TDP-43 in cerebrospinal fluid of patients with frontotemporal lobar degeneration and amyotrophic lateral sclerosis

Abstract

Background: Recently, TAR DNA-binding protein 43 (TDP-43) was identified as the major component of ubiquitin-positive tau-negative neuronal and glial inclusions in the most common form of frontotemporal lobar degeneration (FTLD) and in amyotrophic lateral sclerosis (ALS). It was demonstrated that different TDP-43 profiles correspond to clinical phenotypes of FTLD or ALS subgroups, and the differential diagnostic potential of TDP-43 was suggested.

Objectives: To examine TDP-43 in cerebrospinal fluid (CSF) and to analyze whether it could serve as a diagnostic marker.

Design: We characterized CSF TDP-43 by immunoblot using different TDP-43 antibodies and determined the relative TDP-43 levels in CSF samples from patients.

Setting: Academic research.

Patients: Twelve patients with FTLD, 15 patients with ALS, 9 patients with ALS plus FTLD, 3 patients with ALS plus additional signs of frontal disinhibition, and 13 control subjects.

Main outcome measures: Results of TDP-43 immunoblot.

Results: Polyclonal TDP-43 antibodies recognized a 45-kDa band in all analyzed samples. Two monoclonal and N-terminus-specific antibodies did not detect any specific bands, but C-terminus-specific antibodies detected a 45-kDa band and additional bands at approximately 20 kDa in all CSF samples. Relative quantification of 45-kDa bands revealed significant differences among the diagnostic groups (P =.046). Specifically, patients with ALS (P =.03) and FTLD (P =.02) had higher TDP-43 levels than controls but with a prominent overlap of values.

Conclusion: Although there is no evidence of pathologically altered TDP-43 proteins in CSF, TDP-43 levels in CSF might aid in characterizing subgroups of patients across the ALS and FTLD disease spectrum.

Figure 1

Western immunoblot analyses of TAR DNA-binding protein 43 (TDP-43) applying rabbit polyclonal antibody. Lane 1 is the mouse RIPA (radioimmunoprecipitation assay) buffer homogenate. A band of approximately 46 kDa (upper arrow in A and B) was used as an internal standard for quantification of cerebrospinal fluid (CSF) 45-kDa TDP-43 bands (middle arrow in A and B). A, In mouse brain homogenate, a major band at 46 kDa and a minor band at 42 kDa are visible (lane 2). Lane 4 shows a representative signal in human CSF with a specific TDP-43 band at 45 kDa. A band at 28 kDa was found regularly in CSF (lower asterisk in A and B) but represents unspecific binding of IgG as demonstrated by depletion of CSF (lanes 3 and 4) and by purified human IgG (5 μg; lane 5, lower arrow). Purified human albumin (10 μg, lane 6) demonstrated no immunoreactive band. B, Immunodetection of TDP-43 and IgG in CSF (lanes 2 and 3, middle and lower arrows). In the urea fraction of brain tissue of frontotemporal lobar degeneration with ubiquitin-positive tau-negative inclusions using polyclonal TDP-43 antibody (lane 4), 2 major bands between 47 and 50 kDa and 2 minor bands at 44 and approximately 60 kDa are detected (4 lower asterisks). In addition, a high molecular mass smear is detected (lane 4, upper asterisk).

Figure 2

TAR DNA-binding protein 43 (TDP-43) immunoblot applying monoclonal antibody (Proteintech Group Inc, Chicago, Illinois). In 2 cerebrospinal fluid samples (lanes 1 and 3) and in IgG (lane 2), no specific bands are detected. In the urea fraction of brain tissue of frontotemporal lobar degeneration with ubiquitin-positive tau-negative inclusions (FTLD-U), using monoclonal TDP-43 antibody, signature consisting of pathologically phosphorylated 46- to 50-kDa full-length TDP-43 (second and third asterisks), physiological 43-kDa TDP-43 (fourth asterisk), C-terminus-truncated 26-kDa TDP-43 (fifth asterisk), and a high relative molecular mass smear (first asterisk) are visible. In addition, a weak band at approximately 60 kDa can be seen.

Figure 3

Immunoblots of murine neuroblastoma cell homogenates (lane 1), IgG (lane 7), and 5 cerebrospinal fluid samples per blot (lanes 2-6) from the same individuals in the control group (lane 3), frontotemporal lobar degeneration (FTLD) group (lane 4), amyotrophic lateral sclerosis (ALS) plus FTLD group (lane 5), ALS plus additional signs of frontal disinhibition group (lane 6), and ALS group (lane 7). Immunodetection was performed using polyclonal TAR DNA-binding protein 43 (TDP-43) antibody (A) and monoclonal TDP-43 antibody clone 2E2-D3 (B), N-terminus—specific polyclonal antiserum (C), and C-terminus—specific polyclonal antiserum (D). Using polyclonal TDP-43 antibody, 45-kDa bands are detected in all patients (A, arrow). Neither monoclonal nor N-terminus—specific antibodies detect any specific bands (B and C), whereas bands at 45 kDa (D, upper arrow) and at approximately 20 kDa (D, lower arrow) are detected using C-terminus—specific antibodies.

Figure 4

Densitometric quantification of the 45-kDa TAR DNA-binding protein 43 (TDP-43) band recognized by polyclonal antibodies in immunoblots from 50 μL of cerebrospinal fluid samples from patients with amyotrophic lateral sclerosis (ALS) (n=15), ALS plus frontotemporal lobar degeneration (FTLD) (n=9), and FTLD (n=12) and from control subjects (n=13). The TDP-43 level is expressed in terms of percentages of an internal standard murine neuroblastoma cell preparation. Box plots show median values, 25% and 75% percentile values, 5% and 95% percentile values, and outliers.