Monoclonal antibodies distinguish several differentially phosphorylated states of the two largest rat neurofilament subunits (NF-H and NF-M) and demonstrate their existence in the normal nervous system of adult rats

Abstract

A new panel of greater than 300 monoclonal antibodies (mAbs) was prepared to the high, middle, and low Mr rat neurofilament (NF) subunits (NF-H, NF-M and NF-L, respectively). NF proteins were purified both from native, i.e., phosphorylated rat NFs and from enzymatically dephosphorylated rat NFs. The resulting mAbs were used to biochemically and immunochemically distinguish and characterize distinct and differentially phosphorylated isoforms of NF subunits. By immunoblot, all mAbs specific for NF-L and some mAbs specific for NF-M detected their specific NF subunit regardless of whether or not the NFs had been treated with alkaline phosphatase, and such antibodies were termed "phosphate-independent" or P[ind] mAbs. The other mAbs were specific for NF-M, NF-H, or for both NF-M and NF-H, and they recognized epitopes in the COOH termini of these subunits. Significantly, the latter mAbs could discriminate different isoforms of NF-M and NF-H, depending on the phosphorylation state of each variant. Such mAbs were assigned to one of 4 distinct categories on the basis of their performance in immunoblots of progressively dephosphorylated rat NF samples and by immunohistochemistry of various adult rat nervous tissues: (1) P[-] mAbs preferentially stained neuronal perikarya and dendrites, and they recognized only extensively dephosphorylated (and nonphosphorylated) NF-H; (2) P[+] mAbs stained axons more strongly than perikarya, and primarily blotted phosphorylated, but not nonphosphorylated, forms of NF-H and NF-M; (3) P[++] mAbs stained axons almost to the exclusion of perikarya, and in blots recognized only the extensively phosphorylated forms of NF-H and NF-M (i.e., subunits subjected to limited enzymatic dephosphorylation); (4) P[ ] mAbs also predominantly stained axons, but the briefest alkaline phosphatase treatment abolished the NF-M and NF-H immunobands produced by these mAbs. Two-dimensional gel analysis and immunoblotting of total proteins from adult rat dorsal root ganglion verified mAb specificity in situ, and showed that differentially phosphorylated isoforms of NF-M and NF-H occur in vivo. This provided additional evidence that mAbs can detect all 4 phosphorylation-dependent endogenous isoelectric variants of NF-H and NF-M.(ABSTRACT TRUNCATED AT 400 WORDS)

Ammonium-Induced Impairment of Axonal Growth Is Prevented through Glial Creatine

“…For histological staining, 16-m-thick cryosections were prepared, postfixed 1 hr in 4% paraformaldehyde (PFA) in PBS, and stained by hematoxylin coloration or Bielschowsky's silver impregnation (Cox, 1977). N N-18 (Chemicon International, Temecula, CA; Shaw et al, 1986;Harris et al, 1991) and RMO-44 (Zymed Laboratories, San Francisco, CA) (Lee et al, 1987) monoclonal antibodies were used for immunohistochemistry against N F-M. Axonal growth cones were detected with a monoclonal antibody directed against growth cone-associated protein 43 (GAP43; Chemicon International). Astrocytes and oligodendrocytes were labeled using monoclonal antibodies directed against glial fibrillary acidic protein (GFAP; Chemicon International) and myelin basic protein (MBP; Boehringer Ingelheim).…”

?-Internexin aggregates are abundant in neuronal intermediate filament inclusion disease (NIFID) but rare in other neurodegenerative diseases

“…Anti-NF antibodies used in this study are well characterized and have been used previously to demonstrate epitopes of NF in neuronal inclusions in AD, Lewy body diseases, and MND [6,19,25,26,32,33,34,35]. In addition, antibodies to α-synuclein [10], cytoskeletal, and other proteins associated with abnormal protein aggregates in neurodegenerative diseases were used in this study.…”

“…Recently, we described α-internexin as a major component of the pathological hallmark of NIFID [5]. Although previous studies have demonstrated the co-localization of NF epitopes in AD, PD, DLB, and MND [19,25,26,28,29,32,33,34,35], no study has demonstrated the presence of α-internexin as a component of the pathological inclusions of any neurodegenerative disease other than NIFID.…”

“…All three subunits are phosphorylated and most of the phosphorylation sites are located in the tail domain of NF-H [25,36]. The use of phosphorylation-dependent and -independent antibodies to NF epitopes has enabled the immunohistochemical dissection of these proteins and has revealed that NFs within the perikaryon and proximal segments of axons and dendrites are normally hypophosphorylated, while NFs in axons are heavily phosphorylated.…”

Functional genomics and psychiatric illness

“…One section from each subject was stained with acridine orange to verify the presence of nucleic acids in the tissue (Figure 2). An adjacent section was immunostained with a monoclonal antibody to non-phosphorylated neurofilament (RmdO20; Lee et al, 1987) to identify individual neurons for subsequent single cell analysis. Following in situ transcription, Layer II stellate neurons were dissected and contents were amplified using aRNA amplification (Tecott et al, 1988;VanGelder et al, 1990;Eberwine et al, 1992).…”