|Erythropoietin produced by genetic-modified NIH/3T3 fibroblasts enhances the survival of degenerating neurons.|
Li, YC; Chen, SJ; Chien, CL
Brain and behavior
Erythropoietin (EPO) has potent neuroprotective effects. The short-term delivery of high-dose EPO seemed to improve patients' neuromuscular functions; however, excessive EPO resulted in systematically high hematocrit and thrombotic risk. In our study, we established a cellular material for future in vivo studies of neurodegenerative diseases based on EPO provided regionally at a nontoxic level.A mouse EPO cDNA was subcloned into the pCMS-EGFP vector and transfected into NIH/3T3 fibroblasts to design a biological provider that can regionally release EPO for the treatment of neurological diseases. After G418 selection, a stable EPO-overexpressing cell line, EPO-3T3-EGFP, was established. To further confirm the neuroprotective abilities of secreted EPO from EPO-3T3-EGFP cells, a cell model of neurodegeneration, PC12-INT-EGFP, was applied.The expression level of EPO was highly elevated in EPO-3T3-EGFP cells, and an abundant amount of EPO secreted from EPO-3T3-EGFP cells was detected in the extracellular milieu. After supplementation with conditioned medium prepared from EPO-3T3-EGFP cells, the survival rate of PC12-INT-EGFP cells was significantly enhanced. Surprisingly, a fraction of aggregated cytoskeletal EGFP-tagged α-internexin in PC12-INT-EGFP cells was disaggregated and transported into neurites dynamically. The immunocytochemical distribution of IF proteins, including NF-M, phosphorylated-NF-M, and the α-INT-EGFP fusion protein, were less aggregated in the perikaryal region and transported into neurites after the EPO treatment.The established EPO-overexpressing NIH/3T3 cell line, EPO-3T3-EGFP, may provide a material for future studies of cell-based therapies for neurodegenerative diseases via the secretion of EPO on a short-term, high-dose, regional basis.
|Phosphate-dependent and independent neurofilament protein epitopes are expressed throughout the cell cycle in human medulloblastoma (D283 MED) cells.|
Trojanowski, J Q, et al.
Am. J. Pathol., 135: 747-58 (1989)
The low (NF-L) and middle (NF-M) molecular weight (Mr) neurofilament (NF) subunits are expressed before the high (NF-H) Mr NF subunit in embryonic neurons. Thereafter, NF-M attains its mature state of phosphorylation more rapidly than does NF-H. However, little is known about NF subunit expression during cell division. A rapidly dividing medulloblastoma cell line (D283 MED), therefore, was examined using flow cytometry, immunochemistry, and a large panel of NF subunit-specific polyclonal and monoclonal antibodies. Many of the monoclonal antibodies (MAbs) distinguished NF-H and NF-M in different states of phosphorylation. By flow cytometry, more than 90% of the D283 cells expressed NF-H and NF-M in different states of phosphorylation, and an antiserum specific for the carboxy terminus of NF-L labeled more than 60% of these cells. Furthermore, the fluorescence intensity produced by MAbs that detected phosphorylated versus nonphosphorylated NF-H and/or NF-M epitopes, appropriately decreased or increased, respectively, by preincubating the D283 cells with alkaline phosphatase. In contrast, cell staining with antibodies specific for phosphate-independent NF protein epitopes did not change substantially as a result of enzymatic dephosphorylation. These results agreed closely with those obtained from studies of normal human spinal cord NF extracts. However, NF-H, NF-M, and NF-L were expressed throughout the cell cycle in dual parameter studies of D283 cells labeled with an antibody and propidium iodide. Nevertheless, reductions in the fluorescence intensity produced with most of these antibodies late in the cell cycle suggested that NF proteins may be subject to modifications in their structure or accessibility to antibody probes during different phases of the cell cycle. These data led to the conclusion that NF subunits are expressed throughout the cell cycle in cultured human medulloblastoma cells, but that subtle changes in the immunoreactivity of these proteins occur during cell division.
|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.|
Lee, V M, et al.
J. Neurosci., 7: 3474-88 (1987)
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)
|Two-stage expression of neurofilament polypeptides during rat neurogenesis with early establishment of adult phosphorylation patterns.|
Carden, M J, et al.
J. Neurosci., 7: 3489-504 (1987)
Monoclonal antibodies (mAbs) to rat neurofilament (NF) proteins NF-L, NF-M, and NF-H were used to examine the developmental programs of NF expression in rat embryos. The ability of these mAbs to recognize differentially phosphorylated states of NF-M and NF-H (Lee et al., 1987, the preceding paper) was exploited in order to examine the temporal and spatial patterns of NF phosphorylation during early neuronal development in vivo. NF proteins were first detected on the twelfth day postfertilization (E12) using NF-L- or NF-M-specific mAbs. By E13, the coexpression of NF-L and NF-M was widespread, reflecting dramatic increases of immunoreactivity to both subunits. Partial phosphorylation, denoted P[+], of NF-M was already present in perikarya and neurites of E12 neurons. Extensively phosphorylated, or P[+++], isoforms of NF-M appeared in E13 axons, thereby establishing a proximodistal gradient of NF phosphorylation during the earliest phase of NF expression. Immunoblots of tissue homogenates revealed that most NF-M of E13 embryos exists in a partially phosphorylated, or P[+], isoform. Unequivocal staining for NF-H first appeared at E15, a time at which NF-L and NF-M had already attained their adult patterns of immunocytochemical staining. Levels of NF-H were extremely low at E15 but could be detected in all of its differentially phosphorylated states, i.e., nonphosphorylated P[-], partly P[+], and highly P[+++] phosphorylated isoforms. P[+++] isoforms of NF-H were restricted to the distal portions of E15 axons, although staining of more proximal axons, like those in adult, was noted by E17. Immunoblots of E17 embryos revealed most NF-H as P[-] and P[+] isoforms. Quantities of immunoreactive NF-H increased very slowly and remained well below those of NF-M and NF-L for several weeks beyond birth. These results show that sequential forms of NFs are expressed by developing and maturing neurons throughout the nervous system. An "immature" form of NFs, composed of NF-M and NF-L, appears to function in establishing the neuronal phenotype and in initiating and maintaining neurite outgrowth. Addition of NF-H confers a "mature" state to the NF. This delayed expression of NF-H is a slow and graduated process that coincides in time with the stabilization of neuronal circuitries and may be important in modulating axonal events, such as the slowing of cytoskeletal transport and the growth of axonal caliber.