Identification of a novel interaction of 14-3-3 with p190RhoGEF.

Activation of Rho GTPases by guanine nucleotide exchange factors (GEFs) mediates a broad range of cytoskeletal alterations that determine cell shape. In the nervous system, Rho GTPases are essential for establishing highly asymmetrical neuronal forms and may fine-tune the shape of dendrites in differentiated neurons. p190RhoGEF is a brain-enriched, RhoA-specific GEF whose highly interactive C-terminal domain provides potential linkage to multiple pathways in the cell. In the present study, a yeast two-hybrid screen was used to identify 14-3-3eta and 14-3-3epsilon as additional binding partners of p190RhoGEF. Interactions between p190RhoGEF and 14-3-3eta were confirmed biochemically and by colocalization of the respective proteins when fused to fluorescent markers and transfected in neuronal cells. We also mapped a unique phosphorylation-independent binding site (I(1370)QAIQNL) in p190RhoGEF. Deletion of the binding site abolished interactions in vitro as well as the ability of 14-3-3eta to alter the cytoplasmic aggregation of p190RhoGEF in cotransfected cells. The findings suggest a potential role for 14-3-3 in modulating p190RhoGEF activity or in linking p190RhoGEF to the activities of other pathways in the neuron.

p190RhoGEF Binds to a destabilizing element in the 3' untranslated region of light neurofilament subunit mRNA and alters the stability of the transcript.

Stabilization of neurofilament (NF) mRNAs plays a major role in regulating levels of NF expression and in establishing axonal size and rate of axonal conduction. Previous studies have identified a 68-nucleotide destabilizing element at the junction of the coding region and 3' untranslated region of the light NF subunit (NF-L) mRNA. The present study has used the destabilizing element (probe A) to screen a rat brain cDNA library for interactive proteins. A cDNA clone encoding 1068 nucleotides in the C-terminal domain of p190RhoGEF (clone 39) was found to bind strongly and specifically to the RNA probe. The interaction was confirmed using a glutathione S-transferase/clone 39 fusion protein in Northwestern, gel-shift, and cross-linkage studies. The glutathione S-transferase/clone 39 fusion protein also enhanced the cross-linkage of a major 43-kDa protein in brain extract to the destabilizing element. Functional studies on stably transfected neuronal cells showed that p190RhoGEF expression increased the half-life of a wild-type NF-L mRNA but did not alter the half-life of a mutant NF-L mRNA lacking the destabilizing element. The findings reveal a novel interactive feature of p190RhoGEF that links the exchange factor with NF mRNA stability and regulation of the axonal cytoskeleton.

Similar poly(C)-sensitive RNA-binding complexes regulate the stability of the heavy and light neurofilament mRNAs.

The potential role of RNA processing in regulating neurofilament (NF) subunit expression and in mediating the neuropathic effects of NF transgenes was explored by determining whether similar regulatory elements and cognate binding factors are present in NF mRNAs. Gel-shift studies were used to compare RNA-binding complexes that assemble on the 3'UTR of the heavy (NF-H), mid-sized (NF-M) and light (NF-L) NF mRNAs when radioactive RNA probes are incubated with high-speed supernatants (S100) of rat brain homogenates. RNA-binding complexes were characterized by their rate of migration in non-denaturing gels and by their ability to be competed with specific homoribopolymers. Similar RNA-binding complexes formed on probes to the 3'UTRs of NF-L and NF-H mRNAs. The complexes were competed with poly(C) and are referred to as poly(C)-sensitive complexes. Their binding sites were localized to a 36 nt sequence in the mid-distal region of the NF-H 3'UTR and to a 45 nt sequence at the proximal edge of the 3'UTR of the NF-L transcript. Although the binding sites showed limited sequence homology, the complexes were cross-competed with unlabeled probes and radioactivity in either probe was cross-linked to a 43 kDa protein. The 43 kDa protein also bound directly to NF-L and NF-H probes in Northwestern blots. Functional studies showed that deletion of the binding sites markedly increased expression of a luciferase reporter gene containing the 3'UTR of NF-L or NF-H by stabilizing the fusion transcripts. Point mutations in the NF-H binding site which prevented formation of the poly(C)-sensitive complex also stabilized the fusion mRNA. The findings reveal a common destabilizing element in the 3'UTR of NF-L and NF-H mRNAs that may be important in coordinating NF subunit expression and in mediating the neuropathic effects of the NF-L and NF-H transgenes in transgenic mice.

Mutation in neurofilament transgene implicates RNA processing in the pathogenesis of neurodegenerative disease.

A mouse neurofilament light subunit (NF-L) transgene with a 36 bp c-myc insert at the end of the coding region was found to have neuropathic effects on enteric and motor neurons of transgenic mice. The severity of phenotype was related directly to the levels of transgenic mRNA expression. High levels of transgene expression were lethal to newborn pups, causing profound alterations in the development of the enteric nervous system and extensive vacuolar changes in motor neurons. Lower levels of transgene expression led to a transient stunting of growth and focal alterations of enteric and motor neurons. Because the positioning of the c-myc insert coincided with the location of the major stability determinant of the NF-L mRNA (Cañete-Soler et al., 1998a,b), additional studies were undertaken. These studies showed that the c-myc insert alters the ribonucleoprotein (RNP) complexes that bind to the stability determinant and disrupts their ability to regulate the stability of the transcripts. The findings indicate that expression of an NF-L transgene with a mutant mRNA stability determinant is highly disruptive to enteric and motor neurons and implicate alterations in RNA processing in the pathogenesis of a neurodegenerative condition.

Stability determinants are localized to the 3'-untranslated region and 3'-coding region of the neurofilament light subunit mRNA using a tetracycline-inducible promoter.

The tetracycline-responsive expression system of Bujard was used to compare rates of decay of wild-type and mutant neurofilament (NF) light subunit (NF-L) mRNAs. Optimal conditions for activation and inactivation of the target transgene were determined using a luciferase reporter gene. Analyses of mRNA stability were thereupon conducted on cells that were doubly transfected with transactivator and inducible target genes and derived from pooled clones of transfected cells. Rates of mRNA decay were compared upon inactivation of the transgenes after high levels of mRNA had been induced. Deletion of the 445-nucleotide (nt) 3'-untranslated region (3'-UTR) (L/++(+)-) or 527 nt of the 3'-coding region (3'-CR) (L/++-+) increased the stability of NF-L mRNA compared with the full-length (L/++(++)) transcript in neuronal (N2a and P19 cells) and non-neuronal (L cells) lines. Deletion of both the 3'-UTR and 3'-CR (L/++--) led to a further stabilization of the transcript. A major stability determinant was then localized to a 68-nt sequence that forms the junction between the 3'-CR and 3'-UTR of NF-L and is the binding site of a unique ribonucleoprotein complex (Cañete-Soler, R., Schwartz, M. L., Hua, Y., and Schlaepfer, W. W. (1998) J. Biol. Chem. 273, 12655-12661). The studies establish a novel system for mapping determinants of mRNA stability and have applied the system to localize determinants that regulate the stability of the NF-L mRNA.

Characterization of ribonucleoprotein complexes and their binding sites on the neurofilament light subunit mRNA.

Levels of neurofilament (NF) gene expression are important determinants of basic neuronal properties, but overexpression can lead to motoneuron degeneration in transgenic mice. In a companion study (Cañete-Soler, R., Schwartz, M. L., Hua, Y., and Schlaepfer, W. W. (1998) J. Biol. Chem. 273, 12650-12654), we show that levels of NF expression are regulated by altering mRNA stability and that stability determinants are present in the 3'-coding region (3'-CR) and 3'-untranslated region (3'-UTR) of the NF light subunit (NF-L) transcript. This study characterizes the ribonucleoprotein complexes that bind to the NF-L mRNA when cytoplasmic brain extracts are incubated with radioactive probes. Gel retardation assays reveal ribonucleoprotein complexes that are selectively competed with poly(C) or poly(U))/poly(A) homoribopolymers and are referred to as C-binding and U/A-binding complexes, respectively. The C-binding complex forms on the proximal 45 nucleotides of 3'-UTR, but its assembly is markedly enhanced by 23 nucleotides of flanking 3'-CR sequence. U/A-binding complexes form at multiple binding sites in the 3'-CR and 3'-UTR. A pattern of reciprocal binding suggests that the C-binding and U/A-binding complexes interact and may compete for common components or binding sites. Cross-linking studies reveal unique polypeptides in the C-binding and U/A-binding complexes. The findings provide the basis for probing mechanisms regulating NF-L mRNA stability and the relationship between NF overexpression and motoneuron degeneration in transgenic mice.

Characterization of the mouse neurofilament light (NF-L) gene promoter by in vitro transcription.

We have used in vitro transcription to access the basic sequences and factors required for the transcription of the mouse neurofilament light promoter (pNF-L) in the absence of chromatin structure. Deletion from -1.7 to -154 results in little change in NF-L promoter activity using nuclear extracts from either brain (expressing) or liver (non-expressing) tissues. Further deletion to -29 results in a gradual five-fold drop in promoter activity in both extracts. Only replacement of the entire -148 to -29 region results in a drop in NF-L promoter activity to basal levels. Thus, the NF-L promoter differs from the mouse NF heavy (NF-H) and mid-sized (NF-M) promoters in that no specific sequence within the immediate upstream NF-L promoter region (-154 to -29) appears to be responsible for enhancement or brain-specific transcription. We show that the order of strength of the three NF promoters is NF-H>NF-M>NF-L and identify sequences that can increase or reduce transcription when placed in front of heterologous NF promoters. We conclude that the NF-L promoter is a modular, weak and promiscuous promoter whose regulation differs from NF-H or NF-M. Our data suggest that chromatin structure may play an important role in the regulation of the NF-L promoter.

In vitro activation of the mouse mid-sized neurofilament gene by an NF-1-like transcription factor.

In vitro transcription using nuclear extracts from rat brain and liver were used to assess the tissue-specific and functional elements of the mouse neurofilament mid-sized gene promoter (pNF-M). Deletion from -2.7 to -103 (relative to the start site of transcription) resulted in a small increase (2-fold) in the activity of the NF-M promoter in both extracts. Promoter strength was slightly higher in brain vs. liver extracts. Deletion to -49 resulted in a 10-fold loss of promoter activity in brain extracts and 6-fold drop in liver. Transcription in both extracts was TATA box-dependent. The region between -65 and -40 was shown to contain sequences responsible for high-level NF-M promoter activity in brain and liver extracts. Within this region are Sp1 and NF-1-like binding sites. Mutation of the NF-1-like site (-53/-39) caused a large drop in the activity of the NF-M promoter while mutation of the Sp1 site (-64/-57) possibly slightly diminished promoter activity in brain and liver extracts. Both the Sp1 and NF-1-like sites were shown by gel shift competition and supershift assays to be able to bind their respective factors. We conclude that the basic mouse NF-M promoter is a promiscuous promoter whose activity is modulated by a NF-1-like transcription factor. The lack of tissue specificity in an in vitro system strongly suggests an important role for chromatin structure in the regulation of the mouse NF-M promoter.

Coordinate induction of the three neurofilament genes by the Brn-3a transcription factor.

The POU domain transcription factor Brn-3a is able to stimulate neurite outgrowth when overexpressed in the neuronal ND7 cell line, whereas the closely related Brn-3b factor does not have this effect. We show that Brn-3a overexpression also enhances the expression of the three neurofilament genes at both the mRNA and protein levels, whereas Brn-3b overexpression has no effect. In addition Brn-3a activates the three neurofilament gene promoters in co-transfection assays in both neuronal and non-neuronal cells. As observed for enhanced neurite outgrowth, the stimulation of neurofilament gene expression and activation of the neurofilament gene promoters is observed with the isolated POU domain of Brn-3a. A single amino acid change in the POU homeodomain of Brn-3a to the equivalent amino acid in Brn-3b abolishes its ability to activate the neurofilament promoters, whereas the reciprocal change converts Brn-3b to an activator of these promoters.

Transient expression of the neurofilament proteins NF-L and NF-M by Schwann cells is regulated by axonal contact.

Expression of the genes that encode neurofilament proteins is considered to be confined normally to neurons. However, in demyelinating peripheral nerves Schwann cells upregulate the mRNA for the medium-sized neurofilament protein (NF-M), and cultured Schwann cells of the myelin-forming phenotype can also synthesize and incorporate NF-M protein into their intermediate filament (IF) cytoskeleton. The purpose of this study was to establish how axonal contact might influence glial neurofilament gene expression and regulate the synthesis of neurofilament proteins. We show that the gene encoding NF-M is expressed at early stages of differentiation in myelin-forming Schwann cells in vivo; nevertheless, little NF-M protein can be detected in these cells. The transient induction of NF-M mRNA is also apparent in dedifferentiating Schwann cells during Wallerian degeneration. In these Schwann cells the mRNAs for NF-M and NF-L (the smallest polypeptide), but not NF-H (the largest neurofilament subunit), are coordinately expressed. In contrast to differentiating myelin-forming Schwann cells, the cells of degenerating nerves express both NF-M and NF-L polypeptides. Restoration of axonal contact in the growing nerve stimulates the recapitulation of Schwann cell differentiation including the elevation of NF-M and NF-L mRNA expression. These results demonstrate that the transient induction of neurofilament mRNAs in Schwann cells is a feature of both differentiation and dedifferentiation. However translation of these mRNAs is confined to Schwann cells deprived of axonal contact either by nerve injury or by culture in the absence of axons. These findings suggest that the expression of the NF-M and NF-L polypeptides is an important characteristic of those Schwann cells that will contribute to the repair of damaged peripheral nerves.

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.

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.

Brain-specific enhancement of the mouse neurofilament heavy gene promoter in vitro.

We have investigated the DNA elements responsible for transcription from the proximal portion of the mouse neurofilament heavy gene (NF-H) promoter by in vitro transcription using extracts from expressing (brain) and non-expressing (liver) tissues. We have found that constructs containing 5' region from -1314 to -115 exhibit a 3-5-fold higher level of NF-H promoter activity, relative to the adenovirus major late promoter (pML), in brain versus liver extracts. Deletion to -85 lowers the level of brain transcription by 2-fold, while deletion from -65 through -31 reduces transcription by 5-fold to a relatively strong (10% of pML) basal level. Basal level expression is observed in all deletions transcribed with liver extract. Deletion to -24 (TATA-less) abolishes promoter activity with both extracts. Deletion of the -115 to -65 region from a larger construct reduces transcription in brain extracts to basal levels, suggesting that this region contains the elements necessary for the brain-specific enhancement of promoter function. Mutation of a palindromic sequence within this region abolishes brain-specific enhanced promoter activity. This loss of enhanced transcriptional activity is correlated with the loss of a shifted band in gel shift assays. Our studies suggest that the sequence (-106)GGGGAGGAGG-(15 bp)-CCTCCTCCCC(-72) (where bp = base pairs) is important in brain-specific enhancement of transcription from the mouse NF-H promoter.

Methylation and expression of neurofilament genes in tissues and in cell lines of the mouse.

The light (NF-L), mid-sized (NF-M) and heavy (NF-H) neurofilament (NF) genes were probed with methylation-sensitive restriction enzymes and patterns of methylation and expression of the NF genes were compared in tissues and cell lines of the mouse. The 5' regions of all three NF genes are identified as CpG islands that remain unmethylated in expressing and non-expressing tissues, although partial methylation occurs at -795 in NF-H and at -525 in NF-M. Methylation of the NF CpG islands is associated with the inactivation of NF genes in L cells and with the selective inactivation of NF-L and NF-M in Neuro 2a cells. We also show that methylation diminishes the ability of the NF promoters to drive transcription of a CAT reporter gene. Hence, the presence of CpG islands may be important in determining patterns of NF transcription in vitro. Moreover, the preservation of CpG islands may be an evolutionary link that bears upon the nature of the NF genes and the mechanisms that have evolved to limit NF expression.

Actinomycin prevents the destabilization of neurofilament mRNA in primary sensory neurons.

The levels of light, mid-sized, and heavy neurofilament (NF) mRNAs were compared to that of beta-actin mRNA in primary dissociated cultures of adult rat dorsal root ganglia (DRG). Decreases in the levels of all three NF mRNAs occur after 24 h in culture, mimicking the down-regulation of NF mRNAs in axotomized DRG neurons. The loss of NF mRNAs in DRG cultures is prevented by actinomycin and, to a lesser extent, by cycloheximide. Based on decay curves in actinomycin-treated cultures, the half-lives of NF mRNAs are at least 4 days in DRG neurons, but < 24 h in PC12 cells. Our data support the view that NF mRNAs are stabilized in DRG neurons and that stabilization prevents destabilization by a transcription-dependent process. We further propose that putative stabilizing factor(s) are able to prevent degradation of NF transcripts in intact neurons, but not in axotomized or cultured neurons.

Negative regulatory regions are present upstream in the three mouse neurofilament genes.

We have cloned and examined the 5' flanking regions of the heavy (NF-H), light (NF-L) and mid-sized (NF-M) mouse neurofilament (NF) genes in order to begin to characterize the regions of each gene that regulate NF transcription. Chimeric plasmids bearing the CAT reporter gene and deletion mutants of the upstream NF genes were transiently transfected into neuronal (PC12 and Neuro 2A) and non-neuronal (HeLa) cell lines. Constructs bearing upstream regions to -4000 in NF-H, to -5600 in NF-L and to -4500 in NF-M were expressed at low levels in neuronal and in non-neuronal cells. Progressive deletion of 5' flanking sequence to -385 in NF-H, to -325 in NF-L and to -505 in NF-M caused a several-fold increase of transcription from the transfected plasmids. Increases of transcription by deletion mutants followed a similar pattern in neuronal and in non-neuronal cell lines. Negative upstream regions are located between -1314 and -385 in NF-H, between -936 and -325 in NF-L and between -874 and -505 in NF-M. Additional negative regions are present further upstream in NF-L and in NF-H. The negative regions of NF-H and of NF-L suppress transcription when placed in either orientation in front of the SV40 or a heterologous NF promoter. These studies demonstrate that the three mouse NF genes possess similar functional features, namely, that of a relatively strong and promiscuous promoter with negative upstream elements. The role of the negative elements in regulating NF expression remains unclear.

Two-stage autolysis of the catalytic subunit initiates activation of calpain I.

Calcium-induced autolysis of bovine erythrocyte calpain I occurs in multiple stages. Initially, a 14 amino acid segment is cleaved from the N-terminus of the native 80 kDa catalytic subunit, yielding a 78 kDa form of the subunit. Then, an additional 12 amino acid segment is cleaved from the N-terminus, forming a 76 kDa subunit. The 76 kDa enzyme is the active form of the catalytic subunit that is able to proteolyze the 30 kDa regulatory subunit as well as exogenous substrates. While the initial autolytic step requires high calcium, the 76 kDa enzyme form is active in microM calcium and can cleave the amino termini of native 80 kDa and intermediate 78 kDa enzyme forms at low calcium. Both intramolecular and intermolecular proteolysis of the catalytic subunit appear to yield the same products.

Axonal dependency of the postnatal upregulation in neurofilament expression.

A coordinated up-regulation in the expression of all three neurofilament (NF) proteins occurs during postnatal development in the rat (Schlaepfer and Bruce, J Neurosci Res [in press], 1990a). In the present study, sciatic nerves were transected in neonatal rats in order to determine the effects of axotomy on the postnatal upregulation of NF expression in neurons of rat dorsal root ganglia (DRG). Left sciatic nerves were transected at postnatal day 3 (P3), 6 (P6), 8 (P8), or 10 (P10). mRNA and protein levels of the light (NF-L), mid-sized (NF-M), and heavy (NF-H) NF proteins were compared in L4 and L5 DRGs from the transected (left) vs. control (right) sides of the same animals at varying intervals after transection. When nerves were transected at P10, mRNA levels of all three NF proteins declined markedly in the parent DRG neurons, thereby completely interrupting the postnatal upregulation of NF expression. P10 transections also led to widespread chromatolytic changes in axotomized neurons, indistinguishable from those that occur in adult DRG following sciatic nerve transection (Goldstein et al., J Neurosci 7:1586-1594, 1987). Nerve transections at earlier (e.g., P3) neonatal timepoints also led to a decrease of NF expression, but to a lesser extent than that which resulted from a P10 transection. Also, P3 transections caused only minimal chromatolytic changes in the axotomized neurons. Thus, the postnatal upregulation of NF expression is dependent upon axonal continuity and the extent of axonal dependency increases during early postnatal development. These findings support the hypothesis that the postnatal upregulation of NF expression, the axotomy-induced downregulation of NF expression and the chromatolytic reaction to nerve transection are all dependent upon or responsive to axonal- or target cell-derived signals that are acquired during postnatal maturation.

Neurofilament proteins are distributed in a diminishing proximodistal gradient along rat sciatic nerve.

Neurofilament (NF) proteins are distributed in a diminishing proximodistal gradient along rat sciatic nerve when compared with total noncollagen or other proteins in nerve. About a twofold decline of NF proteins can be detected by quantitating nerve proteins that have been separated by gel electrophoresis. A similar decrease of immunoreactivity to each NF subunit is seen in distal nerve segments when noncollagen nerve proteins are immunoblotted. Parallel decreases occur in all three NF proteins, thereby maintaining neurofilament subunit stoichiometry along the neuraxis. The same NF gradient can be detected when the NF contents in nerve branches to the gluteus and gastrocnemius muscles are compared with each other and with those in nerve segments taken from the same proximodistal levels of the parent sciatic nerve. The gradient of NF proteins increases during postnatal development and is readily detected by postnatal day 16. During the same period of development, the heavy NF subunit appears for the first time and is rapidly incorporated throughout the sciatic nerve. Hence, the NF gradient becomes manifest during the development and maturation of the adult form of the axonal cytoskeleton. The basis for the proximodistal gradient of NF proteins in peripheral nerve is presently unknown. The extent of the gradient cannot be accounted for on the basis of diminishing numbers of nerve fibers or increasing amounts of other nerve proteins, e.g., collagen, in distal nerve. An alternative interpretation is that the gradient reflects a low level of NF protein turnover during axonal transport.