNRNPA1) in subsets
years or duration of smoking was found (51). Recently, of patients with ALS, FTD, inclusion body myopathy
the gene-time-environment model of ALS was proposed, and Paget disease of the bone showed that at least some
in which the genetic component of liability, time and forms of ALS are part of the ‘multisystem proteinopathy’,

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Annals of Translational Medicine, Vol 4, No 2 January 2016 Page 3 of 12

Table 1 Summary of genes linked to amyotrophic lateral sclerosis

Gene Location FALS locus Inheritance Strategy Associated phenotype Protein function References
SOD1 21q22.11 ALS 1 AD, AR LA ALS, PMA, juvenile ALS Superoxide (9)
metabolism
ALS2 2q33.2 ALS 2 AR LA Juvenile ALS, infantile HSP Vesicle trafficking (10)
SETX 9q34.13 ALS 4 AR LA Juvenile ALS, dHMN, AOA2 RNA metabolism (11)
SPG11 15q21.1 ALS 5 AR LA, CGA Juvenile ALS, HSP DNA damage repair (12)
FUS 16p11.2 ALS 6 AD, AR LA, CGA ALS, ALS-FTD, FTD RNA metabolism (13,14)
VAPB 20q13.33 ALS 8 AD LA ALS, PMA Vesicle trafficking (15)
ANG 14q11.1 ALS 9 AD CGA ALS, ALS-FTD Angiogenesis (16,17)
TARDBP 1p36.22 ALS 10 AD LA, CGA ALS, ALS-FTD, FTD RNA metabolism (18-20)
FIG4 6q21 ALS 11 AD, AR CGA ALS, PLS, CMT Vesicle trafficking (21)
OPTN 10p13 ALS 12 AD, AR HM ALS, FTD Vesicle trafficking; (22)
autophagy
ATXN2 12q24.12 ALS 13 − CGA ALS, SCA2 Endocytosis; RNA (23,24)
translation
VCP 9p13.3 ALS 14 AD WES ALS, FTD, IBM, PDB Proteasome; vesicle (25,26)
trafficking
UBQLN2 Xp11.21 ALS 15 XL LA ALS, ALS-FTD, juvenile ALS Proteasome (27)
SIGMAR1 9p13.3 ALS 16 AR HM juvenile ALS, dHMN Proteasome (28,29)
CHMP2B 3p11.2 ALS 17 AD LA, CGA ALS, FTD Vesicle trafficking (30,31)
PFN1 17p13.3 ALS 18 AD WES ALS Cytoskeletal (32)
dynamics
C9orf72 9p21.2 ALF-FTD AD GWAS, LA ALS, FTD, ALS-FTD RNA metabolism (33,34)
MATR3 5q31.2 − AD WES ALS, distal myopathy RNA metabolism (35)
CHCHD10 22q11.23 − AD WES ALS, FTD, cerebellar ataxia, Mitochondrial (36)
myopathy dysfunction
SQSTM1 5q35.3 − AD CGA ALS, FTD, IBM, PDB Ubiquitination; (37)
autophagy
HNRNPA1 12q13.13 − AD WES ALS, FTD, IBM, PDB RNA metabolism (38)
HNRNPA2B1 7p15.2 − AD WES ALS, FTD, IBM, PDB RNA metabolism (38)
TBK1 − AD WES ALS, FTD Autophagy (39)
AD, autosomal dominant; ALS, amyotrophic lateral sclerosis; AOA2, ataxia with oculomotor apraxia type 2; AR, autosomal
recessive; CGA, candidate gene analysis; CMT, Charcot-Marie-Tooth disease; dHMN, distal heredity motor neuropathy; FTD,
frontotemporal dementia; GWAS, genome-wide association studies; HSP, hereditary spastic paraplegia; HM, homozygosity
mapping; IBM, inclusion body myopathy; LA, linkage analysis; PDB, Paget disease of bone; PLS, primary lateral sclerosis; PMA,
progressive muscular atrophy; SCA2, spinocerebellar ataxia type 2; WES, whole exome sequencing; XL, X-linked.

a widespread disease process with muscle, bone and designated as ‘ALS with multisystem degeneration’ (7,55).
neuronal degeneration (54). Thus, there is also a spectrum Different phenotypes of ALS may fit within a
of extramotor involvement in ALS, ranging from classic clinical and pathological continuum, or on the contrary,
ALS with no or mild extramotor involvement to ALS with reflect heterogeneity of underlying pathophysiological
cognitive, extrapyramidal, cerebellar, sensory, autonomic, mechanisms. Therefore, accurate disease categorization
urinary, oculomotor, muscular or bone involvement, may help to explore the underlying pathophysiological

© Annals of Translational Medicine. All rights reserved. www.atmjournal.org Ann Transl Med 2016;4(2):27
Page 4 of 12 Zou et al. Toward precision medicine in ALS

Astrocyte
Mitochondrial
dysfunction
Abnormal
RNA metabolism
Neuroinflammation

Microglial cell Impaired axonal

transport
Proteasome
impairment

Excitotoxicity
Oxidative Endoplasmic
stress reticulum stress

Figure 1 Potential molecular pathways of the pathogenesis of amyotrophic lateral sclerosis.

mechanisms and select candidates for clinical trials. motor neuron function was clearly shown to precede
clinical deficit in ALS carrying SOD1 or C9orf72 mutations
(58-60). Moreover, surprisingly homogenous miRNA
Confirming a presymptomatic period
alterations were found in FALS patients and asymptomatic
It is unclear whether ALS, like many neurodegenerative mutation carriers (60). These findings suggested the
disorders such as Alzheimer’s, Parkinson’s and Huntington’s existence of biomolecular dysfunctions at a cellular level
diseases, is characterized by a presymptomatic period. that are insufficient to cause clinical features and that such
However, the fact that patients with hereditary ALS do not dysfunctions are potentially present and building for years
present clinically until mid- to late-adulthood indicates or decades prior to the onset of clinical disease (53). Further
that either these mutated genes are not ‘switched on’ until studies are needed to confirm the presymptomatic period
later in life or that there are decades of progressive cellular in patients with ALS and to establish presymptomatic
compromise, eventually culminating in catastrophic decline diagnostic tools to identify those at high risk of developing
manifesting as presentation of clinically overt ALS (53). the disease.
Recent progress in the genetic basis of ALS has led to
the identification of increasing numbers of asymptomatic
Studying potential molecular pathways
people at genetic risk for ALS, which will help to define the
presymptomatic phase of the disease. With advances in ALS genetics, several potential
There is a compelling body of evidence to indicate pathophysiological mechanisms have been implicated
that the onset of clinical symptoms is preceded by a long and proposed including oxidative stress, mitochondrial
presymptomatic period. Longitudinal studies reported dysfunction, impairment of axonal transport, excitotoxicity,
reduced motor unit number estimation (MUNE) and protein aggregation, endoplasmic reticulum stress,
increased cortical excitability 3–10 months in advance of abnormal RNA processing and neuroinflammation (61)
symptom onset in SOD1 mutation carriers (56,57). It is (Figure 1). Studies of ALS excitotoxicity mechanisms led
therefore imperative to identify the sensitive biomarkers to discovery of riluzole, the only FDA-proved drug for the
at this preclinical stage by establishing presymptomatic disease. Recently, some additional molecular pathways have
diagnostic tools to identify those at high risk of developing been identified by basic science research and therapeutic
ALS. This would open a potentially important window development efforts. The most notable of these include
for neuroprotective intervention that might allow rescue alterations in RNA metabolism and protein homeostasis.
of dysfunctional, but not yet dead, neurons and might The identification of ALS-causing mutations in the
even enable disease prevention (53). Abnormality of upper genes encoding TDP-43 and the RNA-binding protein

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FUS, both of which are involved in pre-mRNA splicing, to a stress response. More evidence for interference with
RNA transport and RNA translation, led to the proposal normal proteasomal or autophagic protein degradation as
that aberrant RNA metabolism contributes to ALS a factor in ALS pathogenesis comes from the discovery of
pathogenesis (13,14,18,19). Besides TDP-43 and FUS, a ALS-linked mutations affecting proteins that are directly
surprising number of proteins linked to ALS are directly involved in proteostasis or autophagy, such as mutations
or indirectly involved in RNA processing and metabolism. in VCP, UBQLN2, SQSTM1, charged multivesicular
These proteins include TAF15, EWSR1, ANG, SETX, body protein 2b (CHMP2B), optineurin (OPTN), TANK-
ELP3, ataxin-1 and -2, hnRNPA1 and hnRNPA2B1 and binding kinase 1 (TBK1), TDP-43 and FUS (62). Recent
C9orf72 (62). TDP-43 and FUS shuttle between the studies showed that induction of autophagy might have
nucleus and cytoplasm. In response to stressors such as therapeutic benefits for ALS. Molecules targeting mTOR
starvation or oxidative stress, TDP-43 and FUS exit from dependent and mTOR independent autophagy pathways,
the nucleus and exist primarily in the cytoplasm, where including rapamycin (72,73), trehalose (74,75), spermidine,
they are incorporated into stress granules and form stress carbamazepine and tamoxifen (76), prolonged motor
granule-based aggregates. ALS-associated mutations cause a neuron survival or rescued motor dysfunction in mutant
shift in localization of TDP-43 or FUS from the nucleus to SOD1/TDP-43 transgenic mice, an effect correlating
the cytoplasm and increase their propensity for aggregation with increased autophagy. However, a recent phase III
(63,64). This increased cytoplasmic aggregation, a gain- multicenter clinical trial reported no beneficial effects of
of-function, results in nuclear depletion of TDP-43 and lithium, which enhances autophagy, in ALS patient survival
FUS, inducing abnormalities in RNA processing, a loss-of- (77). Because most available drugs target many biological
function (65-67). The identification of C9orf72 mutations processes beyond autophagy, there is a need to explore new
as causing ALS made RNA toxicity a high profile researches autophagy regulators with higher specificity and lower side
focus for the disease. The C9orf72 mutations causing ALS effects.
are GGGGCC hexanucleotide repeat expansions with
several hundreds or even thousands of repeats (33,34).
Developing disease models
C9orf72 mRNA levels were reduced by 50% in ALS
patients with C9orf72 abnormal expansions, suggesting that Laboratory models of ALS help researchers understand
the expanded allele hinders generation of mature mRNA the basic processes of the disease, which is essential for
(33,68). Thus, C9orf72 expansion may represent a loss-of- developing new therapies. Though ALS has been modeled
function mutation. However, the expanded hexanucleotide in cells, worms, flies, fish, mice and rats, no model is a
repeat was shown to form nuclear RNA foci in neurons in perfect representation of the human disease, though each
the frontal cortex and spinal cord in patients with C9orf72 offers advantages for studying particular disease features.
mutations. Pre-mRNA containing the expansion may thus Rodents are especially important for testing potential
also exert a deleterious gain-of-function effect (33). Another therapies because their nervous systems are much larger
possible mechanism of ALS pathogenesis associated with and more complex than those of many other animal
C9orf72 mutations would be repeat-associated non-ATG models. Development of transgenic animal models carrying
(RAN) translation (69). While medications to significantly genetic mutations identified in ALS patients has facilitated
reduce the gain-of-toxic function effect have not yet been studying disease mechanisms and developing therapeutic
discovered, targeting the production of toxic protein or strategies for ALS because these models recapitulate its key
RNA could achieve this aim (see discussion below). histopathological and biochemical features (78).
Aggregates of mutant SOD1, TDP-43 or FUS are Discovery of SOD1 mutations in FALS led to generation
hallmarks of ALS. These aggregates or, more likely, their of the first transgenic mouse model of ALS (79). SOD1
precursor oligomeric complexes, disturb normal protein transgenic models reproduced many features of ALS,
homeostasis and induce cellular stress. Misfolded mutant including motor deficits, reduced survival, fragmented
SOD1 has toxic effects on the cell’s degradation machinery, and insoluble SOD1 aggregates, reactive gliosis and
impairing its two major components, the proteasomal neuronal loss (79-81). SOD1 mouse models have been
pathway and autophagy, thus circumventing this protective used extensively to study disease pathogenesis and for
regulatory process in the cell (70,71). Mutant SOD1 then drug screening, but a substantial number of compounds
accumulates as oligomers and later as aggregates, leading that prolong survival and/or delay onset of paralysis in the

© Annals of Translational Medicine. All rights reserved. www.atmjournal.org Ann Transl Med 2016;4(2):27
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SOD1 mouse model showed disappointing results in clinical therefore, identify promising candidate therapies with
trials. Recently, identification of TARDBP mutations led greater effectiveness in humans (92).
to a number of transgenic mouse models expressing either
wild-type or mutant TDP-43, which have a phenotype
Discovering biomarkers
primarily consisting of cortical abnormalities and minor
lower motor neuron involvement (82,83). Transgenic Biomarkers would facilitate diagnosis and thus might
mice overexpressing wild-type or mutated human FUS expedite initiation of neuroprotective therapies. Biomarkers
developed ALS-like symptoms, with hindlimb paralysis could also help select patients for enrollment in clinical
and shortened life span, along with cytoplasmic FUS trials or identify subgroups that will benefit most from
aggregation (84,85). Very recently, transgenic mice with certain medications. Furthermore, robust biomarkers for
C9orf72 repeat-expansion was created, and mimicked both disease activity might also help assess drug efficacy in trials.
neuropathological and clinical C9orf72 mutated FTD/ Biomarkers that have prognostic value for survival would
ALS phenotypes (86). These mice had neuronal loss, be of value for decision-making and planning of care.
nuclear RNA foci, dipeptide repeat inclusions and TDP-43 Technological developments have led to the discovery of
pathologies in the brain, as well as such behavioral many candidate protein-based, neurophysiological, and
abnormalities as hyperactivity, anxiety, antisocial behavior neuroimaging biomarkers for ALS.
and motor deficits. Given the high prevalence of C9orf72 Dozens of candidate protein-based biomarkers were
mutations in FALS/ALS-FTD, transgenic C9orf72 mouse identified in the blood and/or cerebrospinal fluid (CSF) of
models are likely to contribute to studying the disease patients with ALS [see review in (93)]. CSF neurofilaments
process and testing therapies against this form of the disease. have become leading candidate neurochemical biomarkers
With the number of genetic mouse models now available, of diagnostic and prognostic value (94,95). Very recently,
compounds should be tested in models with various genetic a prospective study showed the positive predictive value
backgrounds to determine the effects of genetic variability of elevated levels of CSF neurofilaments for diagnosis,
on therapeutic efficacy before advancement to clinical trials. distinguishing between patients with ALS and neurological
However, all of these animal models have limitations. disorder controls (94). Neurofilaments NF-L and pNF-H
They do not faithfully translate to human disease and each were at normal levels before onset of symptoms and were
represents only a subset of FALS cases. Thus, most agents increased at early symptom onset in CSF and/or serum (96).
found effective in these models were not found to be of In addition, neurofilament levels correlated moderately
value in clinical trials (78). Reprogramming fibroblasts of with motor neuron disease progression and duration (94).
ALS patients into induced pluripotent stem cells (iPSCs) Thus blood and CSF neurofilament levels were linked to
appears to be a promising opportunity to develop new the symptomatic phase of ALS and might, therefore, serve
SALS models. These cells have been differentiated into as objective markers of structural damage to the nervous
ALS-relevant cell subtypes including motor neurons and system, a promising surrogate in disease monitoring and
astrocytes. Motor neurons derived from SALS patients clinical staging of ALS and an outcome measurement for
recapitulate the major pathological features of the patients future ALS therapeutic trials (97).
they were derived from, including TDP-43 aggregation (87). A number of global physiological features can be
In addition, motor neurons derived from patients carrying assessed that might differentiate ALS from other diseases
TARDBP or C9ORF72 mutations display abnormal and enable disease progression to be monitored. MUNE
physiological properties (88). Thus iPSC-differentiated enables quantification and tracing of motor unit numbers,
neurons from ALS patients offer another platform to test unaffected by compensatory reinnervation during disease
therapeutic candidates. The emergence of powerful gene- progression. MUNIX was shown to be more rapidly
editing tools such as clustered regularly interspaced short recorded and have a better reproducibility compared with
palindromic repeats (CRISPR) enables introduction of other more complex MUNE methods (98). Early studies of
specific mutations into well-characterized iPSC lines and MUNIX showed index values to be reproducible in normal
into endogenous genes in mice. The use of endogenous subjects, whereas those with ALS tended to decline over
mouse genes might help overcome problems arising from time with disease progression. In addition, in ALS patients,
expression of human rather than mouse proteins (89-91). the index values showed greater changes than other metrics
Preclinical testing in these iPSC-based models might, such as compound muscle action potential amplitude, ALS

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Annals of Translational Medicine, Vol 4, No 2 January 2016 Page 7 of 12

functional rating scale (ALSFRS) values or forced vital harbor a mutant gene. Blocking expression of the mutant
capacity (99). Very recently, a multicenter prospective study gene, therefore, stands out as a potentially definitive
performed longitudinal MUNIX measurements in multiple therapy. It might stop the complex cascade of events
muscles of patients with ALS. The study demonstrated leading to motor neuron death before it starts. Antisense
that MUNIX could track the loss of LMNs even when oligonucleotide (ASO) therapy has emerged as a highly
measuring clinically less affected muscles. In addition, promising approach for preventing mutant gene expression
MUNIX could accurately discriminate between faster or in neurodegenerative diseases. An interim analysis from a
slower disease progressions. These results confirmed that phase II clinical trial of ISIS-SMNRx in infants with type I
MUNIX is a reliable electrophysiological biomarker to spinal muscular atrophy demonstrated good tolerability and
track lower motor neuron loss in ALS and might serve improved muscle function (105).
as a prognostic indicator (100). Electrical impedance
myography, which assesses integrity and structure of a
SOD1 targeted therapeutic strategies
muscle, was shown to outperform other measures such as
revised ALSFRS, MUNE and handheld dynamometry, in Increasing evidence has indicated that the ALS-associated
terms of its ability to detect deterioration (101), and might effects of mutant SOD1 are caused by a gain of toxic
therefore serve as a meaningful measure of disease severity function rather than a loss of enzymatic function.
in ALS (102). Therefore, reducing concentrations of the mutant protein
Imaging offers a noninvasive approach to biomarker would be expected to slow progression of SOD1-linked
discovery and disease monitoring. Voxel-based diffusion ALS. In a SOD1-G93A rat model, treatment with ASOs
tensor imaging studies consistently showed reduced at the time of symptom onset significantly slowed disease
fractional anisotropy within the corticospinal tract and progression and increased survival (106). These results
the corpus callosum. Voxel and surface-based MRI have led to an initial phase I clinical trial showing that
morphometry quantification demonstrated thinning of the intrathecal delivery of ASO targeting SOD1 (SOD1Rx) to
primary motor cortex. However, the correlation of these the CNS was well tolerated by patients harboring SOD1
imaging metrics with the absolute level of disability at the mutations and that CSF and plasma concentrations of ASOs
time of MRI, using ALSFRS or the estimated rate of disease were dose dependent, suggesting that intrathecal ASO
progression, was inconsistent (103). delivery in patients is an effective route of delivery for ALS
Further longitudinal studies are necessary to determine therapy (104). The next phase of testing will determine
whether any protein-based candidate biomarkers, in whether SOD1Rx can be given at a sufficiently high dosage
combination with those obtained by neurophysiology and and for a sufficiently long time to significantly reduce SOD1
neuroimaging, will increase sensitivity and accuracy of protein levels, and whether such reduction will impact the
diagnosis, help to monitor disease progression or predict course of disease in SOD1-mutated ALS. In addition to
prognosis in ALS (93). the ASO targeting SOD1, antibodies and small molecules
targeting misfolded SOD1 are in preclinical testing stages (107).

Interventions tailored to individual molecular

drivers C9orf72 targeted gene therapy

Genetic studies and outcomes from experimental models The SOD1Rx trial has supported pursuing potential
have provided the most compelling data on molecular applications of ASOs to other forms of genetically determined
mechanisms. Subsequent multiple clinical trials to test ALS. The likelihood that C9orf72 mutations cause a toxic gain
these proposed disease-altering interventions have been of function makes it a promising candidate for treatment with
attempted but ultimately failed in treating ALS. In fact, ASO. In fact, ASOs targeting C9orf72 were shown to reduce
virtually all such approaches neglected to consider the C9orf72 pathology in iPSC-differentiated neurons from
underlying clinical and biological complexities of this C9orf72-mutated ALS patients, including RNA aggregation,
disease. A precision medicine approach has been overlooked aberrant transcription factor binding, dysregulated expression
until very recently, with some clinical trials aimed at specific of other genes, susceptibility to glutamate excitotoxicity
patient populations (104). As the number of genes linked and neuronal firing abnormalities (108-110). Furthermore,
to ALS has increased, many patients have been found to administering a mouse-specific C9orf72 ASO to the lateral

© Annals of Translational Medicine. All rights reserved. www.atmjournal.org Ann Transl Med 2016;4(2):27
Page 8 of 12 Zou et al. Toward precision medicine in ALS

ventricle of adult mice, using a single intracerebroventricular Conclusions

stereotactic injection, resulted in a significant reduction
Together, the key elements of phenotypic classification,
of C9orf72 RNA levels in the spinal cord and the brain
comprehensive risk assessment, detecting a presymptomatic
3 weeks after injection (110). In this study, the ASO was
period, studying potential molecular pathways, developing
also detected throughout the CNS. However, no functional
disease models, discovering biomarkers and tailoring
or behavioral alterations in strength, motor coordination,
interventions to molecular specifics embody a precision
activity or anxiety were reported even at 17 weeks after the
medicine approach. This approach should provide a
injection (110). These preliminary results indicated that the
strategy for optimal targeting and timing of efforts to
C9orf72 suppression strategy might be safe and long-lasting
prevent, stop or slow progression of ALS. Neuroscientists,
in animal models. Given the high prevalence of C9orf72
neuropathologists and clinical researchers should work
mutations in ALS patients of Caucasian origin, ASO could
closely together to bring phenomics, genomics, proteomics,
represent a promising approach for these C9orf72-mutated
metabolomics and other unbiased approaches to bear on the
ALS patients.
problem, ensuring that future therapeutic strategies will be
based on a solid molecular understanding of pathological
ATXN2 targeted therapeutic strategies mechanisms, identified in robust animal and cellular
Intermediate-length polyQ expansions in ATXN2 were models.
convincingly associated with an increased risk for ALS and
functional screens identified ataxin-2 as a disease-modifying Acknowledgements
factor (23,24,111). Effects of intermediate-length repeat
expansions may occur not only at the protein but also at Funding: The study was supported by the Joint Fund
the RNA level, similar to what has been observed in other of Natural Science Foundation and Health Industrial
disorders caused by expanded repeat regions. Given that Foundation (grant No. 2020174), as well as Key Clinical
therapeutic strategies aimed at targeting polyQ expansions Specialty Discipline Construction Program of Fujian and
have been successfully developed and applied in other Nation P.RC.
disorders, such as Huntington’s disease (112), ataxin-2
is likely to be a promising therapeutic target in ALS. Footnote
ASOs and genome engineering techniques are promising
approaches to target intermediate-length repeats in ATXN2. Conflicts of Interest: The authors have no conflicts of interest
However, because manipulation of ataxin-2 may trigger to declare.
unwanted side effects, such therapeutic approaches would
require extensive preclinical testing (113). References

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103. Bede P, Hardiman O. Lessons of ALS imaging: Pitfalls
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