Paul S. Shneidman

The structure and organization of the human heavy neurofilament subunit (NF-H) and the gene encoding it.

Genomic clones for the largest human neurofilament protein (NF‐H) were isolated, the intron/exon boundaries mapped and the entire protein‐coding regions (exons) sequenced. The predicted protein contains a central region that obeys the structural criteria identified for alpha‐helical ‘rod’ domains typically present in all IF protein components: it is approximately 310 amino acids long, shares amino acid sequence homology with other IF protein rod domains and displays the characteristic heptad repeats of apolar amino acids which facilitate coiled‐coil interaction. Nevertheless, anomalies are noted in the structure of the NF‐H rod which could explain observations of its poor homopolymeric assembly in vitro. The protein segment on the carboxy‐terminal side of the human NF‐H rod is uniquely long (greater than 600 amino acids) compared to other IF proteins and is highly charged (greater than 24% Glu, greater than 25% Lys), rich in proline (greater than 12%) and impoverished in cysteine, methionine and aromatic amino acids. Its most remarkable feature is a repetitive sequence that covers more than half its length and includes the sequence motif, Lys‐Ser‐Pro (KSP) greater than 40 times. Together with the recent identification of the serine in KSP as the main target for NF‐directed protein kinases in vivo, this repetitive character explains the massive phosphorylation of the NF‐H subunit that can occur in axons. The human NF‐H gene has three introns, two of which interrupt the protein‐coding sequence at identical points to introns in the genes for the two smaller NF proteins, NF‐M and NF‐L.(ABSTRACT TRUNCATED AT 250 WORDS)

mRNA levels of all three neurofilament proteins decline following nerve transection

The control of neurofilament (NF) protein gene expression was studied by determining and comparing the levels of mRNA to the heavy (NF-H), mid-sized (NF-M) and light (NF-L) NF protein subunits in rat dorsal root ganglia (DRG) following sciatic nerve transection. mRNA to NF-H (4.5 kb), to NF-M (3.4 kb) and to NF-L (2.5 and 4.0 kb) were identified in Northern blots and quantitated in dot blot analyses, using specific cDNA probes for each NF protein. Following transection and continuing for at least 28 days. The early and co-terminal fall in mRNAs suggests that the 3 NF genes are regulated by common factor(s) and that the function of these factor(s) is influenced by the state of axonal continuity with the target organ.

The structure of the largest murine neurofilament protein (NF-H) as revealed by cDNA and genomic sequences

The complete primary structure of the largest mammalian neurofilament component, NF-H, is predicted from mouse cDNA and genomic clones, revealing a protein of molecular weight ca. 115,000. A central filament-forming domain structurally typical of all intermediate filament proteins is present, but anomalies are noted which may place constraints on the mechanism of NF-H assembly into filaments. The COOH-terminal portion of the protein is extremely long (661 amino acids) by comparison to non-neuronal intermediate filament components and has a remarkably monotonous, highly charged composition (Glu and Lys at 20% each). Its most remarkable feature is a tandem repeat of a 6 amino acid sequence containing the motif Lys-Ser-Pro that extends for more than half the length of the COOH-terminus. The Lys-Ser-Pro motif appears 48 times and since it is now known that the serine therein is a target for in vivo kinases, the massive axonal phosphorylation of NF-H is explained. Comparison of mouse and human NF-H reveals that otherwise conserved proteins have been subjected to evolutionary mutation within their multiphosphorylation repeat domains, although the Lys-Ser-Pro motif has been conserved.

Stabilization of neurofilament transcripts during postnatal development

Neurofilament (NF) mRNAs in primary sensory neurons are long-lived transcripts that undergo transcription-dependent destabilization when placed in primary culture [32]. Destabilization of NF transcripts implies that the transcripts are stabilized in high-expressing neurons and that stabilization may coordinate and increase levels of NF expression. The present study examines the stabilities of the three NF subunit mRNAs in postnatal cultures of dorsal root ganglia (DRG) to determine whether increased stability of NF mRNAs could be responsible for the coordinate postnatal upregulation of the three NF subunits [29]. The studies show that the light (NF-L), mid-sized (NF-M) and heavy (NF-H) NF mRNAs are lost at 8 and 16 h in primary cultures from postnatal day 2 (P2) rats, but much less so in cultures from postnatal day 16 (P16) and day 30 (P30) rats. Losses of each NF mRNAs in P2 cultures occurs simultaneously in the presence or absence of actinomycin. The findings support the view that stabilization of NF transcripts contribute to the high and coordinate level NF expression and that components of the stabilizing process are acquired during postnatal development.

Deletion of 3′-Untranslated Region Alters the Level of mRNA Expression of a Neurofilament Light Subunit Transgene

High levels of neurofilament (NF) mRNA expression are attained during early postnatal development and are a major determinant of axonal size. High level NF expression is also dependent upon axonal continuity since NF mRNA levels are down-regulated after nerve transection. This study shows that both postnatal up-regulation and axotomy-induced down-regulation are altered by deletion of 3′-UTR from the mouse light NF subunit (NF-L). Transgenes with (NF-L+) or without (NF-L−) 3′-UTR display similar patterns of neuron-specific expression but differ in their respective levels of expression. Whereas changes in the level of NF-L+ mRNA parallel those of the endogenous mouse NF-L mRNA, changes in the level of NF-L− mRNA differ from the pattern of endogenous NF-L expression during postnatal up-regulation and axotomy-induced down-regulation. Specifically, the NF-L− transgene undergoes a 3-fold aberrant up-regulation between embryonic days 15 (E15) and 18 (E18) and has lost its susceptibility to axotomy-induced down-regulation. Studies of transfected P19 cells show that 3′-UTR deletion leads to a severalfold stabilization of NF-L mRNA and an increase in steady-state mRNA level. The findings support the working hypothesis that the 3′-UTR contains determinants that alter stability and that stabilization of NF-L mRNA regulates the levels of NF-L mRNA in neuronal tissues and cells.