In this study we have used SELDI-TOF-MS to profile CSF and identify biomarkers for ALS. We report 30 spectral peaks with statistically significant differences in peak intensities between ALS and control subjects (p < 0.01). RL identified 10 m/z peaks from a training set of 24 subjects that predicted ALS disease status in coded test subjects (N = 20) with 80% sensitivity, 60% specificity and 74% accuracy. Increasing the training set to 40 subjects revealed nine additional m/z peaks that increased the specificity to 100% and accuracy to 91% for a separate testing group of 12 subjects. We have determined the identity of three biomarker peaks that RL analysis recognized as having high diagnostic predictive value; as compared to control CSF, two were decreased (TTR and cystatin C) and one was increased (the carboxy-terminal fragment of the neuroendocrine protein 7B2) in ALS CSF. The mass spectrometry results for TTR and cystatin C were confirmed using immunoblot and immunohistochemistry on two separate and distinct cohorts of ALS and control subjects. A total of 112 subjects (57 ALS and 55 healthy and neurologic disease controls) collected at two medical centers were used in this study for biomarker discovery and validation.
We note that SELDI-TOF-MS permits only a limited analysis of proteins of high molecular mass and therefore additional biomarkers that were not identified in this study may be present within this mass range. Additional studies with a larger cohort of control and ALS subjects are necessary to both confirm and validate these initial findings and further establish the disease specificity for the biomarker panel. With increased sample size, our studies may also be extended to distinguish a spectrum of motor neuron diseases.
A diagnosis of ALS is typically made after exhaustive clinical tests over many months to eliminate other potential causes for the presenting symptoms, and patients often exhibit symptoms for months prior to seeking medical evaluation. A panel of predictive biomarkers will aid in a more rapid clinical diagnosis, permit initiation of therapy at or near the onset of clinical symptoms, and avoid unnecessary or improper treatment interventions. It has been reported that up to 10% of ALS diagnoses are false-positive and up to 44% may be false-negative (Brooks 1999
). Therefore a rapid and accurate diagnostic test would be quite beneficial for ALS patients and families. For this study we used CSF samples obtained from subjects during initial clinical diagnostic evaluation or at the time of clinical diagnosis to identify biomarkers near the time of disease onset. However CSF samples were not included in this study until a definitive clinical diagnosis was complete. Of the healthy control subjects misclassified, one subject had leg stiffness with an unknown diagnosis (though multiple sclerosis has been ruled out), and another experienced arm numbness with a family history of neurodegenerative disorders. The biomarker panel failed to correctly predict ALS for two additional test subjects with a time from symptom onset to CSF draw of 1339 and 3913 days (data not shown). This suggests that the biomarker signature pattern may change during disease progression. Longitudinal studies are required to examine how the proteomic biomarker signature pattern changes during disease progression within individual ALS patients.
RL analysis confirmed that no individual biomarker peak provides accurate diagnostic predictions in our training or coded test groups. However RL determined that using only the 7B2CT (3.42 kDa) and TTR (6.88 kDa) biomarker peaks provided 80% sensitivity and 72% specificity for ALS in the small coded test group (N = 12). These data support our contention that a panel of biomarkers is required for diagnostic predictions with greater than 90% accuracy. Overall, no individual or pair-wise combination of biomarkers provides diagnostic predictions with the accuracy of the complete biomarker panel, though the combination of 3.42, 6.88 and 13.38 kDa biomarker peaks exhibited significant diagnostic predictive value and provided the basis for determining the protein identity of these peaks.
The protein level of 7B2CT (3.42 kDa) increases in ALS, whereas levels of both TTR (13.78 kDa) and cystatin C (13.38 kDa) decrease. Human 7B2 protein is localized to the secretory granules of neurons (including motor neurons) and endocrine cells (Marcinkiewicz 1993
; Marcinkiewicz et al. 1994
). It interacts with proprotein convertase 2 (PC2) within the trans
-Golgi network and aids in the maturation of pro-PC2. Mature PC2 then catalyzes the conversion of hormone and neuropeptide precursors into their active forms. In addition, 7B2 has been shown to function as a chaperone in the maturation of growth factors such as IGF-1 (Chaudhuri 1995
). Furin cleaves 7B2 within the Golgi into a 21 kDa fragment and a 3.4 kDa carboxy terminal fragment called 7B2CT. 7B2CT inhibits the maturation and function of PC2 (Zhu 1996
; Hwang and Lindberg 2001
). Thus, 7B2 is an important chaperone in the secretory pathway for the proper maturation and release of numerous hormones, neuropeptides and growth factors, and 7B2CT negatively regulates the function of PC2. Increased levels of 7B2CT in ALS subjects may be a reactive response to altered enzymatic activities that generate or degrade 7B2CT. Alternatively, increased levels of 7B2CT in ALS may result from Golgi fragmentation within motor neurons during ALS (Gonatas 1992
The observation that cystatin C and TTR levels are decreased in ALS CSF near the time of symptom onset is of considerable interest but must be interpreted cautiously. One interpretation is that the protein deficiency is causal, triggering one or more cascades of molecular events that reduce motor neuron viability. On the other hand, reduced protein levels in a neurodegenerative disorder like ALS may represent an expected, secondary consequence of neuronal cell loss.
Cystatin C is a 13.3 kDa secreted protein that belongs to the class of cysteine protease inhibitors and plays an important role in regulating extracellular protein homeostasis in the CNS. The choroid plexus is a major site for the synthesis of cystatin C and CSF concentrations of this protein are ~5.5 times that of serum (Davidsson et al. 1997
). Decreased levels of cystatin C in ALS may indicate increased proteolysis via cysteine proteases. Cystatin C levels are altered in other neurodegenerative diseases such as Alzheimer's disease (AD) and Creutzfeldt–Jakob disease, and in models of pain (Deng 2001
; Levy 2001
; Kalso 2004
; Sanchez 2004
). A recent SELDI-TOF-MS analysis of CSF from AD subjects revealed increased cystatin C levels in AD (Carrette et al. 2003
), suggesting cystatin C protein levels may exhibit distinct alterations in different neurodegenerative disorders. Cystatin C is localized to Bunina bodies, a specific neuropathologic hallmark of ALS contained in degenerating motor neurons (Okamoto 1993
; van Welsem 2002
; Seilhean 2004
). Mutations in the cystatin C gene are associated with a rare hereditary brain amyloid angiopathy that also results in decreased CSF levels of cystatin C and increased amyloid aggregation and deposition (Coria and Rubio 1996
). Further studies will be pursued to explore potential functional links between cystatin C, protein aggregation and ALS.
In our mass spectrometry study, TTR was resolved into a series of m/z peaks and levels of specific TTR m/z peaks were reduced in ALS CSF. TTR is synthesized predominately in cells of the choroid plexus and liver and secreted into the CSF or plasma, respectively (Schreiber 2002
). TTR is also produced in neurons and we report that motor neurons express TTR (). Reduced level of TTR in ALS spinal cord is likely due to a reduction both in the numbers of motor neurons and in the levels of TTR expression within remaining motor neurons. TTR immunoreactivity was also observed in other cell types in the spinal cord, which will be the focus of future studies. We also note that TTR levels are reduced in the CSF of postmortem ALS subjects when compared to healthy controls (data not shown), indicating that reduced levels of TTR spectral peaks in ALS CSF occurs early in the disease process and continues throughout the course of disease. TTR is required for the transport of thyroxine and transport of retinol/vitamin A via interactions with retinol-binding protein (Monaco 2000
; Power 2000
). Decreased levels of TTR have been noted in the CSF of late stage AD patients (Serot et al. 1997
), indicating that decreased TTR levels is not unique to ALS. A recent study indicates that TTR has neuroprotective functions in a transgenic mouse model of AD (Stein et al. 2004
). The basis for the neuroprotective effect has not been delineated. Because TTR is known to form aggregates and bind multiple proteins, it is conceivable that TTR deficiency might lead to inadequate sequestration of abnormally functioning proteins. We also note that polymorphisms of the TTR gene induce increased protein aggregation and result in familial amyloid polyneuropathy and familial amyloid cardiomyopathy (Saraiva 2001
). Considered together, these findings suggest that low levels of TTR in ALS motor neurons could reduce its neuroprotective function and thus be more susceptible to neurodegenerative insults, a hypothesis that merits further analysis.