Specific human astrocyte subtype revealed by affinity purified GFAP antibody; unpurified serum cross-reacts with neurofilament-L in Alzheimer.
The human GFAP splice variants GFAPDelta164 and GFAPDeltaexon6 both result in a GFAP protein isoform with a unique out-of-frame carboxy-terminus that can be detected by the GFAP+1 antibody. We previously reported that GFAP+1 was expressed in astrocytes and in degenerating neurons in Alzheimer's disease brains. In this study we aimed at further investigating the neuronal GFAP+1 expression and we started by affinity purifying the GFAP+1 antibody. The purified antibody resulted in a loss of neuronal GFAP+1 signal, although other antibodies directed against the amino- and carboxy-terminus of GFAPalpha still revealed GFAP-immunopositive neurons, as described before. With an in-depth analysis of a western blot, followed by mass spectrometry we discovered that the previously detected neuronal GFAP+1 expression was due to cross-reactivity of the antibody with neurofilament-L (NF-L). This was confirmed by double-label fluorescent immunohistochemistry and western blotting with the unpurified GFAP+1 antibody and an antibody against NF-L. Our data imply that NF-L can accumulate in some tangle-like structures in Alzheimer brains. More importantly, the purified GFAP+1 antibody clearly revealed a specific subtype of astrocytes in the adult human brain. These large astrocytes are present throughout the brain, e.g., along the subventricular zone, in the hippocampus, in the striatum and in the spinal cord of controls, Alzheimer, and Parkinson patients. The presence of a specific GFAP-isoform suggests a specialized function of these astrocytes.
Middeldorp
Jinte
J
Department of Astrocyte Biology & Neurodegeneration, Netherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences, Amsterdam, The Netherlands.
van den Berge
Simone A
SA
Aronica
Eleonora
E
Speijer
Dave
D
Hol
Elly M
EM
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000544:Alzheimer Disease / Q000276:immunology; D000595:Amino Acid Sequence; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism; D001921:Brain / Q000378:metabolism; D016022:Case-Control Studies; D005260:Female; D005904:Glial Fibrillary Acidic Protein / Q000737:chemistry* / Q000276:immunology; D006624:Hippocampus / Q000378:metabolism; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D016900:Neurofilament Proteins / Q000737:chemistry / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D005904:Glial Fibrillary Acidic Protein; D016900:Neurofilament Proteins; C044882:neurofilament protein L