Two-stage expression of neurofilament polypeptides during rat neurogenesis with early establishment of adult phosphorylation patterns

Abstract

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.

Adult bone marrow stromal stem cells express germline, ectodermal, endodermal, and mesodermal genes prior to neurogenesis

“…Here, we employed NF-M expression as a temporal, quantitative index of neuronal differentiation to help place the present observations in context. NF-M expression is associated with initiation of neuritogenesis, neural process outgrowth, and assumption of the characteristic mature neuronal morphology (Carden et al, 1987). Consistent with our previous findings, NF-M mRNA was undetectable in uncommitted MSCs but was present after 48 hr of neuronal induction.…”

Clusterin secreted by astrocytes enhances neuronal differentiation from human neural precursor cells

“…10 Phosphorylation of neurofilaments during development is fundamental to maturation and stabilization of the neuronal cytoskeleton, as well as an increase in axonal diameter. 11 We show that hippocampal and midbrain ACM also increase the neurite length of pNF-positive neurons, indicating their role in neuronal maturation. Cortical ACM had no such effect.…”

?-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.…”