Review Paper

Early detection of Amyotrophic Lateral Sclerosis using Proteomic and Metabolomic studies to identify biomarkers

Torres Juan C.

UPR Cayey, Department of Biology, PR.

Abstract

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease that affects the motor

neurons. It has no cure, but treatments are being made that extend life expectancy. Biomarkers is

a topic that is associated with the early diagnosis of diseases. Scientists are trying to find

biomarkers that could help in the early diagnosis and prognosis of ALS, and for the utilization in

routine patient checkup. Two fields of science that are studying specific biomarkers for early

diagnosis in ALS are Proteomics and Metabolomics, with progress in finding biomarkers for ALS.

They utilized different techniques to find biomarkers using the same sample collection from

cerebrospinal fluid or plasma. The results for the search of biomarker for ALS using metabolomics

showed that 17 metabolites could be identified as possible biomarkers while only three

biomarkers for proteomics were identified. Of all the biomarker related to ALS, cystatin C is the

closest specific biomarker that scientist have for the moment. Science found some biomarkers in

both of these areas, but those specific biomarkers have still to go through validation and more

studies need to be performed.

Introduction

Amyotrophic lateral sclerosis (ALS) is a

progressive neurodegenerative disease

associated with a life expectancy of

approximately 3 years after symptom onset,

but the range of survival extends from a few

months in some patients to decades for

approximately 5% of the patients diagnosed

with the disease (Gordon 2011). This is the

third most common neurodegenerative

disease after Alzheimer and Parkinson

disease with a prevalence of 4-6 per 100.000

individuals and a mean age of onset of about

56 years. As a motor neuron disease, ALS is

characterized by the selective progressive

degeneration of motor neurons in the brain,

brainstem and spinal cord (Süssmuth et al.

2008). Unfortunately, there is only one drug

currently approved by the FDA to treat ALS,

which is riluzole, and this therapy increases

life span by just two to three months on

average (Wilson et al. 2010). There is a need

in finding an early diagnosis of this disease;

more scientists are looking for biomarkers

that help give an early prognosis of the

disease. Biomarkers in human blood or

cerebrospinal fluid (CSF) are urgently

needed for diagnosis, evaluation, and

effective treatment in ALS and other

neurodegenerative diseases (Wuolikainen et

al. 2009). Two fields in science, Proteomics

and Metabolomics, are searching for more

specific biomarkers for ALS. At the present

time, no verified specific biomarkers exist for

ALS and diagnosis is currently made

following clinical examination,

neurophysiological tests and various simple

chemical tests to exclude other conditions

with similar symptoms. The present

protocols for diagnosis lead to delays and

may result in misdiagnosis (Wuolikainen et

al. 2009). Biomarkers also hold promise to

monitor disease progression and to stratify

patient populations for use in clinical trials.

Biomarkers that monitor disease progression

would aid in the design and execution of

human clinical trials and would provide

novel targets for future drug therapies

Prognostic biomarkers that predict patient

survival would also aid in the design of

clinical trials (Wilson et al. 2010).

Proteomics profiling helps to find biomarkers

in the identification of ALS specific protein

biomarkers that may provide insight into the

nature of the degenerative process.

Additionally, biomarkers found through

proteomics profiling may be useful both in

the diagnosis of ALS and in monitoring the

effect of the therapeutic interventions

(Ranganathan et al. 2005). On the other hand,

metabolomics offers unique possibilities to

screen for molecular biomarkers in human

biofluids and tissues (Wuolikainen et al.

2009). In comparison, proteomics has

advanced more in identifying specific

biomarkers than Metabolomics. They both

use cerebrospinal fluid and plasma for their

experimental trials. From proteomic profiling

three biomarkers were identified that

decreased transthyretin, cystatin C or

increased carboxy-terminal fragment of

neuroendocrine protein 7B2 in ALS CSF

(cerebrospinal fluid) (Ranganathan et al.

2005). Cystatin C is a possible candidate for

biomarker that has been extensively

researched. It is a widely expressed cysteine

protease inhibitor that is approximately five

times more abundant in CSF than in plasma.

The challenge is to make cystatin C clinically

useful as a diagnostic biomarker. To do this

it must also be able to differentiate between

ALS patients and individuals with neurologic

diseases that closely resemble ALS, or ALS

‘‘mimic diseases.’’ (Wilson et al. 2010). In

any event, these findings contribute to the

advancement of science, and point to cystatin

C as a leap forward in the right direction.

Metabolomic Studies on ALS research

Metabolomics is a newborn cousin to

genomics and proteomics. (I would

eliminate this sentence. It is too much to

digest and loses me as a reader.)

Metabolomics is increasingly being used in a

variety of health applications including

pharmacology, pre-clinical drug trials,

toxicology, transplant monitoring, newborn

screening and clinical chemistry. The

growing field called Metabolomics detects

and quantifies the low molecular weight of

molecules, known as metabolites, produced

by active, living cells under different

conditions and times in their life cycles.

NMR (nuclear magnetic resonance) is

playing an important role in metabolomics

because of its ability to observe mixtures of

small molecules in living cells or in cell

extracts (Tyagi et al. 2010). Metabolomics is

concerned with the quantification and

identification of a large number of low

molecular compounds in biological samples.

For example, by means of multiple sample

comparisons, single metabolites or patterns

of metabolites are extracted to see if their

concentration is significantly altered in

relation to the onset and progression of a

specific disease or the response to a specific

treatment (Wuolikainen et al. 2009).

Recently, high-throughput techniques such as

metabolomics have been used to evaluate a

combination of markers in patients with

neurological diseases, such as ALS.

Metabolomic studies have been performed

via different analytical methods such as high

performance liquid chromatography followed

by electrochemical detection or high

resolution 1H NMR spectroscopy. NMR

spectroscopy appears to be cost-effective,

useful in routine care, and screening.

Different kinds of biological fluids have been

screened, but this researcher also confirms

that CSF may have the highest yield of

biomarkers in ALS. Some of the reasons are

because of its direct contact with the brain,

its accessibility, and its dynamic changes

with the cerebral environment (Blasco et al.

2010). A study done by Christian R. Andres

and colleagues also used metabolomics to

identify certain biomarkers. The aim of this

study was to analyze the CSF of patients with

ALS by 1H NMR (Nuclear Magnetic

Resonance) spectroscopy in order to identify

biomarkers in the early stages of the disease,

and to evaluate the biochemical factors

involved in ALS. Their results showed 17

metabolites that could be possible biomarkers

for ALS. That same study focused on the

NMR profile, but they are aware that

accuracy of early diagnosis of ALS will

depend on a combination of several

approaches like imaging,

electrophysiological and biological markers

(Blasco et al. 2010). Further studies with

larger numbers of patients and controls,

including other motor neuron diseases, will

be crucial to validate this model and to assure

its place in routine practice (Blasco et al.

2010). The conclusion of that study is that

CSF screening by NMR spectroscopy could

be a useful, simple and low cost tool to

improve the early diagnosis of ALS. The

results indicate a perturbation of glucose

metabolism, and the need to further explore

cerebral energetic metabolism.

Proteomic studies on ALS research

Proteomics encompasses the study of

expressed proteins, including identification

and elucidation of the structure-function

interrelationships that define healthy and

disease conditions (Wright and Semmes

2003). In ALS, changes in protein

composition of the cerebrospinal fluid (CSF)

or serum may denote corresponding

alterations in protein expression, post-

translational modifications or turnover within

the tissue of the central nervous system.

Because CSF contains proteins and protein

fragments released from ALS-affected

neurons and glia, it seems likely that profiles

of CSF proteins may serve as biomarkers for

the process of motor neuron degeneration in

the spinal cord in this ALS. Indeed, the

identification of ALS specific protein

biomarkers may provide insight into the

nature of the degenerative process.

Additionally, such biomarkers may be useful

both in the diagnosis of this disease and in

monitoring the response of the degenerative

process to therapeutic interventions.

Proteomic analyses have been used to

uncover biomarkers in other CNS disorders

and neurodegenerative diseases such as

multiple sclerosis, schizophrenia,

Alzheimer’s disease, and HIV-1 associated

cognitive impairment (Ranganathan et al.

2005). Robert Bowser used proteomic profile

of CSF from recently diagnosed ALS

patients and control subjects using surface-

enhanced laser desorption/ionization time-of-

flight mass spectrometry (SELDI-TOF-MS)

for his study to find specific biomarkers.

Three biomarkers were identified that could

be possible biomarkers which are

transthyretin, cystatin C and carboxy-

terminal fragment of neuroendocrine protein

7B2 in ALS CSF. The cysteine protease

inhibitor cystatin C has recently gained

interest as a candidate diagnostic biomarker

for ALS, but further studies are required to

fully characterize its biomarker utility.

Cystatin C levels in ALS patients were

significantly elevated in plasma and reduced

in CSF compared to healthy controls.

Cystatin C is also linked to ALS

histopathologically, as it is one of only two

known proteins that localize to Bunina

bodies. Bunina bodies are small

intraneuronal inclusions specific to ALS.

Plasma cystatin C has been extensively

characterized as a peripheral biomarker for

kidney function and as a prognostic indicator

of the risk of morbidity and mortality relating

to cardiovascular disease. However, blood-

borne levels of cystatin C have not been

evaluated as a biomarker candidate for

neurologic disorders such as ALS. (Wilson et

al. 2010).

Conclusion

Amyotrophic lateral sclerosis is a disease that

affects a small portion of the population of

the United States. A cure for the disease has

not been found and the struggle in finding

new therapies or techniques that extend the

life span of the patient are being looked at.

Proteomics and Metabolomics are new

studies that can be applied for an early

detection of amyotrophic lateral sclerosis.

These advances are great, but there is still

progress to be made, the results in those

studies still require validation. Both of these

areas of research have more pros than cons

and are an improvement towards finding the

cause and a cure for ALS then most scientist

imagine. These studies also bring new

techniques and forms to detect biomarkers

for an early prognosis of ALS. Proteomics

and Metabolomics have helped in other type

of diseases such as multiple sclerosis,

schizophrenia, Alzheimer’s disease, and

HIV-1 associated cognitive impairment.

Also, they have been recently helpful in the

area of cancer for early prognosis. In

conclusion, additional studies need to be

conducted so that these techniques could

eventually be performed in a patient’s

routine checkup.

Reference

Blasco H, Corcia P, Moreau C, Veau S, Fournier C, Vourc’h P, Emond Patrick, Gordon P, Pradat PF, Praline J, Devos D, Desbarats L and Andres CR. 2010. H-NMR-Based Metabolomic Profiling of CSF in Early Amyotrophic Lateral Sclerosis. Plos One. 5(10):e13223

Gordon P. Amyotrophic Lateral Sclerosis: Pathophysiology, Diagnosis and Management. 2011. CNS Drugs. 25(1):1-16

Ranganathan S, Williams E, Ganchev P, Gopalakrishnan V, Lacomis D, Urbinelli à L, Newhall K, Cudkowicz ME, Brown Jr. RH and Bowser R. 2005. Proteomic profiling of cerebrospinal fluid identifies biomarkers for amyotrophic lateral sclerosis. Journal of Neurochemestry. 95:1461–1471

Süssmuth S, Brettschneider J, Ludolph A and Tumani Hayrettin. Biochemical Markers in CSF of ALS Patients. 2008. Current Medical Chemistry. 15:1788-1801

Tyagi S, Raghvendra, Singh U, Kalra T and Munjal K. Applications of metabolomics - a systematic study of the unique chemical fingerprints: an overview. 2010. International Journal of Pharmaceutical Sciences Review and Research. 3(1):83-86

Wilson ME, Boumaza I, Lacomis D and Bowser R. Cystatin C: A Candidate Biomarker for Amyotrophic Lateral Sclerosis. 2010. Plos One. 5(12):e15133.

Wright G and Semmes O. Proteomics in Health and Disease. 2003. Journal of Biomedicine and Biotechnology. 4:215-216

Wuolikainen A, Moritz T, Marklund SL, Antti H, Munch P. Disease-Related Changes in the Cerebrospinal Fluid Metabolome in Amyotrophic Lateral Sclerosis Detected by GC/TOFMS. 2011. Plos One. 6(4):e17947

Wuolikainen A, Hedenstro M, Moritz T, Marklund SL, Antti H and Andersen PM. Optimization of procedures for collecting and storing of CSF for studying the metabolome in ALS. 2009. The World Federation Of Neurology Research Group On Motor Neuron Diseases. 10(4):229-236